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Understanding the Anaerobic Digestibility of Lignocellulosic Substrates Using Rumen Content as a Cosubstrate and an Inoculum
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  • Xavier Fonoll
    Xavier Fonoll
    Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
    Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain
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    Orcidhttp://orcid.org/0000-0003-3304-2437
  • Shilva Shrestha
    Shilva Shrestha
    Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
    Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Ma̅noa, Honolulu, Hawaii 96822, United States
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  • Samir Kumar Khanal
    Samir Kumar Khanal
    Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Ma̅noa, Honolulu, Hawaii 96822, United States
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    Orcidhttp://orcid.org/0000-0001-6680-5846
  • Joan Dosta
    Joan Dosta
    Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain
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  • Joan Mata-Alvarez
    Joan Mata-Alvarez
    Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain
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  • Lutgarde Raskin*
    Lutgarde Raskin
    Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
    *Email: [email protected]
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Cite this: ACS EST Engg. 2021, 1, 3, 424–435
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https://doi.org/10.1021/acsestengg.0c00164
Published January 31, 2021

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Abstract

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While rumen microorganisms are known to facilitate the hydrolysis of lignocellulosic substrates in anaerobic digestion (AD), it is unclear how rumen content can be used to maintain rumen microorganisms in continuous-flow AD systems. We used rumen content as either an inoculum or a cosubstrate in two separate AD experiments, and performed microbial and multivariate statistical analyses to study how to best use this resource to enhance AD. Hydrolytic bacteria such as Fibrobacter spp. remained present for two months of reactor operation when the rumen content was used as an inoculum, producing 0.3–0.5 g of short-chain fatty acids (SCFA) as acetic acid per gram of volatile solids (VS)fed. The lack of acetoclastic methanogens in the rumen content initially led to the accumulation of SCFA (10–15 g of SCFA as acetic acid per liter) as well as a low pH (5.9–6.8) and a low methane yield (0.02–0.05 L of CH4 per gram of VSfed). The reactor performed similarly (0.1–0.2 L of CH4 per gram of VSfed) to the control, which was not inoculated with rumen content, toward the end of the experiment, and the microbial analyses showed a washout of the rumen bacteria. Rumen hydrolytic bacteria remained in the reactor during the codigestion experiment. However, the methane yield (0.1–0.2 L CH4 per gram of VSfed) was similar to that of the control reactor, which did not receive the rumen content as a cosubstrate, because the reactor conditions (pH > 6.9) were not favorable for the activity of rumen bacteria. Our analyses suggest that using rumen content as a cosubstrate at a low pH (6.0–6.5) is necessary to maintain rumen hydrolytic bacteria and enhance hydrolysis.

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Introduction

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The anaerobic digestion (AD) of a variety of biomass streams has been implemented worldwide to reduce waste, produce energy, and mitigate greenhouse gas emissions. Approximately 2200 AD plants are operational in the U.S., (1) and regulatory changes in regard to waste management in some U.S. states are expected to further expand AD applications. (2,3) In Europe, approximately 18 000 AD plants are operational, with an installed electric capacity of 10 000 MW. (4) The number of European AD plants has been steadily increasing due to legislation related to the treatment and disposal of organic wastes (European directive 2008/98/EC) as well as the implementation of renewable energy directives (European directive 2009/28/EC). These data suggest that AD is a mature technology that has the potential to continue to expand across the world.
One of the bottlenecks of AD, especially for substrates with high lignocellulosic content, is hydrolysis. (5) The cross-linking between cellulose, hemicellulose, and lignin makes the lignocellulosic biomass highly recalcitrant to microbial degradation. Lignocellulosic biomass streams are readily available as they are produced in urban environments (e.g., food, paper, and park waste streams), food and beverage industries (e.g., fruit and vegetable processing waste), and rural areas (e.g., energy crops and agricultural waste streams). Various physical, chemical, and biological pretreatment methods as well as combinations of these approaches have been applied to enhance hydrolysis. (5) While such pretreatment strategies can be effective, they are often energy intensive and sometimes generate biomass degradation byproducts (e.g., phenolic and furfural compounds), which inhibit AD microbial communities. (5)
Nature continuously demonstrates that the efficient degradation of lignocellulosic substrates is feasible in anaerobic environments without high energy inputs. (5,6) For example, the microbial community in one of the stomachs of ruminants (rumen) degrades lignocellulosic biomass (grass) efficiently under anaerobic conditions. (7) Rumen content has been used as an inoculum and a cosubstrate for different types of laboratory-scale AD systems since the late 1980s. (8−33) Its use as an inoculum in batch experiments demonstrated that rumen populations can enhance hydrolysis in AD. (23−25) However, in continuous-flow systems that used rumen content as an inoculum, rumen microbial populations were typically washed out over time, and hydrolysis was only enhanced at the beginning of the experiments. (16,24) Only Agematu et al. (22) were able to maintain rumen bacteria for 60 days in a continuous-flow bioreactor seeded with rumen content. Using rumen content as a cosubstrate could be a strategy to avoid the wash-out of rumen microbial populations and maintain a high hydrolysis rate in continuous-flow AD systems. Wall et al. (15) examined rumen content and grass silage codigestion; however, they demonstrated a similar performance in terms of methane production and the removal of volatile solids (VS) with and without the addition of rumen content. While the addition of rumen content has the potential to improve hydrolysis in AD, it is unclear which conditions are needed for rumen microbial populations to grow in a continuous-flow bioreactor.
The aim of this study was to evaluate the use of rumen content as an inoculum and a cosubstrate and establish the conditions necessary to enhance the hydrolysis of lignocellulosic biomass in AD. The use of high-throughput 16S rRNA gene sequencing and a multivariate statistical analysis suggested new strategies for maintaining rumen microbial populations and thus for enhancing hydrolysis in AD systems.

Materials and Methods

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Two experiments were conducted to understand how the anaerobic digestibility of lignocellulosic substrates is affected by the availability of rumen microbial populations. In one experiment, rumen content was used as the inoculum. In a second experiment, rumen content was used as a cosubstrate. Both experiments were run in semicontinuously fed stirred-tank reactors.

Inocula

Two types of inocula were used in this study. One inoculum consisted of the effluent from a full-scale AD plant treating food waste and cow manure at Michigan State University, East Lansing, MI, USA, (34) and is referred to as AD inoculum. The second inoculum consisted of rumen content collected from a fistulated cow from a dairy farm at Michigan State University. Solid and liquid fractions of the rumen content were collected separately and then mixed according to the ratio needed to simulate the rumen content generated in a slaughterhouse (total solids (TS) concentration of approximately 120 g L–1). (35,36) Both AD and the rumen content inocula were used and characterized (Table S1) within 1 h of collection using the methods described below.

Substrates

Four different substrates were used for this study. Napier grass (Pennisetum purpureum), a second generation lignocellulosic energy crop, was used as the primary substrate. Napier grass was hand-harvested from the Waimanalo Research Station (Waimanalo, HI, USA), shredded using a cutting mill (Vincent Corporation, Tampa, FL, USA), and air-dried to reduce the moisture content to less than 10% to facilitate downstream processing. The dried substrate was then milled using a laboratory cutting mill (Retch SM2000, Haan, Germany) with a screen size of 2 mm. The second substrate consisted of cow feces collected from the same dairy farm at Michigan State University. The third substrate was cow manure, a mixture of cow feces and urine, that was collected from the same farm. Cow manure was used in this study as a cosubstrate to supplement nutrients and balance the C/N ratio of the mixture. (37) Cow feces and urine were collected when cows were defecating and urinating, respectively. Cow feces and urine were mixed at a mass ratio of 30:70 (wet weight) to represent the concentration of TS in the cow manure fed to the full-scale AD plant at Michigan State University. The fourth substrate consisted of the rumen content collected and mixed as described above. All substrates were characterized (Table S2) using the methods described below and were stored at 4 °C except for the rumen content, which was stored at −20 °C until use. While keeping the rumen content at −20 °C did not affect the microbial fermentation patterns in previous studies, (38) preserving the samples at −20 °C might have decreased the microbial load in the cosubstrate.

Bioreactor Operating Conditions

For both experiments, three 2 L bottles with a working volume of 1.3 L were operated as semicontinuously fed stirred-tank reactors in a shaking water bath (gyrotory water bath shaker g76, New Brunswick Scientific, NJ, USA) at 130 rpm. The reactors were briefly opened once a day after stopping agitation both to remove digestate and for feeding. Even though some air exposure was inevitable, oxygen was expected to be quickly consumed by the facultative microbes without affecting the anaerobic environment. (39) The reactors were operated at 39 °C to simulate the rumen temperature. (36)
For the inoculum experiment, the three reactors were filled with 1.3 L of inocula. Reactor Inoc_AD received the AD inoculum (control), reactor Inoc_RC+AD was filled with a 50:50 (VS/VS) mixture of AD and the rumen content inocula, and reactor Inoc_RC was inoculated with the rumen content only. All reactors were inoculated within 1 h after the inocula were collected and transported to the laboratory. The rumen content was diluted with distilled water so that the VS content of the inoculum was the same in all three reactors (25.2 ± 3.7 g VS L–1). The C/N ratio of the individual substrates was estimated through different studies from the literature. (40−43) To have a C/N ratio between 20 and 30 in the feedstock, the three reactors were fed with a 30:70 (wet weight) mixture of Napier grass and either cow feces (first 80 days) or cow manure (days 81–140). The cow feces and cow manure were diluted with distilled water to reach 60 g VS L–1 and provide an optimal rheology to facilitate analyses and reactor mixing. Initially, buffers were added to Inoc_RC+AD and Inoc_RC to maintain the pH around 7.0. NaHCO3 was added to Inoc_RC on days 1 (4.0 g), 3 (2.0 g), and 4 (2.0 g). However, to avoid Na+ toxicity, (44) NH4HCO3 was added on days 5 (6.0 g), 8 (2.0 g), 22 (5.0 g), and 37 (5.0 g) to Inoc_RC and on days 7 (2.0 g) and 44 (2.0 g) to Inoc_RC+AD. When adding NH4HCO3 in Inoc_RC and Inoc_RC+AD, the C/N ratio changed from 20 to 10; however, the addition of NH4HCO3 was not frequent enough to have a negative effect on the system stability. Starting from day 81, the cow urine was mixed with cow feces (referred to as cow manure) as described above to increase the buffering capacity of the substrate.
For the codigestion experiment, the general operating conditions were similar to those of the inoculum experiment except that all three reactors were inoculated with 1.3 L of AD inoculum and fed with different substrates. Reactor Co_NG+CM (control) was fed with a mixture of Napier grass and cow manure (30:70, wet weight); reactor Co_NG+CM+RC was fed with a mixture of Napier grass, cow manure, and rumen content (25:60:15, wet weight); and reactor Co_NG+RC was fed with a mixture of Napier grass and rumen content (85:15, wet weight). The amount of rumen content that was fed was determined by considering the average production of rumen content in the US and the average amount of lignocellulosic feedstock fed in AD plants (see Section S1). Before feeding the rumen content, it was thawed in a water bath at 39 °C. Again, all substrate proportions generated a feedstock C/N ratio of 20–30 according to the literature data. (40−43) NH4HCO3 was added daily to Co_NG+RC from day 44 to keep the pH at 7.0. The operating conditions of the six reactors are summarized in Table S3.

Chemical Analyses

TS, VS, alkalinity, SCFA, free ammonia, and biogas production and composition were analyzed regularly as described in Section S2.

Biomass Sampling, DNA Extraction, and 16S rRNA Gene Sequencing

Biomass sample preservation and DNA extraction methods are described in Section S3. DNA samples were submitted to the Host Microbiome Initiative (University of Michigan, Ann Arbor, MI, USA) for 16S rRNA gene sequencing via Illumina MiSeq (San Diego, CA, USA). Universal primers F515 and R806 that target the V4 region of the 16S rRNA gene were used for PCR amplification. (45) The sequences obtained were processed using mothur (ver. 1.34.4) following the MiSeq SOP. (46) The SILVA database (Release 119) was used for taxonomic classification, and sequences were grouped into operational taxonomic units (OTUs) using the average neighbor algorithm at a 3% sequence divergence cutoff. Unclassified OTUs were further classified using the basic local alignment search tool (BLAST; NCBI, Bethesda, MD), and only the hits with a query cover of 100% and an identity greater than 90% were selected. Sequences for which no significant match was found in the databases using BLAST were termed “unclassified”. The relative abundance was calculated by dividing the number of reads of a particular OTU or genus by the total number of reads in the sample. The raw sequence data were submitted to NCBI with accession number SUB8879343.

Statistical Analyses

Performance data of the reactors within each study were compared using the Kruskal–Wallis test (nonparametric one-way ANOVA). The comparison of the reactor performance though a Kruskal–Wallis statistical test was performed with data collected for at least 14 days. To allow for comparisons among samples represented by different numbers of sequences, subsampling (as implemented in mothur) was performed using the sample with the lowest number of sequences, i.e., 4729 reads for the inoculum study and 6454 reads for the codigestion study. β-Diversity was represented in a two-dimensional graph using nonmetric multidimensional scaling (NMDS) analyses based on the Bray–Curtis dissimilarity index as implemented in mothur. A canonical correspondence analysis (CCA) was performed to study the relationship between the microbial community structure and various chemical conditions and operational parameters. Parameters with multicorrelations were removed if the variance inflation factor value was higher than 10. (47) The optimum CCA model was selected after applying Akaike’s information criterion, and its significance was tested with ANOVA. (47,48) The variance inflation factor calculation, Akaike’s information criterion, and the CCA were performed using the vegan package (2.4–4) in R 3.3.3. (47) For the CCA inoculum model, the parameter “Rumen content only as inoculum” was introduced to evaluate if mixing the rumen content and the AD inoculum had an effect on the differences among the microbial communities in the reactors. For the CCA codigestion model, the parameter “Rumen content only as cosubstrate” was introduced to evaluate if mixing the rumen content and the cow manure had an effect on the differences among the microbial communities in the reactors. Both parameters were yes or no parameters. A significance level of α ≤ 0.05 was used to interpret the significance of the results in all the statistical tests. While other studies have shown that long-term experiments can help address concerns when operating anaerobic digesters in replicates is not possible, (49,50) we acknowledge that the lack of replicates is a limitation of the statistical analysis.

Results and Discussion

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Using the Rumen Content as an Inoculum Enhances Hydrolysis But Limits Stable Methanogenesis

Performance results of the inoculum experiment for reactors Inoc_AD, Inoc_RC+AD, and Inoc_RC, all three of which were fed the same mixture of Napier grass and cow feces or manure, are presented in Figures 1, S1, and S2. During the first two weeks of operation, the SCFA accumulation (grams of SCFA per gram of VS–1fed) for reactors Inoc_RC and Inoc_RC+AD were significantly higher than for Rreactor Inoc_AD (p = 1.4 × 10–04 for Inoc_RC vs Inoc_AD and p = 3.77 × 10–03 for Inoc_RC+AD vs Inoc_AD) (Figure 1A). Specifically, the reactors inoculated with the rumen content (Inoc_RC and Inoc_RC+AD) produced a maximum of 0.79 g of SCFA as acetic acid (HAc) per gram of VSfed and 0.57 g of SCFA as HAc per gram of VSfed, respectively (Figure 1A). This indicates that the rumen content provided hydrolytic microbes that accelerated the hydrolysis step, producing high levels of SCFA. The SCFA accumulation by Inoc_RC was especially high considering that acidogenic reactors typically produce 0.15–0.55 g of SCFA as HAc per gram of VSfed. (51−53) Moreover, in previous studies that used rumen content as the inoculum and lignocellulosic biomass as the substrate, the SCFA accumulation ranged from 0.18 to 0.22 g of SCFA as HAc per gram of VSfed. (22,23,25) The reason for a higher SCFA accumulation in our study could be our use of a mixture of rumen fluid and solids instead of only rumen fluid like in other studies. Including the solids from the rumen content to accelerate hydrolysis is important because they harbor a higher concentration of hydrolytic microbes than the fluid. (7)

Figure 1

Figure 1. (A) Short-chain fatty acid (SCFA) accumulation, (B) pH, and (C) specific methane production in the reactors. Organic loading rate (OLR) and retention time in Inoc_AD, Inoc_RC+AD (−·−), and Inoc_RC (− −). The vertical line indicates the change from cow feces to cow manure on day 81. The red arrow and green lines with circles indicate the buffer addition in Inoc_RC and Inoc_RC+AD, respectively. The legend refers to the three reactors with the type of inocula used.

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The high hydrolytic activity led to high SCFA levels in reactor Inoc_RC (10.8 ± 3.0 g of SCFA as HAc L–1 in the first 50 days), which affected the stable methanogenesis. The low partial alkalinity (826 ± 142 mg of CaCO3 L–1) and pH (5.4 ± 0.2) in the rumen content (Table S1) and the low pH in Inoc_RC (5.0–6.8 during the first 50 days) were also not favorable for the growth of acetoclastic methanogens. These conditions are not typical in the rumen, where SCFA concentrations are around 7 g of SCFA as HAc L–1 with a hydraulic retention time of 5–20 h and there is the continuous removal of SCFA via absorption through the rumen walls. (36,54) Therefore, a buffer in the form of NaHCO3 or NH4HCO3 was added several times in an attempt to increase the pH and restore the reactor stability (Figure 1B). The frequent addition of NH4HCO3 resulted in an increase in the free ammonia concentration in the reactor; however, it did not reach inhibitory levels for methanogens (<124 mg of NH3–N L–1), and the concentration was still low in comparison to the optimal levels in the rumen (41–248 mg of NH3–N L–1) (Figure S1C). (36,44) However, since the SCFA levels remained high, the pH only increased temporarily (Figures 1B and S1A). The high concentration of SCFA (10–16 g of SCFA as HAc L–1) (Figure S1A) and low pH (5.8–6.8) (Figure 1B) resulted in a very low methane yield (0.02 ± 0.02 L of CH4 per gram of VSfed, 57 ± 4% CH4) (Figure 1C) during the first 50 days of operation for reactor Inoc_RC. The situation changed between days 49 and 56 when the pH increased from 6.3 to 7.4 (Figure 1B), the partial alkalinity increased from 1.7 to 4.2 g of CaCO3 L–1 (Figure S1B), the free ammonia levels increased from 4.9 to 44.6 mg of NH3–N per liter (Figure S1C), the SCFA accumulation decreased (Figure 1A), and the specific methane production increased (Figure 1C). The degradation of SCFA (especially acetate, Figure S2A) seems to be related to an increase in the pH and not an increase in the levels of free ammonia because Inoc_RC+AD and Inoc_AD presented very low acetate (<0.5 g of HAc L–1) and free ammonia levels (<10 mg of NH3–N per liter) in between days 25 to 54.
The specific methane production in reactor Inoc_RC+AD was higher during the first 30 days compared to those of Inoc_RC (p = 5 × 10–07) and Inoc_AD (p = 3 × 10–04) (Figure 1C). This may be explained if the hydrolytic microbes originating from the rumen content hydrolyzed the Napier grass into SCFA, which would be converted to methane by methanogens in the AD inoculum. A previous batch study also reported a higher methane production when the rumen content was mixed with an adapted inoculum from an anaerobic digester. (25) Inoc_RC+AD also exhibited a greater stability with respect to pH (Figure 1B) and SCFA levels (Figure S1A) compared to reactor Inoc_RC; alkalinity as NH4HCO3 was supplemented only on days 7 and 44 when the pH dropped below 6.5 (Figure 1B). Reactor Inoc_AD took 32 days to adapt to the new substrate, as indicated by an increase in the specific methane production to 0.15 L of CH4 per gram of VSfed and SCFA levels of 1.7 g of SCFA per liter, which are adequate for methanogenesis. (55) The performance of reactor Inoc_AD did not differ from the performance of reactor Inoc_RC+AD after day 44 (Figures 1, S1, and S2).
Starting from day 81, cow manure was fed to all three reactors instead of cow feces. This change increased the buffering capacity (Figure S1B), the pH (Figure 1B), and the free ammonia levels without reaching inhibitory conditions (<124 mg of NH3–N per liter) (Figure S1C). Since all three reactors were operating at a steady state for almost 40 days, their retention times were decreased to 20 days to study the performance under a shorter retention time. The retention time was slowly decreased to avoid organic overloading. From day 95, the three reactors demonstrated similar performances with a specific methane production of 0.16 ± 0.02 L of CH4 per gram of VSfed (Figure 1). All parameters remained the same as those reported for the previous conditions except the specific methane production in Inoc_RC. The specific methane production in Inoc_RC was higher before (0.21 ± 0.03 L of CH4 per gram of VSfed from day 61 to 90), probably due to the longer retention time (60 vs 20 days) that allowed a better degradation of the organic matter.

Using the Rumen Content As an Inoculum Provides Hydrolytic Bacteria But They Are Washed Out while Promoting Stable Methanogenesis

On day 7, when the of SCFA accumulation in Reactor Inoc_RC was 0.79 g of SCFA as HAc per gram of VSfed (Figure 1A), >90% of OTUs present in the reactor were also present in the rumen inoculum (Figure 2).

Figure 2

Figure 2. Relative abundances of OTUs grouped by origin in reactors Inoc_RC, Inoc_RC+AD, and Inoc_AD. OTUs were grouped in eight categories depending on whether they were present in the rumen content inoculum only, the AD inoculum only, the cow manure only, various combinations of these three sources, or none of them (“Others”). Samples from day 28 for Inoc_RC+AD and Day 75 for Inoc_AD could not be sequenced.

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However, these OTUs were not exclusively present in that rumen content, as 1.2%, 17.9%, and 8.5% of OTUs were present in the rumen content and the AD inocula, the rumen content inoculum and the cow manure, and all three sources, respectively. The predominant OTUs in Inoc_RC present on day 7 belonged to genera that are commonly found in the rumen and are involved in the hydrolysis of cellulose (Fibrobacter) (56−58) and the fermentation of its products (Treponema, Prevotella, and Succiniclasticum) (Figure S3). (56−60) A recent study reported that Fibrobacter succinogenes produces an array of hydrolytic enzymes that are attached to the cell surface or transported to the extracellular environment by means of outer membrane vesicles, which protect the enzymes from external inhibitors. (57)Fibrobacter was indeed present at a relative abundance higher than 3.0% in reactor Inoc_RC during the first three weeks of the experiment (Figure S3) when hydrolysis was rapid. These results suggest that the addition of the rumen content had a positive effect on hydrolysis and that Fibrobacter likely contributed to this.
Methanobrevibacter, an obligate hydrogenotrophic methanogen that is prevalent in the rumen, was the most abundant archaeal population (relative abundances of 2.5–7.0%) in reactor Inoc_RC until day 34 (Figure S4). The absence of syntrophic bacteria (e.g., Syntrophomonas), syntrophic acetate-oxidizing bacteria (e.g., Thermotoga), and acetoclastic methanogens (e.g., Methanosarcina and Methanothrix) in the rumen content (Figures S3, S4, and S5) explains the accumulation of SCFA and the low pH in reactor Inoc_RC during the first 50 days of operation (Figure 1A and B).
Reactor Inoc_RC experienced two substantial shifts (from day 34 to 56 and from day 56 to 75) in the community structure, as shown in the NMDS plot in Figure S6. The first shift took place between days 34 and 56 and corresponded to a dramatic change in the reactor performance (Figure 1). The OTUs coming from only the rumen content decreased from 45% to 15%, and the OTUs originally found in the AD inoculum increased from 22% to 43% (Figure 2). The decrease in the SCFA accumulation starting from day 55 might be due to the increase in the relative abundance of OTU 108 (Methanosarcina), a methanogen capable of degrading acetate, hydrogen, methylamine, and methanol (Figure S4).
After day 75, the relative abundance of OTUs that were not detected in the biomass sources (“Others”) became a substantial portion of the community (40–48%) in Inoc_RC (Figure 2). These OTUs originated from the cow manure, the AD inoculum, or the rumen content but were not detected in these sources, likely because their relative abundances were low and below the detection limit obtained with the current sequencing depth (Figure S7). These OTUs were detected as the reactor conditions became more favorable for their growth and their relative abundances increased. Most of the abundant OTUs in reactor Inoc_RC (e.g., Alkaliflexus, Petrimonas, and Sedimentibacter) were acidogenic microorganisms involved in the production of SCFA. Interestingly, Sedimentibacter has also been found in other systems that used rumen content as the inoculum. (20,22,24,61)
Reactor Inoc_AD initially had 91.4% of its OTUs associated with the AD inoculum (Figure 2). A community shift took place after day 34 (Figure S6), and OTUs that were not detected previously in any of the biomass sources (“Others”) increased in relative abundance (40–60% of the total OTUs) (Figure 2). The predominant OTUs associated with the AD inoculum remained in the reactor throughout the experiment except for OTU 7 (Sedimentibacter) and OTU 25 (unclassified Firmicutes), which disappeared after day 34 (Figure S5).
The microbial community structure in reactor Inoc_RC+AD was more dynamic than those in the other reactors during the first 50 days (Figure S6). On days 7 and 34, the OTUs coming only from the rumen represented 29.6% and 21.0% of total number of OTUs, respectively. The only parameter in Inoc_RC+AD that was identical on days 7 and 34 but different from days 21 and 56 was pH, which was 6.5 (Figures 1, S1, and S2). On days 7 and 34, the OTUs associated with the rumen content were similar to those in Inoc_RC on day 34 (Figure S3). However, when the pH was 7.0 on day 21, only 6.5% of the OTUs originally present only in rumen content were also in Inoc_RC+AD (Figures 2 and S3). These observations suggest that rumen bacteria are strongly impacted by the pH. The microbial community structure in Inoc_RC+AD became similar to that in Inoc_AD after day 56 (Figure S6). At this time, the OTUs that were not initially detected in the rumen content, cow manure, or AD inoculum (“Others”) became a large portion of the total community (40–68%) in Inoc_RC+AD and Inoc_AD (Figure 2).
At the end of the experiment, the microbial community structures in all three reactors were similar, as shown in the NMDS plot (Figure S6), which is consistent with the observation of similar reactor performance results. In all three reactors, a significant part of the OTUs (45–55%) belonged to the fraction “Others”. Most of the OTUs present at high relative abundances in the three reactors on day 140 were unclassified; however, those that were classified and present at a relative abundance higher than 1.5% were related to Petrimonas (OTU 31), Treponema (OTUs 3, 61, and 81), Alkaliflexus (OTU 6), Bacteroides (OTU 50), and Methanospirillum (OTU 198) (Figures S3 and S5). Bacteroides is a common genus in biogas plants, where species such as Bacteroides cellulosolvens and Bacteroides xylanolyticus are able to degrade cellulose and xylan, respectively. (62)

Using the Rumen Content as the Only Cosubstrate Compromises Methanogenesis Stability But Keeps Rumen Bacteria in the Reactor

In a second experiment, all reactors were inoculated only with an AD inoculum, but the addition of rumen content as a cosubstrate was evaluated. Reactors Co_NG+RC and Co_NG+CM+RC were operated to evaluate whether the hydrolytic activity of the rumen bacteria could be retained through the continuous addition of rumen content. Reactor Co_NG+CM was operated to represent the codigestion of crops and manure, the most common practice in agriculture. (37) Despite the use of rumen content, hydrolysis was not enhanced, as was apparent from the low SCFA accumulation (maximum of 0.07 g of SCFA per gram of VSfed) and methane production (0.12 ± 0.04 L or CH4 per gram of VSfed from days 60–113) (Figure 3A and C).

Figure 3

Figure 3. (A) Short-chain fatty acid (SCFA) accumulation, (B) pH, and (C) methane production in the reactors showing (A) the organic loading rate (OLR) (− −) and (B) retention time (− −) applied in the reactors. The vertical line on day 44 indicates the start of continuous buffer addition.

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Moreover, none of the reactors experienced inhibitory conditions because the pH was never below 6.9 and the free ammonia levels were always below 124 mg of NH3–N per liter (Figures 3 and S8C). Nonetheless, Co_NG+RC had free ammonia levels that were not ideal for the development of rumen microbes (<41 mg of NH3–N per liter) (Figure S8C). To enhance the methane production, the retention time in all three reactors was increased from 20 to 30 days after a steady state was reached for 20 days on day 60 (Table S3). However, increasing the retention time to 30 days (OLR= 2.6 g of VS per liter per day) only enhanced the specific methane production slightly (from 0.10 ± 0.01 to 0.13 ± 0.02 L of CH4 per gram of VSfed) (Figures 3C).
Again, the microbial community structure was analyzed to help understand the reactor performance. During the first 23 days of the experiment, the microbial community structures in all three reactors were similar (Figure S9), with more than 80% of the reactor OTUs initially present in the AD inoculum (Figure 4). In fact, most of the abundant OTUs in the AD inoculum (OTUs 10, 6, 26, 25, 8, 32, 2, and 24) were also prevalent in the reactors (Figure S10).

Figure 4

Figure 4. Relative abundances of OTUs grouped by origin in reactors Co_NG+CM, Co_NG+CM+RC, and Co_NG+RC. OTUs were grouped in eight categories depending on whether they were present in the rumen content only, the AD inoculum only, the cow manure only, a combination of these three sources, or none of them (“Others”). Samples from day 72 for Co_NG+CM and day 51 for Co_NG+RC could not be sequenced.

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From day 23, the pH, partial alkalinity, and free ammonia levels started to decrease only in reactor Co_NG+RC (Figures 3B, S8B, and S8C). Starting from day 44, NH4HCO3 was added daily (100 mg) in Co_NG+RC to maintain the pH above 7.0 and thus avoid the inhibition of methanogens. This strategy allowed the establishment of both acetoclastic and hydrogenotrophic methanogens, promoting stable methanogenesis (Figures 3C and S11) while maintaining a free ammonia concentration below inhibitory levels (<124 mg of NH3–N per liter, Figure S8C). However, the free ammonia levels (5.5 ± 1.7 mg of NH3–N per liter) were still below the optimal level for rumen microbes (41–248 mg of NH3–N per liter), and no more NH4+ was added to maintain the C/N ratio within the optimal range of 20–30 for AD. Nevertheless, low free ammonia levels were not an impediment for rumen microbes to grow in Co_NG+RC, such as in Inoc_RC. The microbial community in reactor Co_NG+RC began to deviate from the communities present in reactors Co_NG+CM and Co_NG+CM+RC (Figure S9). Specifically, the fraction of OTUs associated only with the rumen content in Co_NG+RC increased from 1.7% to 17.8%, and some of the abundant OTUs in Co_NG+RC were present only in the rumen content, e.g., OTUs 28 and 147 (Prevotella), OTU 55 (Treponema), and OTU 71 (Fibrobacter) (Figures 4 and S12). Toward the last 30 days of the experiment, 26–36% of the OTUs in Co_NG+RC belonged only to the rumen content, 29–35% belonged to the AD inoculum and cow manure, and 27–30% belonged to the group “Others” (Figure 4). Again, the fraction “Others” contained OTUs that were present in the cow manure, AD inoculum, or rumen content but were not detected because their relative abundances were low, and they were not captured because of the current sequencing depth (Figure S13). These big changes in the microbial community seemed to be related to the decrease in the pH, partial alkalinity, and free ammonia levels. The decrease of these parameters in Co_NG+RC was linked to the substrate composition. Co_NG+RC was not fed with cow manure, which has a high concentration of free ammonia (2755.9 ± 281.3 mg of NH3–N per liter) and provides high levels of partial alkalinity, promoting a higher pH in Co_NG+CM+RC and Co_NG+CM.
Co_NG+CM+RC was also fed with rumen content in addition to Napier grass and cow manure, and the fraction of OTUs belonging to only the rumen content was only 0.8–3.9% during the last 40 days of operation (Figure 4). Rumen OTUs were low in Co_NG+CM+RC even though the free ammonia levels were within the usual range for the rumen (41–248 mg of NH3–N per liter) (Figure S8C). These results suggest that, contrary to observations for the rumen, free ammonia levels are not that important for maintaining rumen bacteria in AD. When free ammonia levels are low in the rumen, bacterial growth is slow, and the breakdown of carbohydrates is retarded. (36) However, the solid retention time in the rumen is only 60–90 h, whereas the reactors were operated at a minimum retention time of 20 days. Therefore, having concentrations of free ammonia lower than 41 mg of NH3–N per liter seems not to be an impediment to the growth of rumen bacteria. On the other hand, the decrease in free ammonia levels lowered the partial alkalinity and pH levels in Co_NG+RC. The pH seemed to be responsible for the microbial community change in Co_NG+RC. In both Inoc_RC and Co_NG+RC, rumen OTUs were dominant when the pH was below 7.0; meanwhile for Co_NG+CM+RC, which was also fed with the rumen content, the pH was never lower than 7.2 (Figure 3B), and rumen OTUs were present at a lower relative abundance (Figure 4). In fact, among the different studies that used rumen content as an inoculum, (16,19,22,24,27) only Agematu et al. (22) maintained the rumen bacterial populations in the reactor for 50 days. Agematu et al. (22) maintained their reactor pH around 6.5 and produced as much as 5.1 g of SCFA as HAc per liter from cedar. This study thus confirmed that pH was a critical factor for the growth of rumen hydrolytic bacteria in AD, which was further evaluated using a multivariate statistical analysis (below).
During the last 30 days of operation, 70–80% of the OTUs in Co_NG+CM and Co_NG+CM+RC originated from the AD inoculum and cow manure, and 16–25% belonged to the group called “Others” (OTUs not detected in the AD inoculum, rumen content, or cow manure). At the same time, 26–36% of the OTUs in Co_NG+RC belonged to the rumen content only, and hydrolytic rumen bacteria like Fibrobacter were present (Figure 4). These bacteria might be responsible for the higher SCFA levels in Co_NG+RC compared to the SCFA levels in Co_NG+CM+RC (p = 3 × 10–09) and Co_NG+CM (p = 2 × 10–08) (Figures 3A and S14, respectively). However, the specific methane production in Co_NG+RC was similar to that in the other two reactors (p = 0.106). Furthermore, the accumulation of SCFA was also low (maximum of 0.07 g of SCFA as HAc per gram of VSfed) compared to that in Inoc_RC (maximum of 0.79 g of SCFA as HAc per gram of VSfed) in which a higher percentage of the OTUs originated from the rumen content (more than 90% on day 7). This behavior might be due to the fact that the pH in Co_NG+RC was not low enough to support the activity of rumen microorganisms. McDonald et al. reported that the pH in the rumen fluctuates between 5.5 and 7.0, (36) and the optimal pH for F. succinogenes falls between 6.0 and 6.8 with the highest activity at pH values between 6.2 to 6.5. (63−65) In Inoc_RC, the pH was 6.2–6.5 when the accumulation of SCFA was as high as 0.79 g of SCFA as HAc per gram of VSfed, suggesting that these reactor conditions were optimal for the development of rumen hydrolytic bacteria. However, RNA-based analyses would be necessary to further confirm this.

Favorable Conditions for Rumen Bacteria Are Unfavorable for Methanogenesis: pH as the Crucial Factor

The reactor performance and microbial data suggest that pH is an important parameter when considering strategies to maintain rumen bacteria in continuous-flow AD systems. To further evaluate this observation and determine which other factors are favorable for rumen microorganisms, a multivariate statistical analysis using the reactor performance and microbial data was applied for each experiment. Previous studies have demonstrated that such an analysis with long-term time-series experiments can provide results similar to those obtained with anaerobic digesters operated in replicate. (49,50)Tables 1 and 2 show the results of CCA models for the inoculum and codigestion experiments, respectively.
Table 1. CCA model (Akaike’s Information Criterion = 36.57) for the Inoculum Experiment
parameterseigenvalueexplained inertia (%)ANOVA (Pf > F)
acetate concentration0.69330.001
free ammonia0.42200.007
pH0.39190.001
partial alkalinity0.23110.001
propionate concentration0.1990.006
rumen content only as inoculum0.1680.011
residualn.a.n.a. 
Table 2. CCA Model (Akaike’s Information Criterion = 3.95) for the Co-digestion Experiment
parameterseigenvalueexplained inertia (%)ANOVA (Pf > F)
rumen content only as the cosubstrate0.58380.001
pH0.44280.001
partial alkalinity0.23150.001
acetate concentration0.1390.001
propionate concentration0.0960.009
butyrate concentration0.0840.031
residualn.a.n.a. 
The CCA model built for the inoculum experiment (Table 1) was responsible for 55% of the distances between the different microbial communities in the respective reactors. A substantial portion of the distances (83%) was explained by the acetate, free ammonia levels, pH, and partial alkalinity levels. While rumen content populations were abundant during the first 50 days of the inoculum experiment, the acetate concentration (p = 2 × 10–09), pH (p = 1.5 × 10–14), and partial alkalinity (p = 3 × 10–05) were significantly different in Inoc_RC compared to in Inoc_RC+AD and Inoc_AD; however, this was not the case for the free ammonia levels (p = 0.077). The multivariate statistical analysis confirmed the experimental results that maintaining a pH below 7.0 is critically important to maintain rumen microorganisms. In other studies that supported the growth of rumen hydrolytic microorganisms, the pH was between 6.0 and 6.5. (16,19,22,24,27,63−65) When using the rumen content as the inoculum, Fibrobacter was present at a high relative abundance, which accelerated the hydrolysis of the substrate and resulted in a higher production of SCFA (specially acetate and propionate) and a lowered buffering capacity. High levels of SCFA and low levels of partial alkalinity and free ammonia could maintain the pH between 6.0 and 6.5 in Inoc_RC, which promoted the growth of rumen bacteria (Figure 2). For this reason, the parameters of acetate, partial alkalinity and free ammonia levels explained 64% of the CCA model (Table 1). Free ammonia levels and pH had very similar eigenvalues (0.42 for free ammonia and 0.39 for pH). As discussed earlier, free ammonia levels did not seem very important for the growth of rumen hydrolytic microbes, and the levels did not differ statistically between the three reactors (p = 0.077). However, free ammonia appears as an important parameter in Table 1 because the pH is dependent on the free ammonia concentration (their trends were very similar, Figures 1B and S1C). Other parameters in the model, such as the propionate concentration and the “rumen content only as inoculum”, contributed much less to the distances (17% total). The “rumen content only as inoculum” explained only 8% of the variation observed in the microbial community structure. This indicates that, if the pH is within the range of 6.0–6.5, rumen bacteria can still grow in the system whether the AD inoculum is mixed with rumen inoculum at the beginning of the experiment or not. The relative abundance of rumen bacteria in Inoc_RC+AD increased whenever the pH was lower than 6.5 (Figures 2 and S3).
For the codigestion experiment, the same CCA model could have been built using either the pH level or the free ammonia level as a parameter because their trends were similar (Tables 2 and S4). Similar to the inoculum experiment, the pH was dictated by the level of free ammonia. When the pH and free ammonia levels were both considered for the CCA model, the variance inflation factor was 98 for pH and 133 for free ammonia, meaning that these two variables were strongly dependent on each other. Since, as discussed earlier, pH has a more important effect on the growth of rumen microbes, the model was build considering pH. This model (Table 2) was responsible for 73% of the distances between the microbial communities in the different reactors. Parameters such as “rumen content only as cosubstrate”, the pH, and the partial alkalinity explained a large part of the model (81%). The low buffering capacity of the substrate fed in Co_NG+RC lowered the buffering capacity and the pH in the reactor, while the relative abundance of rumen bacteria started to increase (Figures 3B, 4, and S8B). The pH values explained 28% of the total distances observed. Again, the experimental results and the multivariate statistical analysis for the codigestion experiment suggest that pH is a critical factor when developing a system that supports the growth of rumen microorganisms. Furthermore, the factor “rumen content only as cosubstrate” was shown to be an important parameter by the CCA model (explained 38% of the distances). Adding the rumen content as the only cosubstrate with Napier grass could be beneficial because the microbial populations in cow manure could out-compete the rumen bacteria when mixing these two substrates together. Another study in which rumen content was mixed with the effluent of a digester treating municipal solid waste in batch experiments also observed similar out-competition. (14) Unfortunately, the pH in the batch experiments was not reported. Since pH was shown to be a very important factor in this study, mixing cow manure and rumen content is likely detrimental for rumen bacteria not because of the out-competition of rumen microorganisms but because of the increased buffering capacity contributed by the cow manure (Figure S8B), which prevents the reactor pH from being sufficiently low (6.0–6.5, Figure 3B).
The results obtained in this study indicate that the activity of rumen hydrolytic bacteria, such as Fibrobacter, can only be supported at low pH (6.0–6.5) levels, a condition unfavorable for acetoclastic methanogens that are key microorganisms in AD for avoiding the accumulation of acetate. Acetoclastic methanogens are not present in the rumen either, but acetate does not accumulate because it is removed via absorption through the rumen walls. (36) Moreover, free ammonia levels are not as critical for rumen bacteria growth in AD as they are in the rumen, where a minimum of 41 mg of NH3–N per liter is needed, probably because the solid retention times in AD (20–30 days) are higher than those in the rumen (60–90 h). (36) The observations suggest that a two-phase configuration would be beneficial, as it provides the opportunity to promote rumen hydrolytic activity and methanogenesis. Specifically, Napier grass could be fed to a first-phase acidogenic reactor inoculated with rumen content and operated at a low pH. To retain rumen populations in the reactor, either the rumen content would need to be fed continuously as a cosubstrate or the reactor would need to be operated with a solid retention time greater than the hydraulic retention time. Gijzen et al. (8,28) operated such a fermentor designed to simulate rumen conditions; however, they did not perform microbial analyses to evaluate if the rumen hydrolytic bacteria were retained. The first-phase effluent that is rich in SCFA can be diverted to a second-phase reactor for the production of methane or other valuable products, such as medium-chain fatty acids. (66)

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsestengg.0c00164.

  • Calculations to estimate the rumen content availability in the US, description of chemical and microbial analyses, characterization of inocula and substrates, reactor operating parameters, experimental design, and reactor performance data (PDF)

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Author Information

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  • Corresponding Author
    • Lutgarde Raskin - Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States;  Email: [email protected]
  • Authors
    • Xavier Fonoll - Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States;  Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain;  Orcidhttp://orcid.org/0000-0003-3304-2437
    • Shilva Shrestha - Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States;  Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Ma̅noa, Honolulu, Hawaii 96822, United States
    • Samir Kumar Khanal - Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Ma̅noa, Honolulu, Hawaii 96822, United States;  Orcidhttp://orcid.org/0000-0001-6680-5846
    • Joan Dosta - Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain
    • Joan Mata-Alvarez - Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain
  • Author Contributions

    Contributed equally to this study

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by REFRESCH (Global Challenges for the Third Century program, Office of the Provost, University of Michigan), the U.S. National Science Foundation (Sustainability Research Networks 1444745), Fundació Crèdit Andorra, Supplemental Grant, College of Tropical Agriculture and Human Resources (CTAHR), University of Hawaii, and the Sun Grant Western Regional Center at Oregon State University through a grant provided by the United States Department of Agriculture and National Institute of Food and Agriculture under proposal no. 2012-03373.

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    Environmental Technology (2005), 26 (9), 1003-1011CODEN: ENVTEV; ISSN:0959-3330. (Selper Ltd., Publications Division)
    This work studied the use of a simple rumen-fluid-inoculated anaerobic treatment system for the degrdn. of org. waste. Fresh rumen fluid collected from a fistulated sheep was used as the inoculum and fresh grass clippings were used as the waste material for treatment. Studies were carried out on both a 1-stage system where the ligno-cellulosic fraction breaks down into a mixt. of sol. products including volatile fatty acids and a 2-stage system where these products are subsequently mineralized to biogas. In the 1-stage system ∼70% of the org. waste was solubilized and in the 2-stage system ∼60% waste material was solubilized in a week. About 50% of the degrdn. was achieved in 4 days, showing that a 4-day solids retention time would be a suitable operating regime. The max. volatile fatty acid prodn. rate was 327 mg COD/L-h. A higher loading rate of 30 g/L-day was achieved in these systems compared to anaerobic digesters. Microbiol. studies showed an increase in the no. of fungal spores as well as a decrease in the no. of protozoa in the treatment system. These nos. attained stable values over the duration of the expts. The system developed is superior to conventional composting or anaerobic digestion and can be applied for the treatment of ligno-cellulosic agricultural residues.
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  13. 13
    Zhang, M.; Zhang, G.; Zhang, P.; Fan, S.; Jin, S.; Wu, D.; Fang, W. Anaerobic Digestion of Corn Stovers for Methane Production in a Novel Bionic Reactor. Bioresour. Technol. 2014, 166, 606– 609,  DOI: 10.1016/j.biortech.2014.05.067
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    13
    Anaerobic digestion of corn stovers for methane production in a novel bionic reactor
    Zhang, Meixia; Zhang, Guangming; Zhang, Panyue; Fan, Shiyang; Jin, Shuguang; Wu, Dan; Fang, Wei
    Bioresource Technology (2014), 166 (), 606-609CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    To improve the biogas prodn. from corn stovers, a new bionic reactor was designed and constructed. The bionic reactor simulated the rumen digestion of ruminants. The liq. was sepd. from corn stovers and refluxed into corn stovers again, which simulated the undigested particles sepd. from completely digested materials and fed back again for further degrdn. in ruminant stomach. Results showed that the bionic reactor was effective for anaerobic digestion of corn stovers. The liq. amt. and its reflux showed an obvious pos. correlation with biogas prodn. The highest biogas prodn. rate was 21.6 mL/gVS-added d, and the total cumulative biogas prodn. was 256.5 mL/gVS-added. The methane content in biogas ranged from 52.2% to 63.3%. The degrdn. of corn stovers were greatly enhanced through simulating the animal digestion mechanisms in this bionic reactor.
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  14. 14
    Chapleur, O.; Bize, A.; Serain, T.; Mazéas, L.; Bouchez, T. Co-Inoculating Ruminal Content Neither Provides Active Hydrolytic Microbes nor Improves Methanization of 13C-Cellulose in Batch Digesters. FEMS Microbiol. Ecol. 2014, 87 (3), 616– 629,  DOI: 10.1111/1574-6941.12249
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    14
    Co-inoculating ruminal content neither provides active hydrolytic microbes nor improves methanization of 13C-cellulose in batch digesters
    Chapleur, Olivier; Bize, Ariane; Serain, Thibaut; Mazeas, Laurent; Bouchez, Theodore
    FEMS Microbiology Ecology (2014), 87 (3), 616-629CODEN: FMECEZ; ISSN:0168-6496. (Wiley-Blackwell)
    Cellulose hydrolysis often limits the kinetics and efficiency of anaerobic degrdn. in industrial digesters. In animal digestive systems, specialized microorganisms enable cellulose biodegrdn. at significantly higher rates. This study aims to assess the potential of ruminal microbial communities to settle and to express their cellulolytic properties in anaerobic digesters. Cellulose-degrading batch incubations were co-inoculated with municipal solid waste digester sludge and ruminal content. 13C-labeled cellulose degrdn. was described over time with Gas Chromatog.-Combustion-Isotope Ratio Mass Spectrometry. Results were linked to the identification of the microorganisms assimilating 13C and to the monitoring of their relative dynamics. Cellulose degrdn. in co-inoculated incubations was efficient but not significantly improved. Transient disturbances in degrdn. pathways occurred, as revealed by propionate accumulation. Automated Ribosomal Intergenic Spacer Anal. dynamics and pyrosequencing revealed that expected classes of Bacteria and Archaea were active and degraded cellulose. However, despite the favorable co-inoculation conditions, mol. tools also revealed that no ruminal species settled in the bioreactors. Other specific parameters were probably needed for this to happen. This study shows that exploiting the rumen's cellulolytic properties in anaerobic digesters is not straightforward. Co-inoculation can only be successful if ruminal microorganisms manage to thrive in the anaerobic digester and outcompete native microorganisms, which requires specific nutritional and environmental parameters, and a meticulous reprodn. of the selection pressure encountered in the rumen.
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  15. 15
    Wall, D. M.; Straccialini, B.; Allen, E.; Nolan, P.; Herrmann, C.; O’Kiely, P.; Murphy, J. D. Investigation of Effect of Particle Size and Rumen Fluid Addition on Specific Methane Yields of High Lignocellulose Grass Silage. Bioresour. Technol. 2015, 192, 266– 271,  DOI: 10.1016/j.biortech.2015.05.078
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    15
    Investigation of effect of particle size and rumen fluid addition on specific methane yields of high lignocellulose grass silage
    Wall, D. M.; Straccialini, B.; Allen, E.; Nolan, P.; Herrmann, C.; O'Kiely, P.; Murphy, J. D.
    Bioresource Technology (2015), 192 (), 266-271CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    This work examines the digestion of advanced growth stage grass silage. Two variables were investigated: particle size (greater than 3 cm and less than 1 cm) and rumen fluid addn. Batch studies indicated particle size and rumen fluid addn. had little effect on specific methane yields (SMYs). In continuous digestion of 3 cm silage the SMY was 342 and 343 L CH4 kg-1 VS, resp., with and without rumen fluid addn. However, digester operation was significantly affected through silage floating on the liquor surface and its entanglement in the mixing system. Digestion of 1 cm silage with no rumen fluid addn. struggled; volatile fatty acid concns. rose and SMYs dropped. The best case was 1 cm silage with rumen fluid addn., offering higher SMYs of 371 L CH4 kg-1 VS and stable operation throughout. Thus, phys. and biol. treatments benefited continuous digestion of high fiber grass silage.
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  16. 16
    Deng, Y.; Huang, Z.; Ruan, W.; Zhao, M.; Miao, H.; Ren, H. Co-Inoculation of Cellulolytic Rumen Bacteria with Methanogenic Sludge to Enhance Methanogenesis of Rice Straw. Int. Biodeterior. Biodegrad. 2017, 117, 224– 235,  DOI: 10.1016/j.ibiod.2017.01.017
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    16
    Co-inoculation of cellulolytic rumen bacteria with methanogenic sludge to enhance methanogenesis of rice straw
    Deng, Yuying; Huang, Zhenxing; Ruan, Wenquan; Zhao, Mingxing; Miao, Hengfeng; Ren, Hongyan
    International Biodeterioration & Biodegradation (2017), 117 (), 224-235CODEN: IBBIES; ISSN:0964-8305. (Elsevier Ltd.)
    Ruminal microbiota co-inoculated with methanogenic sludge were applied in a 2 L anaerobic system. The expt. lasted 48 days in three stages with different substrate loading rates of 3 g d-1, 7 g d-1 and 14 g d-1. The fermn. performance and microbial characteristics were assessed. There was no corresponding accumulation of volatile fatty acids (VFAs) and pH values varied between 6.8 and 7.68 during the whole anaerobic digestion, indicating system stability. The cellulolytic activities improved significantly (P < 0.05) compared with control samples. Co-inoculation not only added methanogens but also increased the proportion of bacteria contg. GH 5 genes even though the bacterial compn. changed by PCR-DGGE phylogenetic anal. The proportion of cellulolytic genera of Clostridium and Ruminococcus increased, whereas Bacteroides, Fibrobacter and Acetivibrio disappeared due to their inability to acclimatize to in vitro lignocellulosic conditions. The anal. of GH and 16S rRNA genes also provided a better description of cellulolytic function and phylogenetic profile. Mutual cooperation formed between Methanobrevibacter and Ruminococcus was achieved for high cellulolytic activity and methanogenic efficiency in co-inoculated system. Moreover, this hypothesis was further strengthened by observations on morphol. characteristics of cellulolytic consortia.
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  17. 17
    Murali, N.; Fernandez, S.; Ahring, B. K. Fermentation of Wet-Exploded Corn Stover for the Production of Volatile Fatty Acids. Bioresour. Technol. 2017, 227, 197– 204,  DOI: 10.1016/j.biortech.2016.12.012
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    17
    Fermentation of wet-exploded corn stover for the production of volatile fatty acids
    Murali, Nanditha; Fernandez, Sebastian; Ahring, Birgitte Kiaer
    Bioresource Technology (2017), 227 (), 197-204CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    Volatile fatty acids (VFA) have been used as platform mols. for prodn. of biofuels and bioproducts. In the current study, we examine the VFA prodn. from wet-exploded corn stover through anaerobic fermn. using rumen bacteria. The total VFA yield (acetic acid equiv.) was found to increase from 22.8 g/L at 2.5% total solids (TS) to 40.8 g/L at 5% TS. It was found that the acetic acid concn. increased from 10 g/L to 22 g/L at 2.5% and 5% TS, resp. An increased propionic acid prodn. was seen between day 10 and 20 at 5% TS. Valeric acid (4 g/L) was produced at 5% TS and not at 2.5% TS. Compn. anal. showed that 50% of the carbohydrates were converted to VFA at 5% TS and 33% at 2.5% TS. Our results show that rumen fermn. of lignocellulosic biomass after wet explosion can produce high concns. of VFA without addn. of external enzymes of importance for the process economics of lignocellulosic biorefineries.
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  18. 18
    Wall, D. M.; Allen, E.; O’Shea, R.; O’Kiely, P.; Murphy, J. D. Investigating Two-Phase Digestion of Grass Silage for Demand-Driven Biogas Applications: Effect of Particle Size and Rumen Fluid Addition. Renewable Energy 2016, 86, 1215– 1223,  DOI: 10.1016/j.renene.2015.09.049
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    18
    Investigating two-phase digestion of grass silage for demand-driven biogas applications: Effect of particle size and rumen fluid addition
    Wall, D. M.; Allen, E.; O'Shea, R.; O'Kiely, P.; Murphy, J. D.
    Renewable Energy (2016), 86 (), 1215-1223CODEN: RNENE3; ISSN:0960-1481. (Elsevier Ltd.)
    High lignocellulose content grass silage was investigated for two-phase digestion (leaching followed by upflow anaerobic sludge blanket (UASB)) for application to demand-driven biogas prodn. Leaching trials were undertaken investigating the effects of particle size redn. and rumen fluid addn. on the hydrolysis and acidogenesis phases. Reducing grass silage particle size to <1 cm was not suited to leaching as particles could not be fully entrained in the system; this was not an issue at >3 cm particle size. Rumen fluid addn. increased prodn. of volatile fatty acids (VFA) but reduced pH levels, which subsequently hindered hydrolysis of volatile solids (VS). When electricity demand is low, it is recommended to operate in leach only mode with grass silage particle size >3 cm and with rumen fluid addn.; this limits VS destruction to 30% while maintaining a high VFA yield. When electricity demand is high, connection of the UASB generates 61% destruction of VS maximizing biogas prodn. Operation of the SLBR-UASB achieves lower specific methane yields than traditional single-stage digestion but may offer advantages in demand driven biogas systems.
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  19. 19
    Ozbayram, E. G.; Kleinsteuber, S.; Nikolausz, M.; Ince, B.; Ince, O. Enrichment of Lignocellulose-Degrading Microbial Communities from Natural and Engineered Methanogenic Environments. Appl. Microbiol. Biotechnol. 2018, 102 (2), 1035– 1043,  DOI: 10.1007/s00253-017-8632-7
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    19
    Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments
    Ozbayram, Emine Gozde; Kleinsteuber, Sabine; Nikolausz, Marcell; Ince, Bahar; Ince, Orhan
    Applied Microbiology and Biotechnology (2018), 102 (2), 1035-1043CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examd. by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Addnl., the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.
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  20. 20
    Deng, Y.; Huang, Z.; Zhao, M.; Ruan, W.; Miao, H.; Ren, H. Effects of Co-Inoculating Rice Straw with Ruminal Microbiota and Anaerobic Sludge: Digestion Performance and Spatial Distribution of Microbial Communities. Appl. Microbiol. Biotechnol. 2017, 101 (14), 5937– 5948,  DOI: 10.1007/s00253-017-8332-3
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    20
    Effects of co-inoculating rice straw with ruminal microbiota and anaerobic sludge: digestion performance and spatial distribution of microbial communities
    Deng, Yuying; Huang, Zhenxing; Zhao, Mingxing; Ruan, Wenquan; Miao, Hengfeng; Ren, Hongyan
    Applied Microbiology and Biotechnology (2017), 101 (14), 5937-5948CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Ruminal microbiota (RM) were co-inoculated with anaerobic sludge (AS) at different ratios to study the digestion of rice straw in batch expts. The CH4 yield reached 273.64 mL/g volatile solid (VS) at a co-inoculum ratio of 1:1. The xylanase and cellulase activities were 198.88-212.88 and 24.51-29.08 U/mL in co-inoculated samples, resp., and were significantly different compared to the results for single inoculum (p < 0.05). Higher ratios of AS enhanced acetoclastic methanogenesis, and propionate accumulation could be the main reason for the longer lag phase obsd. in samples with a higher RM ratio. The microbial compns. were clearly altered after digestion. Fibrobacter, Ruminococcus and Butyrivibrio from the rumen did not settle in the co-inoculated system, whereas Clostridiales members became the main polysaccharide degraders. Microbial interactions involving hydrolytic bacteria and acetoclastic methanogens in the residue were considered to be significant for hydrolysis activities and methane prodn. Syntrophy involving propionate oxidizers with assocd. methanogens occurred in the liq. phase. Our findings provide a better understanding of the anaerobic digestion of rice straw that is driven by specific microbial populations.
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  21. 21
    Li, K.; Zhu, H.; Zhang, Y.; Zhang, H. Characterization of the Microbial Communities in Rumen Fluid Inoculated Reactors for the Biogas Digestion of Wheat Straw. Sustainability 2017, 9 (2), 243,  DOI: 10.3390/su9020243
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    21
    Characterization of the microbial communities in rumen fluid inoculated reactors for the biogas digestion of wheat straw
    Li, Ke; Zhu, Haoran; Zhang, Yajie; Zhang, Hongxun
    Sustainability (2017), 9 (2), 243/1-243/14CODEN: SUSTDE; ISSN:2071-1050. (MDPI AG)
    The present study investigated the effect of rumen fluid (RF) concn. on the methane prodn. through anaerobic digestion of wheat straw in batch mode, and compared the microbial communities in RF and RF inoculated reactors by 16S rRNA genes sequencing. Six levels of RF concn. including 1%, 5%, 10%, 15%, 20% and 25% (vol./vol.) were used in reactors R1, R5, R10, R15, R20 and R25 resp. The results revealed that lower than or equal to 5% RF concns. resulted in reactor acidification and low methane prodn. The highest methane yield of 106 mL·CH4·g·VS-1 was achieved in R10, whereas higher RF concns. than 10% could not improve the methane prodn. significantly. Methanosarcina barkeri was abundant in the well-working reactors, and Methanobacterium was dominant in the poor-working reactors, implying the archaeal communities in reactors had changed greatly from the Methanobrevibacter-dominated RF. Although the relative abundance of Clostridium and Ruminococcus were greatly different between RF and reactors, the Bacteroidetes and Firmicutes communities were dominant in all the tested samples. The results indicated that the in vitro anaerobic conditions had altered the rumen methanogenic communities significantly and the facultative acetoclastic Methanosarcina was important for the methane prodn. in the RF seeded reactors.
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  22. 22
    Agematu, H.; Takahashi, T.; Hamano, Y. Continuous Volatile Fatty Acid Production from Lignocellulosic Biomass by a Novel Rumen-Mimetic Bioprocess. J. Biosci. Bioeng. 2017, 124 (5), 528– 533,  DOI: 10.1016/j.jbiosc.2017.06.006
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    22
    Continuous volatile fatty acid production from lignocellulosic biomass by a novel rumen-mimetic bioprocess
    Agematu, Hitosi; Takahashi, Takehiko; Hamano, Yoshio
    Journal of Bioscience and Bioengineering (2017), 124 (5), 528-533CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)
    Lignocellulosic biomass is an attractive source of biofuels and biochems., being abundant in various plant sources. However, processing this type of biomass requires hydrolysis of cellulose. The proposed rumen-mimetic bioprocess consists of dry-pulverization of lignocellulosic biomass and pH-controlled continuous cultivation of ruminal bacteria using ammonium as a nitrogen source. In this study, ruminal bacteria were continuously cultivated for over 60 days and used to digest microcryst. cellulose, rice straw, and Japanese cedar to produce volatile fatty acids (VFAs). The ruminal bacteria grew well in the chem. defined medium. The amts. of VFAs produced from 20 g of cellulose, rice straw, and Japanese cedar were 183±29.7, 69.6±12.2, and 21.8±12.9 mmol, resp. Each digestion completed within 24 h. The carbon yield was 60.6% when 180 mmol of VFAs was produced from 20 g of cellulose. During the cultivation, the bacteria were obsd. to form flocs that enfolded the feed particles. These flocs likely contain all of the bacterial species necessary to convert lignocellulosic biomass to VFAs and microbial protein symbiotically. Denaturing gradient gel electrophoresis (DGGE) anal. of PCR-amplified 16S rDNA fragments revealed that the bacterial community was relatively stable after 1 wk in cultivation, though it was different from the original community structure. Furthermore, sequence anal. of the DGGE bands indicates that the microbial community includes a cellulolytic bacterium, a bacterium acting synergistically with cellulolytic bacteria, and a propionate-producing bacterium, as well as other anaerobic bacteria.
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  23. 23
    Ozbayram, E. G.; Akyol; Ince, B.; Karakoç, C.; Ince, O. Rumen Bacteria at Work: Bioaugmentation Strategies to Enhance Biogas Production from Cow Manure. J. Appl. Microbiol. 2018, 124 (2), 491– 502,  DOI: 10.1111/jam.13668
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    23
    Rumen bacteria at work: bioaugmentation strategies to enhance biogas production from cow manure
    Ozbayram, E. G.; Akyol, C.; Ince, B.; Karakoc, C.; Ince, O.
    Journal of Applied Microbiology (2018), 124 (2), 491-502CODEN: JAMIFK; ISSN:1364-5072. (Wiley-Blackwell)
    Aims : To investigate the effects of different bioaugmentation strategies for enhancing the biogas prodn. from cow manure and evaluate microbial community patterns. Methods and Results : Co-inoculation with cow rumen fluid and cow rumen-derived enriched microbial consortia was evaluated in anaerobic batch tests at 36°C and 41°C. Singular addn. of both rumen fluid and enriched bioaugmentation culture had a promising enhancement on methane yields; however, the highest methane yield (311 mL CH4 per g VS at 41°C) was achieved when the anaerobic seed sludge was co-inoculated together with rumen fluid and enriched bioaugmentation culture. Bacterial community profiles were investigated by Ion PGM Platform, and specific lignocellulolytic bacteria dynamics in batch tests were assessed by qPCR. The temp. had minor effects on the abundance of bacterial community; in which Bacteroidetes and Firmicutes were the most abundant phyla in all digesters. There was an important impact of Firmicutes flavefaciens and Ruminococcus albus at 41°C, which in turn pos. affected the methane prodn. Conclusion : The degree of enhancement in biogas prodn. can be upgraded by the co-inoculation of rumen-derived bioaugmentation culture with anaerobic seed sludge with high methanogenic activity. Significance and Impact of the Study : A close look at the biotic interactions and their assocns. with abiotic factors might be valuable for evaluating rumen-related bioaugmentation applications.
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  24. 24
    Deng, Y.; Huang, Z.; Ruan, W.; Miao, H.; Shi, W.; Zhao, M. Enriching Ruminal Polysaccharide-Degrading Consortia via Co-Inoculation with Methanogenic Sludge and Microbial Mechanisms of Acidification across Lignocellulose Loading Gradients. Appl. Microbiol. Biotechnol. 2018, 102 (8), 3819– 3830,  DOI: 10.1007/s00253-018-8877-9
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    24
    Enriching ruminal polysaccharide-degrading consortia via co-inoculation with methanogenic sludge and microbial mechanisms of acidification across lignocellulose loading gradients
    Deng, Yuying; Huang, Zhenxing; Ruan, Wenquan; Miao, Hengfeng; Shi, Wansheng; Zhao, Mingxing
    Applied Microbiology and Biotechnology (2018), 102 (8), 3819-3830CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Using lignocellulosic materials as substrates, ruminal microbiota were co-inoculated with anaerobic sludge at different loading rates (LR) to study the microbial community in the semi-continuous mode. The results indicated that the highest CH4 yield reached 0.22 L/g volatile solid at LR of 4 g/L/day, which obtained 56-58% of the theor. value. In the steady stage with LR of 2-4 g/L/day and slurry recirculation, copies of total archaea increased. Esp. the Methanobacteriales increased significantly (p < 0.05) to 3.30 × 108 copies/mL. The microbial communities were examd. by MiSeq 16S rRNA sequencing. Enriched hydrolytic bacteria mainly belonged to Clostridiales, including Ruminococcus, Ruminiclostridium, and Ruminofilibacter settled in the rumen. High-active cellulase and xylanase were excreted in the co-inoculated system. Acid-producing bacteria by fermn. were affiliated with Lachnospiraceae and Bacteroidales. The acidogen members were mainly Spirochaetaceae and Clostridiales. Syntrophic oxidn. bacteria mainly consisted of Synergistetes, propionate oxidizers (Syntrophobacter and Pelotomaculum), and butyrate oxidizers (Syntrophus and Syntrophomonas). There had no volatile fatty acid (VFA) accumulation and the pH values varied between 6.94 and 7.35. At LR of 6 g/L/day and a recirculation ratio of 1:1, the hardly degradable components and total VFA concns. obviously increased. The total archaea and Methanobacteriales then deceased significantly to 8.56 × 105 copies/mL and 4.14 × 103 copies/mL resp. (p < 0.05), which resulted in the inhibition of methanogenic activities. Subsequently, microbial diversity dropped, and the hydrolytic bacteria and syntrophic oxidizers obviously decreased. In contrast, the abundances of Bacteroidales increased significantly (p < 0.05). Acetate, propionate, and butyrate concns. reached 2.02, 6.54, and 0.53 g/L, resp., which indicated "acidification" in the anaerobic reactor. Our study illustrated that co-inoculated anaerobic sludge enriched the ruminal function consortia and hydrogenotrophic methanogens played an important role in anaerobic digestion of lignocelluloses.
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    Quintero, M.; Castro, L.; Ortiz, C.; Guzmán, C.; Escalante, H. Enhancement of Starting up Anaerobic Digestion of Lignocellulosic Substrate: Fique’s Bagasse as an Example. Bioresour. Technol. 2012, 108, 8– 13,  DOI: 10.1016/j.biortech.2011.12.052
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    25
    Enhancement of starting up anaerobic digestion of lignocellulosic substrate: fique's bagasse as an example
    Quintero, Mabel; Castro, Liliana; Ortiz, Claudia; Guzman, Carolina; Escalante, Humberto
    Bioresource Technology (2012), 108 (), 8-13CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    In Colombia there are 20,000 ha of fique fields (Furcraea sp., family Agavaceae), that produce around 93,400 tons of fique's bagasse per yr. These residuals are disposed into rivers and soil causing pollution. According to physicochem. characteristics, the lignocellulosic residues from fique crops (fique's bagasse) are appropriate carbon source to biogas prodn. Anaerobic digestion from fique's Bagasse (FB) requires a specialized microbial consortium capable of degrading its high lignocellulosic concn. In this study, the capacities of seven microbial consortia for biomethane potential (BMP) from FB were evaluated. Inoculum of ruminal liq. achieved high hydrolytic activity (0.068 g COD/g VSS day), whereas pig waste sludge inoculum showed high methanogenic activity (0.146 g COD/g VSS day). Mixts. of these two inoculums (RL + PWS) showed the best yields for biomethane potential (0.3 m3 CH4/Kg VS ad).
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  26. 26
    Song, H.; Clarke, W. P.; Blackall, L. L. Concurrent Microscopic Observations and Activity Measurements of Cellulose Hydrolyzing and Methanogenic Populations during the Batch Anaerobic Digestion of Crystalline Cellulose. Biotechnol. Bioeng. 2005, 91 (3), 369– 378,  DOI: 10.1002/bit.20517
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    26
    Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose
    Song, Hyohak; Clarke, William P.; Blackall, Linda L.
    Biotechnology and Bioengineering (2005), 91 (3), 369-378CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    This study compares process data with microscopic observations from an anaerobic digestion of org. particles. This article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcryst. cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amt. of cellulose remaining in the reactor, reached a const. value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A const. value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and 1st-order kinetics only apply after this point. The 1st-order hydrolysis rate const., once surfaces were satd. with biofilm, was twice higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegrdn. process exist entirely within the biofilm. For the reactor conditions used in these expts., the predominant methanogens exist in ball-shaped colonies within the biofilm.
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  27. 27
    Li, K.; Zhu, H.; Zhang, Y.; Zhang, H. Characterization of the Microbial Communities in Rumen Fluid Inoculated Reactors for the Biogas Digestion of Wheat Straw. Sustainability 2017, 9 (2), 243,  DOI: 10.3390/su9020243
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    Characterization of the microbial communities in rumen fluid inoculated reactors for the biogas digestion of wheat straw
    Li, Ke; Zhu, Haoran; Zhang, Yajie; Zhang, Hongxun
    Sustainability (2017), 9 (2), 243/1-243/14CODEN: SUSTDE; ISSN:2071-1050. (MDPI AG)
    The present study investigated the effect of rumen fluid (RF) concn. on the methane prodn. through anaerobic digestion of wheat straw in batch mode, and compared the microbial communities in RF and RF inoculated reactors by 16S rRNA genes sequencing. Six levels of RF concn. including 1%, 5%, 10%, 15%, 20% and 25% (vol./vol.) were used in reactors R1, R5, R10, R15, R20 and R25 resp. The results revealed that lower than or equal to 5% RF concns. resulted in reactor acidification and low methane prodn. The highest methane yield of 106 mL·CH4·g·VS-1 was achieved in R10, whereas higher RF concns. than 10% could not improve the methane prodn. significantly. Methanosarcina barkeri was abundant in the well-working reactors, and Methanobacterium was dominant in the poor-working reactors, implying the archaeal communities in reactors had changed greatly from the Methanobrevibacter-dominated RF. Although the relative abundance of Clostridium and Ruminococcus were greatly different between RF and reactors, the Bacteroidetes and Firmicutes communities were dominant in all the tested samples. The results indicated that the in vitro anaerobic conditions had altered the rumen methanogenic communities significantly and the facultative acetoclastic Methanosarcina was important for the methane prodn. in the RF seeded reactors.
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  28. 28
    Gijzen, H. J.; Zwart, K. B.; van Gelder, P. T.; Vogels, G. D. Continuous Cultivation of Rumen Microorganisms, a System with Possible Application to the Anaerobic Degradation of Lignocellulosic Waste Materials. Appl. Microbiol. Biotechnol. 1986, 25, 155– 162,  DOI: 10.1007/BF00938940
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    28
    Continuous cultivation of rumen microorganisms, a system with possible application to the anaerobic degradation of lignocellulosic waste materials
    Gijzen, Huub J.; Zwart, Kor B.; Van Gelder, Pieter T.; Vogels, Godfried D.
    Applied Microbiology and Biotechnology (1986), 25 (2), 155-62CODEN: AMBIDG; ISSN:0175-7598.
    An in-vitro continuous fermn. device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liqs. found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixt. was used as a solid growth substrate. During a test period of 65 days, the artificial rumen fermenter showed stable operation with respect to ciliate nos., fiber degrdn., and volatile fatty acid prodn. The values obtained were comparable to those found in vivo. Optimal fiber degrdn. and volatile fatty acid prodn. were maintained when the hydraulic retention times (HRT) were 11-14 h. At these HRT values, ciliate nos. were maintained at ∼8.5 × 104 cells/mL. The ciliate nos. declined drastically at HRT values of >14 h. A fermenter inoculated with a small vol. of rumen fluid (1:100, vol./vol.) reached normal protozoal nos., fiber degrdn., and volatile fatty acid prodn. after a start-up period of only 8-10 days. The possible application of rumen microorganisms for efficient degrdn. of lignocellulosic waste material in an artificial rumen digester is discussed.
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  29. 29
    Nagler, M.; Kozjek, K.; Etemadi, M.; Insam, H.; Podmirseg, S. M. Simple yet Effective: Microbial and Biotechnological Benefits of Rumen Liquid Addition to Lignocellulose-Degrading Biogas Plants. J. Biotechnol. 2019, 300, 1– 10,  DOI: 10.1016/j.jbiotec.2019.05.004
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    29
    Simple yet effective: Microbial and biotechnological benefits of rumen liquid addition to lignocellulose-degrading biogas plants
    Nagler, Magdalena; Kozjek, Katja; Etemadi, Mohammad; Insam, Heribert; Podmirseg, Sabine Marie
    Journal of Biotechnology (2019), 300 (), 1-10CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    In biogas plants, lignocellulose-rich biomass (LCB) is particularly slowly degraded, causing high hydraulic retention times. This fact lowers the interests for such substrates. To enhance LCB-degrdn., cattle rumen fluid, a highly active microbial resource accruing in the growing meat industry, might be used as a potential source for bioaugmentation. This study compares 0%, 20% and 40% rumen liq. in a batch anaerobic digestion approach. Moreover, it dets. the biogas- and methane-potentials as well as degrdn.-speeds of corn straw, co-digested with cattle manure. It inspects microbial communities via marker-gene sequencing, qPCR and RNA-DGGE and draws attention on possible beneficial effects of rumen addn. on the biogas-producing community. Bioaugmentation with 20% and 40% vol./vol. rumen liq. accelerated methane yields by 5 and 6 days, resp. (i.e. reaching 90% of total methane prodn.). It also enhanced LCB- as well as (hemi)cellulose- and volatile fatty acid degrdn. These results are supported by increased abundances of bacteria, methanogens and anaerobic fungi in treatments with rumen liq. amendment, and point towards the persistence of specific rumen-borne microorganisms esp. during the first phase of the expt. The results suggest that rumen liq. addn. is a promising strategy for enhanced and accelerated exploitation of LCB for biomethanization.
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  30. 30
    Zamorano-López, N.; Borrás, L.; Giménez, J. B.; Seco, A.; Aguado, D. Acclimatised Rumen Culture for Raw Microalgae Conversion into Biogas: Linking Microbial Community Structure and Operational Parameters in Anaerobic Membrane Bioreactors (AnMBR). Bioresour. Technol. 2019, 290 (July), 121787,  DOI: 10.1016/j.biortech.2019.121787
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    Acclimatised rumen culture for raw microalgae conversion into biogas: Linking microbial community structure and operational parameters in anaerobic membrane bioreactors (AnMBR)
    Zamorano-Lopez, Nuria; Borras, Luis; Gimenez, Juan B.; Seco, Aurora; Aguado, Daniel
    Bioresource Technology (2019), 290 (), 121787CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    Ruminal fluid was inoculated in an Anaerobic Membrane Reactor (AnMBR) to produce biogas from raw Scenedesmus. This work explores the microbial ecol. of the system during stable operation at different solids retention times (SRT). The 16S rRNA amplicon anal. revealed that the acclimatised community was mainly composed of Anaerolineaceae, Spirochaetaceae, Lentimicrobiaceae and Cloacimonetes fermentative and hydrolytic members. During the highest biodegradability achieved in the AnMBR (62%) the dominant microorganisms were Fervidobacterium and Methanosaeta. Different microbial community clusters were obsd. at different SRT conditions. Interestingly, syntrophic bacteria Gelria and Smithella were enhanced after increasing 2-fold the org. loading rate, suggesting their importance in continuous systems producing biogas from raw microalgae.
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    Martí-Herrero, J.; Soria-Castellón, G.; Diaz-de-Basurto, A.; Alvarez, R.; Chemisana, D. Biogas from a Full Scale Digester Operated in Psychrophilic Conditions and Fed Only with Fruit and Vegetable Waste. Renewable Energy 2019, 133, 676– 684,  DOI: 10.1016/j.renene.2018.10.030
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    Biogas from a full scale digester operated in psychrophilic conditions and fed only with fruit and vegetable waste
    Marti-Herrero, J.; Soria-Castellon, G.; Diaz-de-Basurto, A.; Alvarez, R.; Chemisana, D.
    Renewable Energy (2019), 133 (), 676-684CODEN: RNENE3; ISSN:0960-1481. (Elsevier Ltd.)
    The aim of this work was to evaluate the feasibility of treating fruit and vegetable waste from a municipal market in a full-scale anaerobic digester with min. implementation and operational costs, i.e. without pre-treatment, clean water consumption, active heating or mixing. For this purpose, a 13.9 m3 digester that forces the submersion of solids, gains heat through solar radiation and recirculates effluent was monitored during one year of real operational and weather conditions in Bolivia. The digester was initially loaded with cow rumen (inoculum source), and after eight weeks with only fruit and vegetable waste. The digester operated at an org. loading rate (OLR) of 1 kgVS/(m3d). The normalized specific biogas prodn. averaged 0.26 m3/kgVS and increased to 0.34 m3/kgVS when OLR was halved to 0.57 kgVS/(m3d), while the passive solar design allowed an av. digester temp. of 21°C (4°C above the av. ambient temp.).
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    Ferraro, A.; Massini, G.; Mazzurco Miritana, V.; Rosa, S.; Signorini, A.; Fabbricino, M. A Novel Enrichment Approach for Anaerobic Digestion of Lignocellulosic Biomass: Process Performance Enhancement through an Inoculum Habitat Selection. Bioresour. Technol. 2020, 313 (June), 123703,  DOI: 10.1016/j.biortech.2020.123703
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    A novel enrichment approach for anaerobic digestion of lignocellulosic biomass: Process performance enhancement through an inoculum habitat selection
    Ferraro, Alberto; Massini, Giulia; Mazzurco Miritana, Valentina; Rosa, Silvia; Signorini, Antonella; Fabbricino, Massimiliano
    Bioresource Technology (2020), 313 (), 123703CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    Inocula enrichment was performed using an innovative habitat-based selection approach to improve wheat straw (WS) anaerobic digestion (AD) efficiency. The procedure was carried out by sequentially re-inoculating the primary microbial community seven times in subsequent anaerobic reactors contg. untreated WS. Re-inocula were performed at different re-inoculum times (24, 48, and 96 h) by moving a porous support mimicking a rumen structure from one batch to the next (S-tests) or re-inoculating only the culture medium (C-tests). Highest H2 prodn. yields were obsd. after four and five re-inocula (0.08 ± 0.02 NmL h-1 gVS-1 and 0.09 ± 0.02 NmL h-1 gVS-1) for S-24 and S-48, resp. For S-96, higher CH4 yields were obsd. after the start-up test and sixth re-inoculum (0.05 ± 0.003 NmL h-1 gVS-1 and 0.04 ± 0.005 NmL h-1 gVS-1, resp.). Accordingly, S-96 showed the highest active Archaea component (7%). C-test microbial communities were dominated by fermenting, hydrogen-producing bacteria and showed lower microbial community diversity than S-tests.
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    Abbas, Y.; Jamil, F.; Rafiq, S.; Ghauri, M.; Khurram, M. S.; Aslam, M.; Bokhari, A.; Faisal, A.; Rashid, U.; Yun, S. Valorization of Solid Waste Biomass by Inoculation for the Enhanced Yield of Biogas. Clean Technol. Environ. Policy 2020, 22 (2), 513– 522,  DOI: 10.1007/s10098-019-01799-6
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    Valorization of solid waste biomass by inoculation for the enhanced yield of biogas
    Abbas, Yasir; Jamil, Farrukh; Rafiq, Sikander; Ghauri, Moinuddin; Khurram, M. Shahzad; Aslam, Muhammad; Bokhari, Awais; Faisal, Abrar; Rashid, Umer; Yun, Sining; Mubeen, Muhammad
    Clean Technologies and Environmental Policy (2020), 22 (2), 513-522CODEN: CTEPAI; ISSN:1618-954X. (Springer)
    The current work reports exptl. outcomes about anaerobic digestion (AD) of the org. fraction of catering solid waste to produce biogas and methane yield and solidity of the entire system. The possibility of developing AD as an efficient technique for food waste management has also been considered. Inoculation is an established technique to enhance biogas yield through digested sludge, rumen fluid, and slurry recirculation; although this method is proven on cattle manure, however, it is not well studied on food waste. The current study investigates the use of animal intestinal waste as inoculums to enhance biogas yield of food waste. The findings are benchmarked with cattle manure employing known digester materials of carbon steel, aluminum alloy, and at anaerobic mesophilic conditions. The inoculation was varied up to 30% by mass of the substrate materials in each case. The research finds that food waste inoculation was up to 30% using carbon steel reactor under mesophilic temp. conditions (30 ± 1°). The food waste produced biogas at a faster rate with a higher yield compared to cattle manure in all digester materials. The biogas yield kept increasing with increasing inoculum ratio reaching optimal at 30% in the case of both food waste and cattle manure with all the digester materials.
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    Anaerobic Digestion Research and Education Center (ADREC). South Campus Anaerobic Digester. Michigan State University. https://www.egr.msu.edu/bae/adrec/ (accessed 2017-05-12).
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    Tritt, W. P.; Schuchardt, F. Materials Flow and Possibilities of Treating Liquid and Solid Wastes from Slaughterhouses in Germany. A Review. Bioresour. Technol. 1992, 41 (3), 235– 245,  DOI: 10.1016/0960-8524(92)90008-L
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    Materials flow and possibilities of treating liquid and solid wastes from slaughterhouses in Germany. A review.
    Tritt, W. P.; Schuchardt, F.
    Bioresource Technology (1992), 41 (3), 235-45CODEN: BIRTEB; ISSN:0960-8524.
    A review, with 53 refs., on treatment of slaughterhouse wastewater and solid wastes in Germany.
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    McDonald, P.; Edwards, R. A.; Greenhalgh, J. F. D.; Morgan, C. A.; Sinclair, L. A.; Wilkinson, R. G. Animal Nutrition, 7th ed.; Benjamin-Cummings Publishing Company: San Francisco, CA, 2011.
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    Mata-Alvarez, J.; Dosta, J.; Romero-Güiza, M. S.; Fonoll, X.; Peces, M.; Astals, S. A Critical Review on Anaerobic Co-Digestion Achievements between 2010 and 2013. Renewable Sustainable Energy Rev. 2014, 36, 412– 427,  DOI: 10.1016/j.rser.2014.04.039
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    A critical review on anaerobic co-digestion achievements between 2010 and 2013
    Mata-Alvarez, J.; Dosta, J.; Romero-Guiza, M. S.; Fonoll, X.; Peces, M.; Astals, S.
    Renewable & Sustainable Energy Reviews (2014), 36 (), 412-427CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)
    Anaerobic digestion is a com. reality for several kinds of waste. Nonetheless, anaerobic digestion of single substrates presents some drawbacks linked to substrate characteristics. Anaerobic co-digestion, the simultaneous digestion of two or more substrates, is a feasible option to overcome the drawbacks of mono-digestion and to improve plant's economic feasibility. At present, since 50% of the publication has been published in the last two years, anaerobic co-digestion can be considered the most relevant topic within anaerobic digestion research. The aim of this paper is to present a review of the achievements and perspectives of anaerobic co-digestion within the period 2010-2013, which represents a continuation of the previous review made by the authors [3]. In the present review, the publications have been classified as for the main substrate, i.e., animal manures, sewage sludge and biowaste. Animal manures stand as the most reported substrate, agro-industrial waste and the org. fraction of the municipal solid waste being the most reported co-substrate. Special emphasis has been made to the effect of the co-digestion over digestate quality, since land application seems to be the best option for digestate recycling. Traditionally, anaerobic co-digestion between sewage sludge and the org. fraction of the municipal solid waste has been the most reported co-digestion mixt. However, between 2010 and 2013 the publications dealing with fats, oils and greases and algae as sludge co-substrate have increased. This is because both co-substrates can be obtained at the same wastewater treatment plant. In contrast, biowaste as a main substrate has not been as studied as manures or sewage sludge. Finally, three interdisciplinary sections have been written for addressing novelty aspects in anaerobic co-digestion, i.e., pre-treatments, microbial dynamics and modeling. However, much effort needs to be done in these later aspects to better understand and predict anaerobic co-digestion.
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    Prates, A.; de Oliveira, J. A.; Abecia, L.; Fondevila, M. Effects of Preservation Procedures of Rumen Inoculum on in Vitro Microbial Diversity and Fermentation. Anim. Feed Sci. Technol. 2010, 155 (2–4), 186– 193,  DOI: 10.1016/j.anifeedsci.2009.12.005
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    Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation
    Prates, A.; de Oliveira, J. A.; Abecia, L.; Fondevila, M.
    Animal Feed Science and Technology (2010), 155 (2-4), 186-193CODEN: AFSTDH; ISSN:0377-8401. (Elsevier B.V.)
    Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermn. pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at -20 °C (FRZ), frozen in liq. N (FLN) or added with 0.04 glycerol and frozen in liq. N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 mL per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilization of added glycerol after thawing in FGL increased total gas prodn. (P<0.05) and 24 h volatile fatty acid (VFA) prodn. (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas prodn. (as ml/g dry matter per h), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermn. at 8 and 12 h, resp. Differences in gas prodn. did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690-0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas prodn. studies does not affect microbial fermn. pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to vol. ratio. Freezing in liq. N is more appropriate than at -20 °C.
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    Nguyen, D.; Wu, Z.; Shrestha, S.; Lee, P.-H.; Raskin, L.; Khanal, S. K. Intermittent Micro-Aeration: New Strategy to Control Volatile Fatty Acid Accumulation in High Organic Loading Anaerobic Digestion. Water Res. 2019, 166, 115080,  DOI: 10.1016/j.watres.2019.115080
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    Intermittent micro-aeration: New strategy to control volatile fatty acid accumulation in high organic loading anaerobic digestion
    Nguyen, Duc; Wu, Zhuoying; Shrestha, Shilva; Lee, Po-Heng; Raskin, Lutgarde; Khanal, Samir Kumar
    Water Research (2019), 166 (), 115080CODEN: WATRAG; ISSN:0043-1354. (Elsevier Ltd.)
    This study developed an intermittent oxidn.-redn. potential (ORP)-controlled micro-aeration system for high solids anaerobic digestion (AD) of lignocellulosic biomass without volatile fatty acids (VFA) accumulation at high org. loading rate (OLR). Traditional AD of Napier grass, a model lignocellulosic biomass, at an OLR of 5 g volatile solids (VS)/L/day resulted in an accumulation of total VFA concn. up to 9.2 g/L as acetic acid (HAc) equiv., causing rapid drops in pH and methane yield, and driving the digester to the verge of failure. Once intermittent (every 24 h) ORP-controlled micro-aeration (at ORP of -470 mV) was initiated, the total VFA concn. rapidly decreased to 3.0 g HAc/L and the methane yield improved, resulting in stable digester performance without the need for alky. supplementation or OLR redn. By combining reactor performance results, mass balance analyses, microbial community characterization data, and a bioenergetic evaluation, this study suggested that rapid VFA conversion and CH4 prodn. were carried out by facultative anaerobes and hydrogenotrophic methanogens under micro-aerobic conditions. This novel operating approach can be applied as an effective control strategy for high OLR AD processes esp. in the event of VFA accumulation.
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    Sanaei-Moghadam, A.; Abbaspour-Fard, M. H.; Aghel, H.; Aghkhani, M. H.; Abedini-Torghabeh, J. Enhancement of Biogas Production by Co-Digestion of Potato Pulp with Cow Manure in a CSTR System. Appl. Biochem. Biotechnol. 2014, 173, 1858,  DOI: 10.1007/s12010-014-0972-5
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    Enhancement of Biogas Production by Co-digestion of Potato Pulp with Cow Manure in a CSTR System
    Sanaei-Moghadam, Akbar; Abbaspour-Fard, Mohammad Hossein; Aghel, Hasan; Aghkhani, Mohammad Hossein; Abedini-Torghabeh, Javad
    Applied Biochemistry and Biotechnology (2014), 173 (7), 1858-1869CODEN: ABIBDL; ISSN:0273-2289. (Springer)
    Anaerobic digestion (AD) process is a well-established method to generate energy from the org. wastes both from the environmental and economical perspectives. The purpose of present study is to evaluate energy prodn. from potato wastes by incorporating cow manure into the process. Firstly, a lab. pilot of one-stage biogas prodn. was designed and built according to continuously stirred tank reactor (CSTR) system. The setup was able to automatically control the environmental conditions of the process including temp., duration, and rate of stirring. AD expt. was exclusively performed on co-digestion of potato peel (PP) and cow manure (CM) in three levels of mixing ratio including 20:80, 50:50, 80:20 (PP:CM), and 0:100 as control treatment based on the volatile solid (VS) wt. without adding initial inoculums. After hydraulic retention time (HRT) of 50 days on av. 193, 256, 348, and 149 norm liter (LN) (kg VS)-1, methane was produced for different mixing ratios, resp. Statistical anal. shows that these gas productions are significantly different. The av. energy was detd. based on the produced methane which was about 2.8 kWh (kg VS)-1, implying a significant energy prodn. potential. The av. COD (COD) removal of treatments was about 61 %, showing that it can be leached significantly with high org. matter by the employed pilot. The energy efficiency of 92 % of the process also showed the optimum control of the process by the pilot.
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  41. 41
    Estevez, M. M.; Sapci, Z.; Linjordet, R.; Morken, J. Incorporation of Fish By-Product into the Semi-Continuous Anaerobic Co-Digestion of Pre-Treated Lignocellulose and Cow Manure, with Recovery of Digestate’s Nutrients. Renewable Energy 2014, 66, 550– 558,  DOI: 10.1016/j.renene.2014.01.001
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    Incorporation of fish by-product into the semi-continuous anaerobic co-digestion of pre-treated lignocellulose and cow manure, with recovery of digestate's nutrients
    Estevez, Maria M.; Sapci, Zehra; Linjordet, Roar; Morken, John
    Renewable Energy (2014), 66 (), 550-558CODEN: RNENE3; ISSN:0960-1481. (Elsevier Ltd.)
    Norway's fish processing industry generates large amts. of fish waste every year. The high-risk waste fraction with most of its oil removed has not yet been tested for energy prodn. The stability of an anaerobic digestion process that incorporates this material with steam exploded Salix and cow manure was tested using mesophilic, semi-continuous lab.-scale digesters. The effects of recycling the liq. digestate fraction were also investigated. The removal of ammonium (NH4+) and phosphate (PO43-) from the rejected digestate using struvite pptn. and bentonite adsorption were tested to generate a nutrient-enriched, final solid fertiliser. Adding 20% fish byproduct (volatile solids basis) increased methane yields by 35%, while recycling the digestate caused a slight increase. The NH4+-N levels reached 4-5 g l-1 in the reactors with recirculation and fish feed. Although these levels may threaten methanogenesis, the stability of the process was maintained during the entire period due to the good balance between the lignocellulose, proteins and fats provided by the co-digestion mixt. and the pos. effects of recirculation. The NH4+ and PO43- were successfully removed from the rejected liq. digestate. The redns. using struvite reached 87% and 60% (pH 9.5 and Mg2+:NH4+:PO43- ratio of 1.2:1:1), while bentonite achieved 82% and 52%, resp.
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    Rekha, B. N.; Pandit, A. B. Performance Enhancement of Batch Anaerobic Digestion of Napier Grass by Alkali Pre-Treatment. Int. J. ChemTech Res. 2013, 5 (2), 558– 564
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    Performance enhancement of batch anaerobic digestion of napier grass by alkali pre-treatment
    Rekha, B. N.; Pandit, Aniruddha B.
    International Journal of ChemTech Research (2013), 5 (2), 558-564CODEN: IJCRGG; ISSN:0974-4290. (Sphinx Knowledge House)
    The objective of this research was to develop an alkali pre-treatment process prior to anaerobic digestion (AD) of napier grass for improving solubilization of the lignocellulosic biomass and subsequent enhancement in biogas productivity. Mild concns. of NaOH soln. were used to pretreat napier grass which and was later subjected to anaerobic digestion for prodn. of biogas. Lab.-scale batch expts. were carried out in 0.5 L bottles with 0.3 L working vol. Optimal concn. of NaOH soln. for orgs. solubilization in the step of pre-treatment was 0.6% (w/v) i.e. 11.2g of NaOH/100g TS of napier grass. Under this condition, the sol. COD of the hydrolyzate was increased by 93%, which subsequently increased the prodn. of volatile fatty acids (VFA) during anaerobic digestion. The biogas prodn. of napier grass with and without pre-treatment was evaluated. The highest methane yield under optimal pre-treatment condition was found to be 0.158 m3 CH4/kg TS, as compared to 0.047 m3 CH4/kg TS for untreated napier grass when subjected to anaerobic digestion for a period of 8 days. These results indicated that alkali pre-treatment could be an effective method for increasing biodegradability and improving methane yield of napier grass.
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    Frigon, J. C.; Roy, C.; Guiot, S. R. Anaerobic Co-Digestion of Dairy Manure with Mulched Switchgrass for Improvement of the Methane Yield. Bioprocess Biosyst. Eng. 2012, 35 (3), 341– 349,  DOI: 10.1007/s00449-011-0572-5
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    Anaerobic co-digestion of dairy manure with mulched switchgrass for improvement of the methane yield
    Frigon, Jean-Claude; Roy, Caroline; Guiot, Serge R.
    Bioprocess and Biosystems Engineering (2012), 35 (3), 341-349CODEN: BBEIBV; ISSN:1615-7591. (Springer)
    The owners of farm-scale anaerobic digesters are relying on off-farm wastes or energy crops as a co-digestion feedstock with animal manure to increase their prodn. of methane and thus revenues. Switchgrass represents an interesting feedstock for Canadian digesters owners as it is a high-yielding low-maintenance perennial crop, well adapted to northern climate. Methane potential assays in batch tests showed methane prodn. of 19.4 ± 3.6, 28.3 ± 1.7, 37.3 ± 7.1 and 45.7 ± 0.8 L kg-1, for raw manure, blended manure, manure and mulched switchgrass, manure and pretreated switchgrass, resp. Two 6-L lab-scale anaerobic digesters were operated for 130 days to assess the benefit of co-digesting switchgrass with bovine manure (digester #2), at a 20% wet mass fraction, compared with a manure-only operation (digester #1) The digesters were operated at an hydraulic retention time of 37 ± 6 days and at loads of 2.4 ± 0.6 and 2.6 ± 0.6 kg total volatile solids (TVS) L-1 day-1 for digesters #1 (D1) and #2 (D2), resp. The TVS degrdn. reached 25 and 39%, which resulted in a methane prodn. of 1.18 ± 0.18 and 2.19 ± 0.31 L day-1 for D1 and D2, resp. The addn. of 20% on a wet mass ratio of switchgrass to a manure digester increased its methane prodn. by 86%. The co-digestion of switchgrass in a 500 m3 manure digester could yield up to 10.2 GJ day-1 of purified methane or 1.1 MWh day-1 of electricity.
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    Chen, Y.; Cheng, J. J.; Creamer, K. S. Inhibition of Anaerobic Digestion Process: A Review. Bioresour. Technol. 2008, 99 (10), 4044– 4064,  DOI: 10.1016/j.biortech.2007.01.057
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    Inhibition of anaerobic digestion process: A review
    Chen, Ye; Cheng, Jay J.; Creamer, Kurt S.
    Bioresource Technology (2008), 99 (10), 4044-4064CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    A review is given. Anaerobic digestion is an attractive waste treatment practice in which both pollution control and energy recovery can be achieved. Many agricultural and industrial wastes are ideal candidates for anaerobic digestion because they contain high levels of easily biodegradable materials. Problems such as low methane yield and process instability are often encountered in anaerobic digestion, preventing this technique from being widely applied. A wide variety of inhibitory substances are the primary cause of anaerobic digester upset or failure since they are present in substantial concns. in wastes. Considerable research efforts have been made to identify the mechanism and the controlling factors of inhibition. This review provides a detailed summary of the research conducted on the inhibition of anaerobic processes. The inhibitors commonly present in anaerobic digesters include ammonia, sulfide, light metal ions, heavy metals, and orgs. Due to the difference in anaerobic inocula, waste compn., and exptl. methods and conditions, literature results on inhibition caused by specific toxicants vary widely. Co-digestion with other waste, adaptation of microorganisms to inhibitory substances, and incorporation of methods to remove or counteract toxicants before anaerobic digestion can significantly improve the waste treatment efficiency.
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    Caporaso, J. G.; Lauber, C. L.; Walters, W. A.; Berg-Lyons, D.; Lozupone, C. A.; Turnbaugh, P. J.; Fierer, N.; Knight, R. Global Patterns of 16S RRNA Diversity at a Depth of Millions of Sequences per Sample. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 4516– 4522,  DOI: 10.1073/pnas.1000080107
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    Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample
    Caporaso, J. Gregory; Lauber, Christian L.; Walters, William A.; Berg-Lyons, Donna; Lozupone, Catherine A.; Turnbaugh, Peter J.; Fierer, Noah; Knight, Rob
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (Suppl. 1), 4516-4522, S4516/1-S4516/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The ongoing revolution in high-throughput sequencing continues to democratize the ability of small groups of investigators to map the microbial component of the biosphere. In particular, the coevolution of new sequencing platforms and new software tools allows data acquisition-and anal. on an unprecedented scale. Here we report the next stage in this coevolutionary arms race, using the Illumina GAIIx platform to sequence a diverse array of 25 environmental samples and three known "mock communities" at a depth averaging 3.1 million reads per sample. We demonstrate excellent consistency in taxonomic recovery and recapture diversity patterns that were previously reported on the basis of meta-anal. of many studies from the literature (notably, the saline/nonsaline split in environmental samples and the split between host-assocd. and free-living communities). We also demonstrate that 2,000 Illumina single-end reads are sufficient to recapture the same relationships among samples that we observe with the full dataset. The results thus open up the possibility of conducting large-scale studies analyzing thousands of samples simultaneously to survey microbial communities at an unprecedented spatial and temporal resoln.
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    Kozich, J. J.; Westcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Appl. Environ. Microbiol. 2013, 79 (17), 5112– 5120,  DOI: 10.1128/AEM.01043-13
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    Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq illumina sequencing platform
    Kozich, James J.; Westcott, Sarah L.; Baxter, Nielson T.; Highlander, Sarah K.; Schloss, Patrick D.
    Applied and Environmental Microbiology (2013), 79 (17), 5112-5120CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
    Rapid advances in sequencing technol. have changed the exptl. landscape of microbial ecol. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is crit. to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concn. of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large nos. of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
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    Oksanen, J. Multivariate Analysis of Ecological Communities in R: Vegan Tutorial. http://phylodiversity.net/azanne/csfar/images/8/85/Vegan.pdf (accessed 2018-02-07).
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    Werner, J. J.; Garcia, M. L.; Perkins, S. D.; Yarasheski, K. E.; Smith, S. R.; Muegge, B. D.; Stadermann, F. J.; Derito, C. M.; Floss, C.; Madsen, E. L. Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation. Appl. Environ. Microbiol. 2014, 80 (11), 3375– 3383,  DOI: 10.1128/AEM.00166-14
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    Microbial community dynamics and stability during an ammonia-induced shift to syntrophic acetate oxidation
    Werner, Jeffrey J.; Garcia, Marcelo L.; Perkins, Sarah D.; Yarasheski, Kevin E.; Smith, Samuel R.; Muegge, Brian D.; Stadermann, Frank J.; DeRito, Christopher M.; Floss, Christine; Madsen, Eugene L.; Gordon, Jeffrey I.; Angenent, Largus T.
    Applied and Environmental Microbiology (2014), 80 (11), 3375-3383, 9 pp.CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
    Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, the authors experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidn. In comparison with control digesters, the authors obsd. shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the digesters' 3-yr operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When the authors perturbed the digesters by increasing the ammonia concn. or temp., the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidn. had allowed the exptl. bioreactors to regain stable operation. The emergence of syntrophic acetate oxidn. coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. The authors' 16S rRNA gene anal. also revealed that acetate-fed enrichment expts. resulted in communities that did not represent the bioreactor community. Anal. of shotgun sequencing of community DNA suggests that syntrophic acetate oxidn. was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.
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    Vanwonterghem, I.; Jensen, P. D.; Dennis, P. G.; Hugenholtz, P.; Rabaey, K.; Tyson, G. W. Deterministic Processes Guide Long-Term Synchronised Population Dynamics in Replicate Anaerobic Digesters. ISME J. 2014, 8 (10), 2015– 2028,  DOI: 10.1038/ismej.2014.50
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    Deterministic processes guide long-term synchronised population dynamics in replicate anaerobic digesters
    Vanwonterghem, Inka; Jensen, Paul D.; Dennis, Paul G.; Hugenholtz, Philip; Rabaey, Korneel; Tyson, Gene W.
    ISME Journal (2014), 8 (10), 2015-2028CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)
    A replicate long-term expt. was conducted using anaerobic digestion (AD) as a model process to det. the relative role of niche and neutral theory on microbial community assembly, and to link community dynamics to system performance. AD is performed by a complex network of microorganisms and process stability relies entirely on the synergistic interactions between populations belonging to different functional guilds. In this study, three independent replicate anaerobic digesters were seeded with the same diverse inoculum, supplied with a model substrate, α-cellulose, and operated for 362 days at a 10-day hydraulic residence time under mesophilic conditions. Selective pressure imposed by the operational conditions and model substrate caused large reproducible changes in community compn. including an overall decrease in richness in the first month of operation, followed by synchronised population dynamics that correlated with changes in reactor performance. This included the synchronised emergence and decline of distinct Ruminococcus phylotypes at day 148, and emergence of a Clostridium and Methanosaeta phylotype at day 178, when performance became stable in all reactors. These data suggest that many dynamic functional niches are predictably filled by phylogenetically coherent populations over long time scales. Neutral theory would predict that a complex community with a high degree of recognized functional redundancy would lead to stochastic changes in populations and community divergence over time. We conclude that deterministic processes may play a larger role in microbial community dynamics than currently appreciated, and under controlled conditions it may be possible to reliably predict community structural and functional changes over time.
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    Sträuber, H.; Schröder, M.; Kleinsteuber, S. Metabolic and Microbial Community Dynamics during the Hydrolytic and Acidogenic Fermentation in a Leach-Bed Process. Energy. Sustain. Soc. 2012, 2 (1), 13,  DOI: 10.1186/2192-0567-2-13
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    Meng, Y.; Mumme, J.; Xu, H.; Wang, K. A Biologically Inspired Variable-PH Strategy for Enhancing Short-Chain Fatty Acids (SCFAs) Accumulation in Maize Straw Fermentation. Bioresour. Technol. 2016, 201, 329– 336,  DOI: 10.1016/j.biortech.2015.11.064
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    A biologically inspired variable-pH strategy for enhancing short-chain fatty acids (SCFAs) accumulation in maize straw fermentation
    Meng, Yao; Mumme, Jan; Xu, Heng; Wang, Kaijun
    Bioresource Technology (2016), 201 (), 329-336CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    This study investigates the feasibility of varying the pH to enhance the accumulation of short-chain fatty acids (SCFAs) in the in vitro fermn. of maize straw. The corresponding hydrolysis rate and the net SCFA yield increased as inoculum ratio (VSinoculum/VSsubstrate) increased from 0.09 to 0.79. The pH were maintained at 5.3, 5.8, 6.3, 6.8, 7.3, and 7.8, resp. A neutral pH of approx. 6.8 was optimal for hydrolysis. The net SCFA yield decreased by 34.9% for a pH of less than 5.8, but remained const. at approx. 721 ± 5 mg/gvs for a pH between 5.8 and 7.8. In addn., results were obtained for variable and const. pH levels at initial substrate concns. of 10, 30 and 50 g/L. A variable pH increased the net SCFA yield by 23.6%, 29.0%, and 36.6% for concns. of 10, 30 and 50 g/L. Therefore, a variable pH enhanced SCFA accumulation in maize straw fermn.
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    Zhang, M.; Zhang, G.; Zhang, P.; Fan, S.; Jin, S.; Wu, D.; Fang, W. Anaerobic Digestion of Corn Stovers for Methane Production in a Novel Bionic Reactor. Bioresour. Technol. 2014, 166, 606– 609,  DOI: 10.1016/j.biortech.2014.05.067
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    Anaerobic digestion of corn stovers for methane production in a novel bionic reactor
    Zhang, Meixia; Zhang, Guangming; Zhang, Panyue; Fan, Shiyang; Jin, Shuguang; Wu, Dan; Fang, Wei
    Bioresource Technology (2014), 166 (), 606-609CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    To improve the biogas prodn. from corn stovers, a new bionic reactor was designed and constructed. The bionic reactor simulated the rumen digestion of ruminants. The liq. was sepd. from corn stovers and refluxed into corn stovers again, which simulated the undigested particles sepd. from completely digested materials and fed back again for further degrdn. in ruminant stomach. Results showed that the bionic reactor was effective for anaerobic digestion of corn stovers. The liq. amt. and its reflux showed an obvious pos. correlation with biogas prodn. The highest biogas prodn. rate was 21.6 mL/gVS-added d, and the total cumulative biogas prodn. was 256.5 mL/gVS-added. The methane content in biogas ranged from 52.2% to 63.3%. The degrdn. of corn stovers were greatly enhanced through simulating the animal digestion mechanisms in this bionic reactor.
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    Ziemer, C. J.; Sharp, R.; Stern, M. D.; Cotta, M. A.; Whitehead, T. R.; Stahl, D. A. Comparison of Microbial Populations in Model and Natural Rumens Using 16S Ribosomal RNA-Targeted Probes. Environ. Microbiol. 2000, 2 (6), 632– 643,  DOI: 10.1046/j.1462-2920.2000.00146.x
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    Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes
    Ziemer C J; Sharp R; Stern M D; Cotta M A; Whitehead T R; Stahl D A
    Environmental microbiology (2000), 2 (6), 632-43 ISSN:1462-2912.
    A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.
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    Yenigün, O.; Demirel, B. Ammonia Inhibition in Anaerobic Digestion: A Review. Process Biochem. 2013, 48 (5–6), 901– 911,  DOI: 10.1016/j.procbio.2013.04.012
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    Ammonia inhibition in anaerobic digestion: A review
    Yenigun, Orhan; Demirel, Burak
    Process Biochemistry (Oxford, United Kingdom) (2013), 48 (5-6), 901-911CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)
    A review. Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis during anaerobic digestion process if it is available at high concns. Therefore, ammonia is regarded as a potential inhibitor during anaerobic digestion, particularly when dealing with complex type of substrates such as manure or the org. fraction of municipal solid waste. Ammonia is produced through biol. degrdn. of nitrogenous matter. NH4+ and free NH3 are the 2 principal forms of inorg. ammonia-N. Both forms can directly and indirectly cause inhibition in an anaerobic digestion system. Particularly, free NH3 is a powerful inhibitor in an anaerobic digester above threshold concns. Process inhibition is related to the particular characteristics of the substrate to be anaerobically digested, pH, process temp. (mesophilic or thermophilic), type of the seed sludge (inoculum), the reactor configuration, and to the concns. of NH4+ and NH3. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts after inhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial population available in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.
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    Ransom-Jones, E.; Jones, D. L.; McCarthy, A. J.; McDonald, J. E. The Fibrobacteres: An Important Phylum of Cellulose-Degrading Bacteria. Microb. Ecol. 2012, 63 (2), 267– 281,  DOI: 10.1007/s00248-011-9998-1
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    The Fibrobacteres: an Important Phylum of Cellulose-Degrading Bacteria
    Ransom-Jones, Emma; Jones, David L.; McCarthy, Alan J.; McDonald, James E.
    Microbial Ecology (2012), 63 (2), 267-281CODEN: MCBEBU; ISSN:0095-3628. (Springer)
    A review. The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognized as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted mol. approaches have demonstrated that novel centers of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degrdn. beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a "third" way of attacking the most abundant form of org. carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilize membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecol. and potential as a source of novel glycosyl hydrolases of biotechnol. importance.
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    Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity
    Arntzen, Magnus O.; Varnai, Aniko; Mackie, Roderick I.; Eijsink, Vincent G. H.; Pope, Phillip B.
    Environmental Microbiology (2017), 19 (7), 2701-2714CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
    Summary: Fibrobacter succinogenes is an anaerobic bacterium naturally colonizing the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilizing the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a com. cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to cryst. cellulose. We hypothesize that this complex has a function in plant cell wall degrdn., either by catalyzing polysaccharide degrdn. itself, or by targeting the vesicles to plant biomass.
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    Weimer, P. J.; Russell, J. B.; Muck, R. E. Lessons from the Cow: What the Ruminant Animal Can Teach Us about Consolidated Bioprocessing of Cellulosic Biomass. Bioresour. Technol. 2009, 100 (21), 5323– 5331,  DOI: 10.1016/j.biortech.2009.04.075
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    Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass
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    A review. Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermn. pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermn. is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective phys. pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermn. of nearly all plant components; efficient recovery of primary fermn. end-products; and prodn. of useful co-products. Ruminal fermn. does not produce significant amts. of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other org. compds., particularly transportation fuels.
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    Glucose metabolism of Treponema bryantii, an anaerobic rumen spirochete
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    The pathway of glucose metab. by T. bryantii, an obligately anaerobic spirochete isolated from bovine rumen contents, was studied. Washed cell suspensions of the spirochete consumed glucose and CO2 and produced equimolar amts. of acetate, formate, and succinate. CO2 was essential for glucose metab. Detn. of radioactivity in products formed from 14C-labeled glucose and NaH14CO3 and assays of enzyme activities in cell-free exts. were used to det. the pathway of glucose metab. T. bryantii Catabolized glucose to pyruvate via the Embden-Meyerhof-Parnas pathway. The spirochete used a coliform pyruvate-formate lyase to degrade pyruvate and produce formate and acetate. Succinate was formed by a pathway which involved the condensation of CO2 with pyruvate (or phospho(enol)pyruvate) formed from the breakdown of glucose.
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    To assess the contribution of individual bacterial species to the overall process of cellulose digestion in the rumen, cellulolytic bacteria (Bacteroides succinogenes and Ruminococcus albus) were tested as pure cultures and as cocultures with noncellulolytic T. bryantii. In studies of in vitro barley straw digestion, Treponema cocultures surpassed pure cultures of the cellulolytic organisms in dry matter disappearance, volatile fatty acid generation, and in the prodn. of succinic acid, lactic acid, and EtOH. Morphol. examn., by electron microscopy, showed that cells of T. bryantii assoc. with the plant cell wall materials in straw, but that cellulose digestion occurs only when these organisms are present with cellulolytic species such as B. succinogenes. These results show that cellulolytic bacteria interact with noncellulolytic Treponema to promote the digestion of cellulosic materials.
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    Hahnke, S.; Langer, T.; Koeck, D. E.; Klocke, M. Description of Proteiniphilum Saccharofermentans Sp. Nov., Petrimonas Mucosa Sp. Nov. and Fermentimonas Caenicola Gen. Nov., Sp. Nov., Isolated from Mesophilic Laboratory-Scale Biogas Reactors, and Emended Description of the Genus Proteiniphilum. Int. J. Syst. Evol. Microbiol. 2016, 66 (3), 1466– 1475,  DOI: 10.1099/ijsem.0.000902
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    Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov., isolated from mesophilic laboratory-scale biogas reactors, and emended description of the genus Proteiniphilum
    Hahnke, Sarah; Langer, Thomas; Koeck, Daniela E.; Klocke, Michael
    International Journal of Systematic and Evolutionary Microbiology (2016), 66 (3), 1466-1475CODEN: ISEMF5; ISSN:1466-5026. (Society for General Microbiology)
    Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic lab.-scale biogas reactors. The strains were Gram-neg. rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1-7.8. The main fermn. products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA-DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3-45.6 and 23.8-25.7 % relatedness, resp., which supports the conclusion that both isolates represent novel species. Phylogenetic anal. and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiol., genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addn., an emended description of the genus Proteiniphilum is provided.
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    Azman, S.; Khadem, A. F.; Van Lier, J. B.; Zeeman, G.; Plugge, C. M. Presence and Role of Anaerobic Hydrolytic Microbes in Conversion of Lignocellulosic Biomass for Biogas Production. Crit. Rev. Environ. Sci. Technol. 2015, 45 (23), 2523– 2564,  DOI: 10.1080/10643389.2015.1053727
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    Presence and Role of Anaerobic Hydrolytic Microbes in Conversion of Lignocellulosic Biomass for Biogas Production
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    Critical Reviews in Environmental Science and Technology (2015), 45 (23), 2523-2564CODEN: CRETEK; ISSN:1064-3389. (Taylor & Francis, Inc.)
    In recent years, biogas prodn. from complex biomass has received great interest. Therefore, many studies have been conducted to understand the anaerobic digestion process and to characterize responsible microbes for the biochem. conversions. Although the knowledge about biogas prodn. in general is rapidly increasing, less information is available about hydrolytic microbes within anaerobic bioreactors. Here, the authors pinpoint the urgent need for solid fundamental knowledge about hydrolytic bacteria within biogas plants. In this review, current knowledge about anaerobic hydrolytic microbes is presented, including their abundance in biogas plants, and the factors impacting their activity.
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    Effect of pH and monensin on glucose transport by Fibrobacter succinogenes, a cellulolytic ruminal bacterium
    Chow, Jo May; Russell, James B.
    Applied and Environmental Microbiology (1992), 58 (4), 1115-20CODEN: AEMIDF; ISSN:0099-2240.
    F. succinogenes S85, a cellulolytic ruminal bacterium, required Na for growth and glucose uptake. Cells which were deenergized with iodoacetate (500 μM) could not take up [14C]glucose. However, deenergized cells which were treated with valinomycin, loaded with K, and dild. into Na or Na plus K to create an artificial elec. gradient (Δψ) plus a chem. gradient of Na (ΔpNa) or ΔpNa alone transported glucose at a rapid rate. Cells which were loaded with Na plus K and dild. into Na (Δψ with Na, but no ΔpNa) also took up glucose at a rapid rate. K-loaded cells that were dild. into buffers which did not contain Na (Δψ without Na) could not take up glucose. An artificial ZΔpH which was created by acetate diffusion could not drive glucose transport even if Na was present. The max. rate and affinity of glucose transport (pH 6.7) were 62.5 nmol/mg protein per min and 0.51 mM, resp. S85 was unable to grow at pH <5.5, and there was little glucose transport at this pH. When the extracellular pH was decreased, the glucose carrier was inhibited, intracellular pH declined, the cells were no longer able to metabolite glucose, and Δψ declined. Monensin (1 μM) or lasalocid (5 μM) decreased intracellular ATP and dissipated both the Δψ and ΔpNa. Since there was no driving force for transport, glucose transport was inhibited. These results indicated that F. succinogenes used a pH-sensitive Na symport mechanism to take up glucose and that either a Δψ or a ΔpNa was required for glucose transport.
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    Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture
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    Applied and environmental microbiology (1980), 39 (3), 604-10 ISSN:0099-2240.
    A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.
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    Weimer, P. J. Effects of Dilution Rate and PH on the Ruminal Cellulolytic Bacterium Fibrobacter Succinogenes S85 in Cellulose-Fed Continuous Culture. Arch. Microbiol. 1993, 160, 288– 294,  DOI: 10.1007/BF00292079
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    Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture
    Weimer, Paul J.
    Archives of Microbiology (1993), 160 (4), 288-94CODEN: AMICCW; ISSN:0302-8933.
    F. succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of diln. rate (D, 0.014-0.076/h) and extracellular pH (6.11-6.84). Effects of pH and D on the fermn. were detd. by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and sol. sugar concn. to response surface anal. The extent of cellulose conversion decreased with increasing D. First-order rate consts. at rapid growth rates were estd. as 0.07-0.11/h, and decreased with decreasing pH. Apparent decreases in the rate const. with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of sol. sugars (0.04-0.18 g/L, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11-0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to det. that maintenance coeff. (0.04-0.06 g cellulose/g cells-h) and true growth yield (0.23-0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermn. end product, were ≤1.15 mol/mol anhydroglucose fermented and were slightly affected by diln. rate but were not affected by pH. Comparison of the fermn. data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of cryst. cellulose and efficient growth, despite a lower μmax on microcryst. cellulose.
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    Kleerebezem, R.; Joosse, B.; Rozendal, R.; Van Loosdrecht, M. C. M. Anaerobic Digestion without Biogas?. Rev. Environ. Sci. Bio/Technol. 2015, 14, 787,  DOI: 10.1007/s11157-015-9374-6
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    Anaerobic digestion without biogas?
    Kleerebezem, Robbert; Joosse, Bart; Rozendal, Rene; Van Loosdrecht, Mark C. M.
    Reviews in Environmental Science and Bio/Technology (2015), 14 (4), 787-801CODEN: RESBC6; ISSN:1569-1705. (Springer)
    Anaerobic digestion for the prodn. of methane contg. biogas is the classic example of a resource recovery process that combines stabilization of particulate org. matter or wastewater treatment with the prodn. of a valuable end-product. Attractive features of the process include the prodn. of a single end-product from a heterogeneous feedstock, and in-situ product sepn. of the gaseous end-product. Despite these intrinsic attractive properties of the process, the economic added value of the biogas produced is limited, enabling the development of alternative processes that yield higher-value end-products. Typically the prodn. of higher value end-products from low value feedstock and industrial wastewater proceeds via intermediate prodn. of org. acids (and carbon dioxide and mol. hydrogen). Optimization of org. acid prodn. from particulate feedstocks and wastewater for development of the org. acid based resource recovery route receives significant research attention. The org. acid stream generated as such, has no economic value, but if org. acids can either be concd. via membrane sepn. or (bio)converted to an end-product that can easily be sepd. from the liq., an attractive biomass processing scheme can be developed. Attractive end-products of org. acid processing include polyhydroxyalkanoates, medium chain length fatty acids, or other org. mols. using bio-electrochem. systems. Overall we suggest that these novel bioprocessing routes for conversion of low value feedstock to higher added value products will contribute to a sustainable future and will change the economic status of org. waste.
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  21. Xavier Fonoll, Kuang Zhu, Lucy Aley, Shilva Shrestha, Lutgarde Raskin. Simulating Rumen Conditions using an Anaerobic Dynamic Membrane Bioreactor to Enhance Hydrolysis of Lignocellulosic Biomass. 2023https://doi.org/10.1101/2023.02.20.529314
  22. Laura E. Walls, Peter Otoupal, Rodrigo Ledesma-Amaro, Sharon B. Velasquez-Orta, John M. Gladden, Leonardo Rios-Solis. Bioconversion of cellulose into bisabolene using Ruminococcus flavefaciens and Rhodosporidium toruloides. Bioresource Technology 2023, 368 , 128216. https://doi.org/10.1016/j.biortech.2022.128216
  23. Diana Cestari Bon, Dagoberto Yukio Okada, Cassiana Maria Reganhan Coneglian. Benefits of biological additive inoculation in the treatment of effluent from the paper recycling industry. Journal of Water Process Engineering 2022, 50 , 103269. https://doi.org/10.1016/j.jwpe.2022.103269
  24. Muhammad Sohail, Alam Khan, Malik Badshah, Allan Degen, Guo Yang, Hu Liu, Jianwei Zhou, Ruijun Long. Yak rumen fluid inoculum increases biogas production from sheep manure substrate. Bioresource Technology 2022, 362 , 127801. https://doi.org/10.1016/j.biortech.2022.127801
  25. Jinsong Liang, Wei Fang, Jianning Chang, Guangming Zhang, Weifang Ma, Mohammad Nabi, Muhammad Zubair, Ru Zhang, Le Chen, Jianghao Huang, Panyue Zhang. Long-term rumen microorganism fermentation of corn stover in vitro for volatile fatty acid production. Bioresource Technology 2022, 358 , 127447. https://doi.org/10.1016/j.biortech.2022.127447
  26. Tianwei Hao, Yihang Xiao, Sunita Varjani. Transiting from the inhibited steady-state to the steady-state through the ammonium bicarbonate mediation in the anaerobic digestion of low-C/N-ratio food wastes. Bioresource Technology 2022, 351 , 127046. https://doi.org/10.1016/j.biortech.2022.127046
  27. Yeadam Jo, Chaeyoung Rhee, Hyungmin Choi, Juhee Shin, Seung Gu Shin, Changsoo Lee. Long-term effectiveness of bioaugmentation with rumen culture in continuous anaerobic digestion of food and vegetable wastes under feed composition fluctuations. Bioresource Technology 2021, 338 , 125500. https://doi.org/10.1016/j.biortech.2021.125500
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  • Abstract

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    Figure 1

    Figure 1. (A) Short-chain fatty acid (SCFA) accumulation, (B) pH, and (C) specific methane production in the reactors. Organic loading rate (OLR) and retention time in Inoc_AD, Inoc_RC+AD (−·−), and Inoc_RC (− −). The vertical line indicates the change from cow feces to cow manure on day 81. The red arrow and green lines with circles indicate the buffer addition in Inoc_RC and Inoc_RC+AD, respectively. The legend refers to the three reactors with the type of inocula used.

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    Figure 2

    Figure 2. Relative abundances of OTUs grouped by origin in reactors Inoc_RC, Inoc_RC+AD, and Inoc_AD. OTUs were grouped in eight categories depending on whether they were present in the rumen content inoculum only, the AD inoculum only, the cow manure only, various combinations of these three sources, or none of them (“Others”). Samples from day 28 for Inoc_RC+AD and Day 75 for Inoc_AD could not be sequenced.

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    Figure 3

    Figure 3. (A) Short-chain fatty acid (SCFA) accumulation, (B) pH, and (C) methane production in the reactors showing (A) the organic loading rate (OLR) (− −) and (B) retention time (− −) applied in the reactors. The vertical line on day 44 indicates the start of continuous buffer addition.

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    Figure 4

    Figure 4. Relative abundances of OTUs grouped by origin in reactors Co_NG+CM, Co_NG+CM+RC, and Co_NG+RC. OTUs were grouped in eight categories depending on whether they were present in the rumen content only, the AD inoculum only, the cow manure only, a combination of these three sources, or none of them (“Others”). Samples from day 72 for Co_NG+CM and day 51 for Co_NG+RC could not be sequenced.

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  • References

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      A review. Herbivorous mammals and wood-eating insects are fairly effective in the digestion of plant polymers, such as lignocellulosics. In order to improve methane prodn. from the lignocellulosic biomass, several kinds of anaerobic digestion processes derived from animal models have been devised. However, the rates of biodegrdn. occurring in the anaerobic bioreactors still remain lower than in animal guts. The effectiveness of the digestive systems of those animals results from the concerted action of the various enzymes (e.g. cellulases, xylanases, esterases, ligninases) produced in their guts as well as their integration with mech. and chem. actions. Powerful pretreatment (prefermentation) operations are integrated to and support efficiently the microbial fermn. system, e.g. the rumination (i.e. mech.) in ruminants and the secretion of endogenous cellulases (i.e. enzymic) or the alk. treatment (chem.) at mid-way in xylophagous insects. The oxygen gradients along the gastrointestinal tract may also stimulate the hydrolytic activities of some microbial populations. In addn., the solid retention time, the digesta flow and the removal of the end-products are well ordered to enable animals to thrive on a complex polymer such as lignocellulose. At the same time, technologies were developed to degrade lignocellulosic biomass, such as the rumen derived anaerobic digestion (RUDAD) process and the rumen simulating technique (RUSITEC), more elaborated and using rumen microbial consortia. Overall, they showed that the fermn. taking place in the rumen fermn. and even in the hindgut are biol. processes that go beyond the limited environmental conditions generally found in anaerobic digesters. Hence, knowledge on herbivores' digestion mechanisms might be better exploited in the design and operation of anaerobic digesters. This literature review is a cross-anal. of the relevant information about the digestive strategies of herbivorous and wood-eating animals and the bioengineering techniques in lignocelluloses degrdn. The aim is to highlight strategies of animals' digestion simulation for more effective anaerobic digestion of lignocellulosic compds. and other solid residues.
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      Yue, Z.-B.; Li, W.-W.; Yu, H.-Q. Application of Rumen Microorganisms for Anaerobic Bioconversion of Lignocellulosic Biomass. Bioresour. Technol. 2013, 128, 738– 744,  DOI: 10.1016/j.biortech.2012.11.073
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      Application of rumen microorganisms for anaerobic bioconversion of lignocellulosic biomass
      Yue, Zheng-Bo; Li, Wen-Wei; Yu, Han-Qing
      Bioresource Technology (2013), 128 (), 738-744CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      A review. Rumen in the mammalian animals is a natural cellulose-degrading system and the microorganisms inside were found to be able to effectively digest lignocellulosic biomass. Furthermore, methane or volatile fatty acids, which could be further converted to other biofuels, are the two major products in such a system. This paper offers an overview of recent development in the application of rumen microorganisms for lignocellulosic biomass conversion. Application of recent mol. tools in the anal. of rumen microbial community, progress in the development of artificial rumen reactors, the latest research results about characterizing rumen-dominated anaerobic digestion process and energy products are summarized. Also, the potential application of such a rumen-dominated process is discussed.
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      High-rate two-phase process for the anaerobic degradation of cellulose, employing rumen microorganisms for an efficient acidogenesis
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      A novel 2-stage anaerobic process for the microbial conversion of cellulose to biogas was developed. In the 1st phase, a mixed population of rumen bacteria and ciliates was used in the hydrolysis and fermn. of cellulose. The volatile fatty acids (VFA) produced in this acidogenic reactor were subsequently converted into biogas in UASB-type methanogenic reactor. A stepwise increase of the loading rate from 11.9 to 25.8 g volatile solids/L reactor vol.-day (g VS/L/day) did not affect the degrdn. efficiency in the acidogenic reactor, whereas the methanogenic reactor appeared to be overloaded at the highest loading rate. Cellulose digestion was almost complete at all loading rates applied. The 2-stage anaerobic process was also tested with a closed fluid circuit. In this instance total CH4 prodn. was 0.438 L/g VS added, which is equiv. to 98% of the theor. value. The application of rumen microorganisms in combination with a high-rate CH4 reactor is proposed as a means of efficient anaerobic degrdn. of cellulosic residues to CH4.
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      Conversion of cereal residues into biogas in a rumen-derived process
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      A recently developed high-rate, 2-phase process, which employs rumen microorganisms for efficient acidogenesis, was tested for anaerobic degrdn. of barley straw, rye straw, and maize stove. Under conditions similar to those of the rumen and loading rates varying between 9.8 and 26.0 g org. matter/L-day in the 1st phase (acidogenic reactor), total fiber degrdn. efficiencies ranged 42-57%, irresp. of the loading rate applied. Av. sp. prodn. of volatile fatty acids and biogas/g volatile solids digested in the acidogenic reactor were 6.9-11.2 mmol and 0.10-0.25 L, resp. The effect of varying solid retention times on the extent of degrdn. of barley straw was examd. Changing of retention times in the range of 60-156 h had no effect on degrdn. efficiency, but a decrease in efficiency was obsd. at retention times <60 h. By connecting the acidogenic reactor in series to an Upflow Anaerobic Sludge Blanket (UASB) methanogenic reactor, the volatile fatty acids were converted to biogas. Av. CH4 contents of the gases produced in the acidogenic reactor and in the UASB reactor were 30 and 78%, resp.
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      Nair, S.; Kuang, Y.; Pullammanappallil, P. Enhanced Degradation of Waste Grass Clippings in One and Two Stage Anaerobic Systems. Environ. Technol. 2005, 26 (9), 1003– 1011,  DOI: 10.1080/09593332608618488
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      Enhanced degradation of waste grass clippings in one and two stage anaerobic systems
      Nair, S.; Kuang, Y.; Pullammanappallil, P.
      Environmental Technology (2005), 26 (9), 1003-1011CODEN: ENVTEV; ISSN:0959-3330. (Selper Ltd., Publications Division)
      This work studied the use of a simple rumen-fluid-inoculated anaerobic treatment system for the degrdn. of org. waste. Fresh rumen fluid collected from a fistulated sheep was used as the inoculum and fresh grass clippings were used as the waste material for treatment. Studies were carried out on both a 1-stage system where the ligno-cellulosic fraction breaks down into a mixt. of sol. products including volatile fatty acids and a 2-stage system where these products are subsequently mineralized to biogas. In the 1-stage system ∼70% of the org. waste was solubilized and in the 2-stage system ∼60% waste material was solubilized in a week. About 50% of the degrdn. was achieved in 4 days, showing that a 4-day solids retention time would be a suitable operating regime. The max. volatile fatty acid prodn. rate was 327 mg COD/L-h. A higher loading rate of 30 g/L-day was achieved in these systems compared to anaerobic digesters. Microbiol. studies showed an increase in the no. of fungal spores as well as a decrease in the no. of protozoa in the treatment system. These nos. attained stable values over the duration of the expts. The system developed is superior to conventional composting or anaerobic digestion and can be applied for the treatment of ligno-cellulosic agricultural residues.
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      Zhang, M.; Zhang, G.; Zhang, P.; Fan, S.; Jin, S.; Wu, D.; Fang, W. Anaerobic Digestion of Corn Stovers for Methane Production in a Novel Bionic Reactor. Bioresour. Technol. 2014, 166, 606– 609,  DOI: 10.1016/j.biortech.2014.05.067
      13
      Anaerobic digestion of corn stovers for methane production in a novel bionic reactor
      Zhang, Meixia; Zhang, Guangming; Zhang, Panyue; Fan, Shiyang; Jin, Shuguang; Wu, Dan; Fang, Wei
      Bioresource Technology (2014), 166 (), 606-609CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      To improve the biogas prodn. from corn stovers, a new bionic reactor was designed and constructed. The bionic reactor simulated the rumen digestion of ruminants. The liq. was sepd. from corn stovers and refluxed into corn stovers again, which simulated the undigested particles sepd. from completely digested materials and fed back again for further degrdn. in ruminant stomach. Results showed that the bionic reactor was effective for anaerobic digestion of corn stovers. The liq. amt. and its reflux showed an obvious pos. correlation with biogas prodn. The highest biogas prodn. rate was 21.6 mL/gVS-added d, and the total cumulative biogas prodn. was 256.5 mL/gVS-added. The methane content in biogas ranged from 52.2% to 63.3%. The degrdn. of corn stovers were greatly enhanced through simulating the animal digestion mechanisms in this bionic reactor.
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    14. 14
      Chapleur, O.; Bize, A.; Serain, T.; Mazéas, L.; Bouchez, T. Co-Inoculating Ruminal Content Neither Provides Active Hydrolytic Microbes nor Improves Methanization of 13C-Cellulose in Batch Digesters. FEMS Microbiol. Ecol. 2014, 87 (3), 616– 629,  DOI: 10.1111/1574-6941.12249
      14
      Co-inoculating ruminal content neither provides active hydrolytic microbes nor improves methanization of 13C-cellulose in batch digesters
      Chapleur, Olivier; Bize, Ariane; Serain, Thibaut; Mazeas, Laurent; Bouchez, Theodore
      FEMS Microbiology Ecology (2014), 87 (3), 616-629CODEN: FMECEZ; ISSN:0168-6496. (Wiley-Blackwell)
      Cellulose hydrolysis often limits the kinetics and efficiency of anaerobic degrdn. in industrial digesters. In animal digestive systems, specialized microorganisms enable cellulose biodegrdn. at significantly higher rates. This study aims to assess the potential of ruminal microbial communities to settle and to express their cellulolytic properties in anaerobic digesters. Cellulose-degrading batch incubations were co-inoculated with municipal solid waste digester sludge and ruminal content. 13C-labeled cellulose degrdn. was described over time with Gas Chromatog.-Combustion-Isotope Ratio Mass Spectrometry. Results were linked to the identification of the microorganisms assimilating 13C and to the monitoring of their relative dynamics. Cellulose degrdn. in co-inoculated incubations was efficient but not significantly improved. Transient disturbances in degrdn. pathways occurred, as revealed by propionate accumulation. Automated Ribosomal Intergenic Spacer Anal. dynamics and pyrosequencing revealed that expected classes of Bacteria and Archaea were active and degraded cellulose. However, despite the favorable co-inoculation conditions, mol. tools also revealed that no ruminal species settled in the bioreactors. Other specific parameters were probably needed for this to happen. This study shows that exploiting the rumen's cellulolytic properties in anaerobic digesters is not straightforward. Co-inoculation can only be successful if ruminal microorganisms manage to thrive in the anaerobic digester and outcompete native microorganisms, which requires specific nutritional and environmental parameters, and a meticulous reprodn. of the selection pressure encountered in the rumen.
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    15. 15
      Wall, D. M.; Straccialini, B.; Allen, E.; Nolan, P.; Herrmann, C.; O’Kiely, P.; Murphy, J. D. Investigation of Effect of Particle Size and Rumen Fluid Addition on Specific Methane Yields of High Lignocellulose Grass Silage. Bioresour. Technol. 2015, 192, 266– 271,  DOI: 10.1016/j.biortech.2015.05.078
      15
      Investigation of effect of particle size and rumen fluid addition on specific methane yields of high lignocellulose grass silage
      Wall, D. M.; Straccialini, B.; Allen, E.; Nolan, P.; Herrmann, C.; O'Kiely, P.; Murphy, J. D.
      Bioresource Technology (2015), 192 (), 266-271CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      This work examines the digestion of advanced growth stage grass silage. Two variables were investigated: particle size (greater than 3 cm and less than 1 cm) and rumen fluid addn. Batch studies indicated particle size and rumen fluid addn. had little effect on specific methane yields (SMYs). In continuous digestion of 3 cm silage the SMY was 342 and 343 L CH4 kg-1 VS, resp., with and without rumen fluid addn. However, digester operation was significantly affected through silage floating on the liquor surface and its entanglement in the mixing system. Digestion of 1 cm silage with no rumen fluid addn. struggled; volatile fatty acid concns. rose and SMYs dropped. The best case was 1 cm silage with rumen fluid addn., offering higher SMYs of 371 L CH4 kg-1 VS and stable operation throughout. Thus, phys. and biol. treatments benefited continuous digestion of high fiber grass silage.
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    16. 16
      Deng, Y.; Huang, Z.; Ruan, W.; Zhao, M.; Miao, H.; Ren, H. Co-Inoculation of Cellulolytic Rumen Bacteria with Methanogenic Sludge to Enhance Methanogenesis of Rice Straw. Int. Biodeterior. Biodegrad. 2017, 117, 224– 235,  DOI: 10.1016/j.ibiod.2017.01.017
      16
      Co-inoculation of cellulolytic rumen bacteria with methanogenic sludge to enhance methanogenesis of rice straw
      Deng, Yuying; Huang, Zhenxing; Ruan, Wenquan; Zhao, Mingxing; Miao, Hengfeng; Ren, Hongyan
      International Biodeterioration & Biodegradation (2017), 117 (), 224-235CODEN: IBBIES; ISSN:0964-8305. (Elsevier Ltd.)
      Ruminal microbiota co-inoculated with methanogenic sludge were applied in a 2 L anaerobic system. The expt. lasted 48 days in three stages with different substrate loading rates of 3 g d-1, 7 g d-1 and 14 g d-1. The fermn. performance and microbial characteristics were assessed. There was no corresponding accumulation of volatile fatty acids (VFAs) and pH values varied between 6.8 and 7.68 during the whole anaerobic digestion, indicating system stability. The cellulolytic activities improved significantly (P < 0.05) compared with control samples. Co-inoculation not only added methanogens but also increased the proportion of bacteria contg. GH 5 genes even though the bacterial compn. changed by PCR-DGGE phylogenetic anal. The proportion of cellulolytic genera of Clostridium and Ruminococcus increased, whereas Bacteroides, Fibrobacter and Acetivibrio disappeared due to their inability to acclimatize to in vitro lignocellulosic conditions. The anal. of GH and 16S rRNA genes also provided a better description of cellulolytic function and phylogenetic profile. Mutual cooperation formed between Methanobrevibacter and Ruminococcus was achieved for high cellulolytic activity and methanogenic efficiency in co-inoculated system. Moreover, this hypothesis was further strengthened by observations on morphol. characteristics of cellulolytic consortia.
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    17. 17
      Murali, N.; Fernandez, S.; Ahring, B. K. Fermentation of Wet-Exploded Corn Stover for the Production of Volatile Fatty Acids. Bioresour. Technol. 2017, 227, 197– 204,  DOI: 10.1016/j.biortech.2016.12.012
      17
      Fermentation of wet-exploded corn stover for the production of volatile fatty acids
      Murali, Nanditha; Fernandez, Sebastian; Ahring, Birgitte Kiaer
      Bioresource Technology (2017), 227 (), 197-204CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      Volatile fatty acids (VFA) have been used as platform mols. for prodn. of biofuels and bioproducts. In the current study, we examine the VFA prodn. from wet-exploded corn stover through anaerobic fermn. using rumen bacteria. The total VFA yield (acetic acid equiv.) was found to increase from 22.8 g/L at 2.5% total solids (TS) to 40.8 g/L at 5% TS. It was found that the acetic acid concn. increased from 10 g/L to 22 g/L at 2.5% and 5% TS, resp. An increased propionic acid prodn. was seen between day 10 and 20 at 5% TS. Valeric acid (4 g/L) was produced at 5% TS and not at 2.5% TS. Compn. anal. showed that 50% of the carbohydrates were converted to VFA at 5% TS and 33% at 2.5% TS. Our results show that rumen fermn. of lignocellulosic biomass after wet explosion can produce high concns. of VFA without addn. of external enzymes of importance for the process economics of lignocellulosic biorefineries.
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    18. 18
      Wall, D. M.; Allen, E.; O’Shea, R.; O’Kiely, P.; Murphy, J. D. Investigating Two-Phase Digestion of Grass Silage for Demand-Driven Biogas Applications: Effect of Particle Size and Rumen Fluid Addition. Renewable Energy 2016, 86, 1215– 1223,  DOI: 10.1016/j.renene.2015.09.049
      18
      Investigating two-phase digestion of grass silage for demand-driven biogas applications: Effect of particle size and rumen fluid addition
      Wall, D. M.; Allen, E.; O'Shea, R.; O'Kiely, P.; Murphy, J. D.
      Renewable Energy (2016), 86 (), 1215-1223CODEN: RNENE3; ISSN:0960-1481. (Elsevier Ltd.)
      High lignocellulose content grass silage was investigated for two-phase digestion (leaching followed by upflow anaerobic sludge blanket (UASB)) for application to demand-driven biogas prodn. Leaching trials were undertaken investigating the effects of particle size redn. and rumen fluid addn. on the hydrolysis and acidogenesis phases. Reducing grass silage particle size to <1 cm was not suited to leaching as particles could not be fully entrained in the system; this was not an issue at >3 cm particle size. Rumen fluid addn. increased prodn. of volatile fatty acids (VFA) but reduced pH levels, which subsequently hindered hydrolysis of volatile solids (VS). When electricity demand is low, it is recommended to operate in leach only mode with grass silage particle size >3 cm and with rumen fluid addn.; this limits VS destruction to 30% while maintaining a high VFA yield. When electricity demand is high, connection of the UASB generates 61% destruction of VS maximizing biogas prodn. Operation of the SLBR-UASB achieves lower specific methane yields than traditional single-stage digestion but may offer advantages in demand driven biogas systems.
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    19. 19
      Ozbayram, E. G.; Kleinsteuber, S.; Nikolausz, M.; Ince, B.; Ince, O. Enrichment of Lignocellulose-Degrading Microbial Communities from Natural and Engineered Methanogenic Environments. Appl. Microbiol. Biotechnol. 2018, 102 (2), 1035– 1043,  DOI: 10.1007/s00253-017-8632-7
      19
      Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments
      Ozbayram, Emine Gozde; Kleinsteuber, Sabine; Nikolausz, Marcell; Ince, Bahar; Ince, Orhan
      Applied Microbiology and Biotechnology (2018), 102 (2), 1035-1043CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examd. by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Addnl., the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.
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    20. 20
      Deng, Y.; Huang, Z.; Zhao, M.; Ruan, W.; Miao, H.; Ren, H. Effects of Co-Inoculating Rice Straw with Ruminal Microbiota and Anaerobic Sludge: Digestion Performance and Spatial Distribution of Microbial Communities. Appl. Microbiol. Biotechnol. 2017, 101 (14), 5937– 5948,  DOI: 10.1007/s00253-017-8332-3
      20
      Effects of co-inoculating rice straw with ruminal microbiota and anaerobic sludge: digestion performance and spatial distribution of microbial communities
      Deng, Yuying; Huang, Zhenxing; Zhao, Mingxing; Ruan, Wenquan; Miao, Hengfeng; Ren, Hongyan
      Applied Microbiology and Biotechnology (2017), 101 (14), 5937-5948CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Ruminal microbiota (RM) were co-inoculated with anaerobic sludge (AS) at different ratios to study the digestion of rice straw in batch expts. The CH4 yield reached 273.64 mL/g volatile solid (VS) at a co-inoculum ratio of 1:1. The xylanase and cellulase activities were 198.88-212.88 and 24.51-29.08 U/mL in co-inoculated samples, resp., and were significantly different compared to the results for single inoculum (p < 0.05). Higher ratios of AS enhanced acetoclastic methanogenesis, and propionate accumulation could be the main reason for the longer lag phase obsd. in samples with a higher RM ratio. The microbial compns. were clearly altered after digestion. Fibrobacter, Ruminococcus and Butyrivibrio from the rumen did not settle in the co-inoculated system, whereas Clostridiales members became the main polysaccharide degraders. Microbial interactions involving hydrolytic bacteria and acetoclastic methanogens in the residue were considered to be significant for hydrolysis activities and methane prodn. Syntrophy involving propionate oxidizers with assocd. methanogens occurred in the liq. phase. Our findings provide a better understanding of the anaerobic digestion of rice straw that is driven by specific microbial populations.
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    21. 21
      Li, K.; Zhu, H.; Zhang, Y.; Zhang, H. Characterization of the Microbial Communities in Rumen Fluid Inoculated Reactors for the Biogas Digestion of Wheat Straw. Sustainability 2017, 9 (2), 243,  DOI: 10.3390/su9020243
      21
      Characterization of the microbial communities in rumen fluid inoculated reactors for the biogas digestion of wheat straw
      Li, Ke; Zhu, Haoran; Zhang, Yajie; Zhang, Hongxun
      Sustainability (2017), 9 (2), 243/1-243/14CODEN: SUSTDE; ISSN:2071-1050. (MDPI AG)
      The present study investigated the effect of rumen fluid (RF) concn. on the methane prodn. through anaerobic digestion of wheat straw in batch mode, and compared the microbial communities in RF and RF inoculated reactors by 16S rRNA genes sequencing. Six levels of RF concn. including 1%, 5%, 10%, 15%, 20% and 25% (vol./vol.) were used in reactors R1, R5, R10, R15, R20 and R25 resp. The results revealed that lower than or equal to 5% RF concns. resulted in reactor acidification and low methane prodn. The highest methane yield of 106 mL·CH4·g·VS-1 was achieved in R10, whereas higher RF concns. than 10% could not improve the methane prodn. significantly. Methanosarcina barkeri was abundant in the well-working reactors, and Methanobacterium was dominant in the poor-working reactors, implying the archaeal communities in reactors had changed greatly from the Methanobrevibacter-dominated RF. Although the relative abundance of Clostridium and Ruminococcus were greatly different between RF and reactors, the Bacteroidetes and Firmicutes communities were dominant in all the tested samples. The results indicated that the in vitro anaerobic conditions had altered the rumen methanogenic communities significantly and the facultative acetoclastic Methanosarcina was important for the methane prodn. in the RF seeded reactors.
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    22. 22
      Agematu, H.; Takahashi, T.; Hamano, Y. Continuous Volatile Fatty Acid Production from Lignocellulosic Biomass by a Novel Rumen-Mimetic Bioprocess. J. Biosci. Bioeng. 2017, 124 (5), 528– 533,  DOI: 10.1016/j.jbiosc.2017.06.006
      22
      Continuous volatile fatty acid production from lignocellulosic biomass by a novel rumen-mimetic bioprocess
      Agematu, Hitosi; Takahashi, Takehiko; Hamano, Yoshio
      Journal of Bioscience and Bioengineering (2017), 124 (5), 528-533CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)
      Lignocellulosic biomass is an attractive source of biofuels and biochems., being abundant in various plant sources. However, processing this type of biomass requires hydrolysis of cellulose. The proposed rumen-mimetic bioprocess consists of dry-pulverization of lignocellulosic biomass and pH-controlled continuous cultivation of ruminal bacteria using ammonium as a nitrogen source. In this study, ruminal bacteria were continuously cultivated for over 60 days and used to digest microcryst. cellulose, rice straw, and Japanese cedar to produce volatile fatty acids (VFAs). The ruminal bacteria grew well in the chem. defined medium. The amts. of VFAs produced from 20 g of cellulose, rice straw, and Japanese cedar were 183±29.7, 69.6±12.2, and 21.8±12.9 mmol, resp. Each digestion completed within 24 h. The carbon yield was 60.6% when 180 mmol of VFAs was produced from 20 g of cellulose. During the cultivation, the bacteria were obsd. to form flocs that enfolded the feed particles. These flocs likely contain all of the bacterial species necessary to convert lignocellulosic biomass to VFAs and microbial protein symbiotically. Denaturing gradient gel electrophoresis (DGGE) anal. of PCR-amplified 16S rDNA fragments revealed that the bacterial community was relatively stable after 1 wk in cultivation, though it was different from the original community structure. Furthermore, sequence anal. of the DGGE bands indicates that the microbial community includes a cellulolytic bacterium, a bacterium acting synergistically with cellulolytic bacteria, and a propionate-producing bacterium, as well as other anaerobic bacteria.
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    23. 23
      Ozbayram, E. G.; Akyol; Ince, B.; Karakoç, C.; Ince, O. Rumen Bacteria at Work: Bioaugmentation Strategies to Enhance Biogas Production from Cow Manure. J. Appl. Microbiol. 2018, 124 (2), 491– 502,  DOI: 10.1111/jam.13668
      23
      Rumen bacteria at work: bioaugmentation strategies to enhance biogas production from cow manure
      Ozbayram, E. G.; Akyol, C.; Ince, B.; Karakoc, C.; Ince, O.
      Journal of Applied Microbiology (2018), 124 (2), 491-502CODEN: JAMIFK; ISSN:1364-5072. (Wiley-Blackwell)
      Aims : To investigate the effects of different bioaugmentation strategies for enhancing the biogas prodn. from cow manure and evaluate microbial community patterns. Methods and Results : Co-inoculation with cow rumen fluid and cow rumen-derived enriched microbial consortia was evaluated in anaerobic batch tests at 36°C and 41°C. Singular addn. of both rumen fluid and enriched bioaugmentation culture had a promising enhancement on methane yields; however, the highest methane yield (311 mL CH4 per g VS at 41°C) was achieved when the anaerobic seed sludge was co-inoculated together with rumen fluid and enriched bioaugmentation culture. Bacterial community profiles were investigated by Ion PGM Platform, and specific lignocellulolytic bacteria dynamics in batch tests were assessed by qPCR. The temp. had minor effects on the abundance of bacterial community; in which Bacteroidetes and Firmicutes were the most abundant phyla in all digesters. There was an important impact of Firmicutes flavefaciens and Ruminococcus albus at 41°C, which in turn pos. affected the methane prodn. Conclusion : The degree of enhancement in biogas prodn. can be upgraded by the co-inoculation of rumen-derived bioaugmentation culture with anaerobic seed sludge with high methanogenic activity. Significance and Impact of the Study : A close look at the biotic interactions and their assocns. with abiotic factors might be valuable for evaluating rumen-related bioaugmentation applications.
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    24. 24
      Deng, Y.; Huang, Z.; Ruan, W.; Miao, H.; Shi, W.; Zhao, M. Enriching Ruminal Polysaccharide-Degrading Consortia via Co-Inoculation with Methanogenic Sludge and Microbial Mechanisms of Acidification across Lignocellulose Loading Gradients. Appl. Microbiol. Biotechnol. 2018, 102 (8), 3819– 3830,  DOI: 10.1007/s00253-018-8877-9
      24
      Enriching ruminal polysaccharide-degrading consortia via co-inoculation with methanogenic sludge and microbial mechanisms of acidification across lignocellulose loading gradients
      Deng, Yuying; Huang, Zhenxing; Ruan, Wenquan; Miao, Hengfeng; Shi, Wansheng; Zhao, Mingxing
      Applied Microbiology and Biotechnology (2018), 102 (8), 3819-3830CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Using lignocellulosic materials as substrates, ruminal microbiota were co-inoculated with anaerobic sludge at different loading rates (LR) to study the microbial community in the semi-continuous mode. The results indicated that the highest CH4 yield reached 0.22 L/g volatile solid at LR of 4 g/L/day, which obtained 56-58% of the theor. value. In the steady stage with LR of 2-4 g/L/day and slurry recirculation, copies of total archaea increased. Esp. the Methanobacteriales increased significantly (p < 0.05) to 3.30 × 108 copies/mL. The microbial communities were examd. by MiSeq 16S rRNA sequencing. Enriched hydrolytic bacteria mainly belonged to Clostridiales, including Ruminococcus, Ruminiclostridium, and Ruminofilibacter settled in the rumen. High-active cellulase and xylanase were excreted in the co-inoculated system. Acid-producing bacteria by fermn. were affiliated with Lachnospiraceae and Bacteroidales. The acidogen members were mainly Spirochaetaceae and Clostridiales. Syntrophic oxidn. bacteria mainly consisted of Synergistetes, propionate oxidizers (Syntrophobacter and Pelotomaculum), and butyrate oxidizers (Syntrophus and Syntrophomonas). There had no volatile fatty acid (VFA) accumulation and the pH values varied between 6.94 and 7.35. At LR of 6 g/L/day and a recirculation ratio of 1:1, the hardly degradable components and total VFA concns. obviously increased. The total archaea and Methanobacteriales then deceased significantly to 8.56 × 105 copies/mL and 4.14 × 103 copies/mL resp. (p < 0.05), which resulted in the inhibition of methanogenic activities. Subsequently, microbial diversity dropped, and the hydrolytic bacteria and syntrophic oxidizers obviously decreased. In contrast, the abundances of Bacteroidales increased significantly (p < 0.05). Acetate, propionate, and butyrate concns. reached 2.02, 6.54, and 0.53 g/L, resp., which indicated "acidification" in the anaerobic reactor. Our study illustrated that co-inoculated anaerobic sludge enriched the ruminal function consortia and hydrogenotrophic methanogens played an important role in anaerobic digestion of lignocelluloses.
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    25. 25
      Quintero, M.; Castro, L.; Ortiz, C.; Guzmán, C.; Escalante, H. Enhancement of Starting up Anaerobic Digestion of Lignocellulosic Substrate: Fique’s Bagasse as an Example. Bioresour. Technol. 2012, 108, 8– 13,  DOI: 10.1016/j.biortech.2011.12.052
      25
      Enhancement of starting up anaerobic digestion of lignocellulosic substrate: fique's bagasse as an example
      Quintero, Mabel; Castro, Liliana; Ortiz, Claudia; Guzman, Carolina; Escalante, Humberto
      Bioresource Technology (2012), 108 (), 8-13CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      In Colombia there are 20,000 ha of fique fields (Furcraea sp., family Agavaceae), that produce around 93,400 tons of fique's bagasse per yr. These residuals are disposed into rivers and soil causing pollution. According to physicochem. characteristics, the lignocellulosic residues from fique crops (fique's bagasse) are appropriate carbon source to biogas prodn. Anaerobic digestion from fique's Bagasse (FB) requires a specialized microbial consortium capable of degrading its high lignocellulosic concn. In this study, the capacities of seven microbial consortia for biomethane potential (BMP) from FB were evaluated. Inoculum of ruminal liq. achieved high hydrolytic activity (0.068 g COD/g VSS day), whereas pig waste sludge inoculum showed high methanogenic activity (0.146 g COD/g VSS day). Mixts. of these two inoculums (RL + PWS) showed the best yields for biomethane potential (0.3 m3 CH4/Kg VS ad).
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    26. 26
      Song, H.; Clarke, W. P.; Blackall, L. L. Concurrent Microscopic Observations and Activity Measurements of Cellulose Hydrolyzing and Methanogenic Populations during the Batch Anaerobic Digestion of Crystalline Cellulose. Biotechnol. Bioeng. 2005, 91 (3), 369– 378,  DOI: 10.1002/bit.20517
      26
      Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose
      Song, Hyohak; Clarke, William P.; Blackall, Linda L.
      Biotechnology and Bioengineering (2005), 91 (3), 369-378CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
      This study compares process data with microscopic observations from an anaerobic digestion of org. particles. This article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcryst. cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amt. of cellulose remaining in the reactor, reached a const. value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A const. value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and 1st-order kinetics only apply after this point. The 1st-order hydrolysis rate const., once surfaces were satd. with biofilm, was twice higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegrdn. process exist entirely within the biofilm. For the reactor conditions used in these expts., the predominant methanogens exist in ball-shaped colonies within the biofilm.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXntlCnsL8%253D&md5=15bb9c6c8e7813b97a9890e57aa829d8
    27. 27
      Li, K.; Zhu, H.; Zhang, Y.; Zhang, H. Characterization of the Microbial Communities in Rumen Fluid Inoculated Reactors for the Biogas Digestion of Wheat Straw. Sustainability 2017, 9 (2), 243,  DOI: 10.3390/su9020243
      27
      Characterization of the microbial communities in rumen fluid inoculated reactors for the biogas digestion of wheat straw
      Li, Ke; Zhu, Haoran; Zhang, Yajie; Zhang, Hongxun
      Sustainability (2017), 9 (2), 243/1-243/14CODEN: SUSTDE; ISSN:2071-1050. (MDPI AG)
      The present study investigated the effect of rumen fluid (RF) concn. on the methane prodn. through anaerobic digestion of wheat straw in batch mode, and compared the microbial communities in RF and RF inoculated reactors by 16S rRNA genes sequencing. Six levels of RF concn. including 1%, 5%, 10%, 15%, 20% and 25% (vol./vol.) were used in reactors R1, R5, R10, R15, R20 and R25 resp. The results revealed that lower than or equal to 5% RF concns. resulted in reactor acidification and low methane prodn. The highest methane yield of 106 mL·CH4·g·VS-1 was achieved in R10, whereas higher RF concns. than 10% could not improve the methane prodn. significantly. Methanosarcina barkeri was abundant in the well-working reactors, and Methanobacterium was dominant in the poor-working reactors, implying the archaeal communities in reactors had changed greatly from the Methanobrevibacter-dominated RF. Although the relative abundance of Clostridium and Ruminococcus were greatly different between RF and reactors, the Bacteroidetes and Firmicutes communities were dominant in all the tested samples. The results indicated that the in vitro anaerobic conditions had altered the rumen methanogenic communities significantly and the facultative acetoclastic Methanosarcina was important for the methane prodn. in the RF seeded reactors.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFemtbjP&md5=6fb9930eb46809dc40401055c4eec0e7
    28. 28
      Gijzen, H. J.; Zwart, K. B.; van Gelder, P. T.; Vogels, G. D. Continuous Cultivation of Rumen Microorganisms, a System with Possible Application to the Anaerobic Degradation of Lignocellulosic Waste Materials. Appl. Microbiol. Biotechnol. 1986, 25, 155– 162,  DOI: 10.1007/BF00938940
      28
      Continuous cultivation of rumen microorganisms, a system with possible application to the anaerobic degradation of lignocellulosic waste materials
      Gijzen, Huub J.; Zwart, Kor B.; Van Gelder, Pieter T.; Vogels, Godfried D.
      Applied Microbiology and Biotechnology (1986), 25 (2), 155-62CODEN: AMBIDG; ISSN:0175-7598.
      An in-vitro continuous fermn. device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liqs. found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixt. was used as a solid growth substrate. During a test period of 65 days, the artificial rumen fermenter showed stable operation with respect to ciliate nos., fiber degrdn., and volatile fatty acid prodn. The values obtained were comparable to those found in vivo. Optimal fiber degrdn. and volatile fatty acid prodn. were maintained when the hydraulic retention times (HRT) were 11-14 h. At these HRT values, ciliate nos. were maintained at ∼8.5 × 104 cells/mL. The ciliate nos. declined drastically at HRT values of >14 h. A fermenter inoculated with a small vol. of rumen fluid (1:100, vol./vol.) reached normal protozoal nos., fiber degrdn., and volatile fatty acid prodn. after a start-up period of only 8-10 days. The possible application of rumen microorganisms for efficient degrdn. of lignocellulosic waste material in an artificial rumen digester is discussed.
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    29. 29
      Nagler, M.; Kozjek, K.; Etemadi, M.; Insam, H.; Podmirseg, S. M. Simple yet Effective: Microbial and Biotechnological Benefits of Rumen Liquid Addition to Lignocellulose-Degrading Biogas Plants. J. Biotechnol. 2019, 300, 1– 10,  DOI: 10.1016/j.jbiotec.2019.05.004
      29
      Simple yet effective: Microbial and biotechnological benefits of rumen liquid addition to lignocellulose-degrading biogas plants
      Nagler, Magdalena; Kozjek, Katja; Etemadi, Mohammad; Insam, Heribert; Podmirseg, Sabine Marie
      Journal of Biotechnology (2019), 300 (), 1-10CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
      In biogas plants, lignocellulose-rich biomass (LCB) is particularly slowly degraded, causing high hydraulic retention times. This fact lowers the interests for such substrates. To enhance LCB-degrdn., cattle rumen fluid, a highly active microbial resource accruing in the growing meat industry, might be used as a potential source for bioaugmentation. This study compares 0%, 20% and 40% rumen liq. in a batch anaerobic digestion approach. Moreover, it dets. the biogas- and methane-potentials as well as degrdn.-speeds of corn straw, co-digested with cattle manure. It inspects microbial communities via marker-gene sequencing, qPCR and RNA-DGGE and draws attention on possible beneficial effects of rumen addn. on the biogas-producing community. Bioaugmentation with 20% and 40% vol./vol. rumen liq. accelerated methane yields by 5 and 6 days, resp. (i.e. reaching 90% of total methane prodn.). It also enhanced LCB- as well as (hemi)cellulose- and volatile fatty acid degrdn. These results are supported by increased abundances of bacteria, methanogens and anaerobic fungi in treatments with rumen liq. amendment, and point towards the persistence of specific rumen-borne microorganisms esp. during the first phase of the expt. The results suggest that rumen liq. addn. is a promising strategy for enhanced and accelerated exploitation of LCB for biomethanization.
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    30. 30
      Zamorano-López, N.; Borrás, L.; Giménez, J. B.; Seco, A.; Aguado, D. Acclimatised Rumen Culture for Raw Microalgae Conversion into Biogas: Linking Microbial Community Structure and Operational Parameters in Anaerobic Membrane Bioreactors (AnMBR). Bioresour. Technol. 2019, 290 (July), 121787,  DOI: 10.1016/j.biortech.2019.121787
      30
      Acclimatised rumen culture for raw microalgae conversion into biogas: Linking microbial community structure and operational parameters in anaerobic membrane bioreactors (AnMBR)
      Zamorano-Lopez, Nuria; Borras, Luis; Gimenez, Juan B.; Seco, Aurora; Aguado, Daniel
      Bioresource Technology (2019), 290 (), 121787CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      Ruminal fluid was inoculated in an Anaerobic Membrane Reactor (AnMBR) to produce biogas from raw Scenedesmus. This work explores the microbial ecol. of the system during stable operation at different solids retention times (SRT). The 16S rRNA amplicon anal. revealed that the acclimatised community was mainly composed of Anaerolineaceae, Spirochaetaceae, Lentimicrobiaceae and Cloacimonetes fermentative and hydrolytic members. During the highest biodegradability achieved in the AnMBR (62%) the dominant microorganisms were Fervidobacterium and Methanosaeta. Different microbial community clusters were obsd. at different SRT conditions. Interestingly, syntrophic bacteria Gelria and Smithella were enhanced after increasing 2-fold the org. loading rate, suggesting their importance in continuous systems producing biogas from raw microalgae.
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    31. 31
      Martí-Herrero, J.; Soria-Castellón, G.; Diaz-de-Basurto, A.; Alvarez, R.; Chemisana, D. Biogas from a Full Scale Digester Operated in Psychrophilic Conditions and Fed Only with Fruit and Vegetable Waste. Renewable Energy 2019, 133, 676– 684,  DOI: 10.1016/j.renene.2018.10.030
      31
      Biogas from a full scale digester operated in psychrophilic conditions and fed only with fruit and vegetable waste
      Marti-Herrero, J.; Soria-Castellon, G.; Diaz-de-Basurto, A.; Alvarez, R.; Chemisana, D.
      Renewable Energy (2019), 133 (), 676-684CODEN: RNENE3; ISSN:0960-1481. (Elsevier Ltd.)
      The aim of this work was to evaluate the feasibility of treating fruit and vegetable waste from a municipal market in a full-scale anaerobic digester with min. implementation and operational costs, i.e. without pre-treatment, clean water consumption, active heating or mixing. For this purpose, a 13.9 m3 digester that forces the submersion of solids, gains heat through solar radiation and recirculates effluent was monitored during one year of real operational and weather conditions in Bolivia. The digester was initially loaded with cow rumen (inoculum source), and after eight weeks with only fruit and vegetable waste. The digester operated at an org. loading rate (OLR) of 1 kgVS/(m3d). The normalized specific biogas prodn. averaged 0.26 m3/kgVS and increased to 0.34 m3/kgVS when OLR was halved to 0.57 kgVS/(m3d), while the passive solar design allowed an av. digester temp. of 21°C (4°C above the av. ambient temp.).
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    32. 32
      Ferraro, A.; Massini, G.; Mazzurco Miritana, V.; Rosa, S.; Signorini, A.; Fabbricino, M. A Novel Enrichment Approach for Anaerobic Digestion of Lignocellulosic Biomass: Process Performance Enhancement through an Inoculum Habitat Selection. Bioresour. Technol. 2020, 313 (June), 123703,  DOI: 10.1016/j.biortech.2020.123703
      32
      A novel enrichment approach for anaerobic digestion of lignocellulosic biomass: Process performance enhancement through an inoculum habitat selection
      Ferraro, Alberto; Massini, Giulia; Mazzurco Miritana, Valentina; Rosa, Silvia; Signorini, Antonella; Fabbricino, Massimiliano
      Bioresource Technology (2020), 313 (), 123703CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      Inocula enrichment was performed using an innovative habitat-based selection approach to improve wheat straw (WS) anaerobic digestion (AD) efficiency. The procedure was carried out by sequentially re-inoculating the primary microbial community seven times in subsequent anaerobic reactors contg. untreated WS. Re-inocula were performed at different re-inoculum times (24, 48, and 96 h) by moving a porous support mimicking a rumen structure from one batch to the next (S-tests) or re-inoculating only the culture medium (C-tests). Highest H2 prodn. yields were obsd. after four and five re-inocula (0.08 ± 0.02 NmL h-1 gVS-1 and 0.09 ± 0.02 NmL h-1 gVS-1) for S-24 and S-48, resp. For S-96, higher CH4 yields were obsd. after the start-up test and sixth re-inoculum (0.05 ± 0.003 NmL h-1 gVS-1 and 0.04 ± 0.005 NmL h-1 gVS-1, resp.). Accordingly, S-96 showed the highest active Archaea component (7%). C-test microbial communities were dominated by fermenting, hydrogen-producing bacteria and showed lower microbial community diversity than S-tests.
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    33. 33
      Abbas, Y.; Jamil, F.; Rafiq, S.; Ghauri, M.; Khurram, M. S.; Aslam, M.; Bokhari, A.; Faisal, A.; Rashid, U.; Yun, S. Valorization of Solid Waste Biomass by Inoculation for the Enhanced Yield of Biogas. Clean Technol. Environ. Policy 2020, 22 (2), 513– 522,  DOI: 10.1007/s10098-019-01799-6
      33
      Valorization of solid waste biomass by inoculation for the enhanced yield of biogas
      Abbas, Yasir; Jamil, Farrukh; Rafiq, Sikander; Ghauri, Moinuddin; Khurram, M. Shahzad; Aslam, Muhammad; Bokhari, Awais; Faisal, Abrar; Rashid, Umer; Yun, Sining; Mubeen, Muhammad
      Clean Technologies and Environmental Policy (2020), 22 (2), 513-522CODEN: CTEPAI; ISSN:1618-954X. (Springer)
      The current work reports exptl. outcomes about anaerobic digestion (AD) of the org. fraction of catering solid waste to produce biogas and methane yield and solidity of the entire system. The possibility of developing AD as an efficient technique for food waste management has also been considered. Inoculation is an established technique to enhance biogas yield through digested sludge, rumen fluid, and slurry recirculation; although this method is proven on cattle manure, however, it is not well studied on food waste. The current study investigates the use of animal intestinal waste as inoculums to enhance biogas yield of food waste. The findings are benchmarked with cattle manure employing known digester materials of carbon steel, aluminum alloy, and at anaerobic mesophilic conditions. The inoculation was varied up to 30% by mass of the substrate materials in each case. The research finds that food waste inoculation was up to 30% using carbon steel reactor under mesophilic temp. conditions (30 ± 1°). The food waste produced biogas at a faster rate with a higher yield compared to cattle manure in all digester materials. The biogas yield kept increasing with increasing inoculum ratio reaching optimal at 30% in the case of both food waste and cattle manure with all the digester materials.
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    34. 34
      Anaerobic Digestion Research and Education Center (ADREC). South Campus Anaerobic Digester. Michigan State University. https://www.egr.msu.edu/bae/adrec/ (accessed 2017-05-12).
      There is no corresponding record for this reference.
    35. 35
      Tritt, W. P.; Schuchardt, F. Materials Flow and Possibilities of Treating Liquid and Solid Wastes from Slaughterhouses in Germany. A Review. Bioresour. Technol. 1992, 41 (3), 235– 245,  DOI: 10.1016/0960-8524(92)90008-L
      35
      Materials flow and possibilities of treating liquid and solid wastes from slaughterhouses in Germany. A review.
      Tritt, W. P.; Schuchardt, F.
      Bioresource Technology (1992), 41 (3), 235-45CODEN: BIRTEB; ISSN:0960-8524.
      A review, with 53 refs., on treatment of slaughterhouse wastewater and solid wastes in Germany.
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    36. 36
      McDonald, P.; Edwards, R. A.; Greenhalgh, J. F. D.; Morgan, C. A.; Sinclair, L. A.; Wilkinson, R. G. Animal Nutrition, 7th ed.; Benjamin-Cummings Publishing Company: San Francisco, CA, 2011.
      There is no corresponding record for this reference.
    37. 37
      Mata-Alvarez, J.; Dosta, J.; Romero-Güiza, M. S.; Fonoll, X.; Peces, M.; Astals, S. A Critical Review on Anaerobic Co-Digestion Achievements between 2010 and 2013. Renewable Sustainable Energy Rev. 2014, 36, 412– 427,  DOI: 10.1016/j.rser.2014.04.039
      37
      A critical review on anaerobic co-digestion achievements between 2010 and 2013
      Mata-Alvarez, J.; Dosta, J.; Romero-Guiza, M. S.; Fonoll, X.; Peces, M.; Astals, S.
      Renewable & Sustainable Energy Reviews (2014), 36 (), 412-427CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)
      Anaerobic digestion is a com. reality for several kinds of waste. Nonetheless, anaerobic digestion of single substrates presents some drawbacks linked to substrate characteristics. Anaerobic co-digestion, the simultaneous digestion of two or more substrates, is a feasible option to overcome the drawbacks of mono-digestion and to improve plant's economic feasibility. At present, since 50% of the publication has been published in the last two years, anaerobic co-digestion can be considered the most relevant topic within anaerobic digestion research. The aim of this paper is to present a review of the achievements and perspectives of anaerobic co-digestion within the period 2010-2013, which represents a continuation of the previous review made by the authors [3]. In the present review, the publications have been classified as for the main substrate, i.e., animal manures, sewage sludge and biowaste. Animal manures stand as the most reported substrate, agro-industrial waste and the org. fraction of the municipal solid waste being the most reported co-substrate. Special emphasis has been made to the effect of the co-digestion over digestate quality, since land application seems to be the best option for digestate recycling. Traditionally, anaerobic co-digestion between sewage sludge and the org. fraction of the municipal solid waste has been the most reported co-digestion mixt. However, between 2010 and 2013 the publications dealing with fats, oils and greases and algae as sludge co-substrate have increased. This is because both co-substrates can be obtained at the same wastewater treatment plant. In contrast, biowaste as a main substrate has not been as studied as manures or sewage sludge. Finally, three interdisciplinary sections have been written for addressing novelty aspects in anaerobic co-digestion, i.e., pre-treatments, microbial dynamics and modeling. However, much effort needs to be done in these later aspects to better understand and predict anaerobic co-digestion.
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    38. 38
      Prates, A.; de Oliveira, J. A.; Abecia, L.; Fondevila, M. Effects of Preservation Procedures of Rumen Inoculum on in Vitro Microbial Diversity and Fermentation. Anim. Feed Sci. Technol. 2010, 155 (2–4), 186– 193,  DOI: 10.1016/j.anifeedsci.2009.12.005
      38
      Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation
      Prates, A.; de Oliveira, J. A.; Abecia, L.; Fondevila, M.
      Animal Feed Science and Technology (2010), 155 (2-4), 186-193CODEN: AFSTDH; ISSN:0377-8401. (Elsevier B.V.)
      Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermn. pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at -20 °C (FRZ), frozen in liq. N (FLN) or added with 0.04 glycerol and frozen in liq. N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 mL per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilization of added glycerol after thawing in FGL increased total gas prodn. (P<0.05) and 24 h volatile fatty acid (VFA) prodn. (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas prodn. (as ml/g dry matter per h), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermn. at 8 and 12 h, resp. Differences in gas prodn. did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690-0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas prodn. studies does not affect microbial fermn. pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to vol. ratio. Freezing in liq. N is more appropriate than at -20 °C.
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    39. 39
      Nguyen, D.; Wu, Z.; Shrestha, S.; Lee, P.-H.; Raskin, L.; Khanal, S. K. Intermittent Micro-Aeration: New Strategy to Control Volatile Fatty Acid Accumulation in High Organic Loading Anaerobic Digestion. Water Res. 2019, 166, 115080,  DOI: 10.1016/j.watres.2019.115080
      39
      Intermittent micro-aeration: New strategy to control volatile fatty acid accumulation in high organic loading anaerobic digestion
      Nguyen, Duc; Wu, Zhuoying; Shrestha, Shilva; Lee, Po-Heng; Raskin, Lutgarde; Khanal, Samir Kumar
      Water Research (2019), 166 (), 115080CODEN: WATRAG; ISSN:0043-1354. (Elsevier Ltd.)
      This study developed an intermittent oxidn.-redn. potential (ORP)-controlled micro-aeration system for high solids anaerobic digestion (AD) of lignocellulosic biomass without volatile fatty acids (VFA) accumulation at high org. loading rate (OLR). Traditional AD of Napier grass, a model lignocellulosic biomass, at an OLR of 5 g volatile solids (VS)/L/day resulted in an accumulation of total VFA concn. up to 9.2 g/L as acetic acid (HAc) equiv., causing rapid drops in pH and methane yield, and driving the digester to the verge of failure. Once intermittent (every 24 h) ORP-controlled micro-aeration (at ORP of -470 mV) was initiated, the total VFA concn. rapidly decreased to 3.0 g HAc/L and the methane yield improved, resulting in stable digester performance without the need for alky. supplementation or OLR redn. By combining reactor performance results, mass balance analyses, microbial community characterization data, and a bioenergetic evaluation, this study suggested that rapid VFA conversion and CH4 prodn. were carried out by facultative anaerobes and hydrogenotrophic methanogens under micro-aerobic conditions. This novel operating approach can be applied as an effective control strategy for high OLR AD processes esp. in the event of VFA accumulation.
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    40. 40
      Sanaei-Moghadam, A.; Abbaspour-Fard, M. H.; Aghel, H.; Aghkhani, M. H.; Abedini-Torghabeh, J. Enhancement of Biogas Production by Co-Digestion of Potato Pulp with Cow Manure in a CSTR System. Appl. Biochem. Biotechnol. 2014, 173, 1858,  DOI: 10.1007/s12010-014-0972-5
      40
      Enhancement of Biogas Production by Co-digestion of Potato Pulp with Cow Manure in a CSTR System
      Sanaei-Moghadam, Akbar; Abbaspour-Fard, Mohammad Hossein; Aghel, Hasan; Aghkhani, Mohammad Hossein; Abedini-Torghabeh, Javad
      Applied Biochemistry and Biotechnology (2014), 173 (7), 1858-1869CODEN: ABIBDL; ISSN:0273-2289. (Springer)
      Anaerobic digestion (AD) process is a well-established method to generate energy from the org. wastes both from the environmental and economical perspectives. The purpose of present study is to evaluate energy prodn. from potato wastes by incorporating cow manure into the process. Firstly, a lab. pilot of one-stage biogas prodn. was designed and built according to continuously stirred tank reactor (CSTR) system. The setup was able to automatically control the environmental conditions of the process including temp., duration, and rate of stirring. AD expt. was exclusively performed on co-digestion of potato peel (PP) and cow manure (CM) in three levels of mixing ratio including 20:80, 50:50, 80:20 (PP:CM), and 0:100 as control treatment based on the volatile solid (VS) wt. without adding initial inoculums. After hydraulic retention time (HRT) of 50 days on av. 193, 256, 348, and 149 norm liter (LN) (kg VS)-1, methane was produced for different mixing ratios, resp. Statistical anal. shows that these gas productions are significantly different. The av. energy was detd. based on the produced methane which was about 2.8 kWh (kg VS)-1, implying a significant energy prodn. potential. The av. COD (COD) removal of treatments was about 61 %, showing that it can be leached significantly with high org. matter by the employed pilot. The energy efficiency of 92 % of the process also showed the optimum control of the process by the pilot.
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    41. 41
      Estevez, M. M.; Sapci, Z.; Linjordet, R.; Morken, J. Incorporation of Fish By-Product into the Semi-Continuous Anaerobic Co-Digestion of Pre-Treated Lignocellulose and Cow Manure, with Recovery of Digestate’s Nutrients. Renewable Energy 2014, 66, 550– 558,  DOI: 10.1016/j.renene.2014.01.001
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      Incorporation of fish by-product into the semi-continuous anaerobic co-digestion of pre-treated lignocellulose and cow manure, with recovery of digestate's nutrients
      Estevez, Maria M.; Sapci, Zehra; Linjordet, Roar; Morken, John
      Renewable Energy (2014), 66 (), 550-558CODEN: RNENE3; ISSN:0960-1481. (Elsevier Ltd.)
      Norway's fish processing industry generates large amts. of fish waste every year. The high-risk waste fraction with most of its oil removed has not yet been tested for energy prodn. The stability of an anaerobic digestion process that incorporates this material with steam exploded Salix and cow manure was tested using mesophilic, semi-continuous lab.-scale digesters. The effects of recycling the liq. digestate fraction were also investigated. The removal of ammonium (NH4+) and phosphate (PO43-) from the rejected digestate using struvite pptn. and bentonite adsorption were tested to generate a nutrient-enriched, final solid fertiliser. Adding 20% fish byproduct (volatile solids basis) increased methane yields by 35%, while recycling the digestate caused a slight increase. The NH4+-N levels reached 4-5 g l-1 in the reactors with recirculation and fish feed. Although these levels may threaten methanogenesis, the stability of the process was maintained during the entire period due to the good balance between the lignocellulose, proteins and fats provided by the co-digestion mixt. and the pos. effects of recirculation. The NH4+ and PO43- were successfully removed from the rejected liq. digestate. The redns. using struvite reached 87% and 60% (pH 9.5 and Mg2+:NH4+:PO43- ratio of 1.2:1:1), while bentonite achieved 82% and 52%, resp.
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    42. 42
      Rekha, B. N.; Pandit, A. B. Performance Enhancement of Batch Anaerobic Digestion of Napier Grass by Alkali Pre-Treatment. Int. J. ChemTech Res. 2013, 5 (2), 558– 564
      42
      Performance enhancement of batch anaerobic digestion of napier grass by alkali pre-treatment
      Rekha, B. N.; Pandit, Aniruddha B.
      International Journal of ChemTech Research (2013), 5 (2), 558-564CODEN: IJCRGG; ISSN:0974-4290. (Sphinx Knowledge House)
      The objective of this research was to develop an alkali pre-treatment process prior to anaerobic digestion (AD) of napier grass for improving solubilization of the lignocellulosic biomass and subsequent enhancement in biogas productivity. Mild concns. of NaOH soln. were used to pretreat napier grass which and was later subjected to anaerobic digestion for prodn. of biogas. Lab.-scale batch expts. were carried out in 0.5 L bottles with 0.3 L working vol. Optimal concn. of NaOH soln. for orgs. solubilization in the step of pre-treatment was 0.6% (w/v) i.e. 11.2g of NaOH/100g TS of napier grass. Under this condition, the sol. COD of the hydrolyzate was increased by 93%, which subsequently increased the prodn. of volatile fatty acids (VFA) during anaerobic digestion. The biogas prodn. of napier grass with and without pre-treatment was evaluated. The highest methane yield under optimal pre-treatment condition was found to be 0.158 m3 CH4/kg TS, as compared to 0.047 m3 CH4/kg TS for untreated napier grass when subjected to anaerobic digestion for a period of 8 days. These results indicated that alkali pre-treatment could be an effective method for increasing biodegradability and improving methane yield of napier grass.
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    43. 43
      Frigon, J. C.; Roy, C.; Guiot, S. R. Anaerobic Co-Digestion of Dairy Manure with Mulched Switchgrass for Improvement of the Methane Yield. Bioprocess Biosyst. Eng. 2012, 35 (3), 341– 349,  DOI: 10.1007/s00449-011-0572-5
      43
      Anaerobic co-digestion of dairy manure with mulched switchgrass for improvement of the methane yield
      Frigon, Jean-Claude; Roy, Caroline; Guiot, Serge R.
      Bioprocess and Biosystems Engineering (2012), 35 (3), 341-349CODEN: BBEIBV; ISSN:1615-7591. (Springer)
      The owners of farm-scale anaerobic digesters are relying on off-farm wastes or energy crops as a co-digestion feedstock with animal manure to increase their prodn. of methane and thus revenues. Switchgrass represents an interesting feedstock for Canadian digesters owners as it is a high-yielding low-maintenance perennial crop, well adapted to northern climate. Methane potential assays in batch tests showed methane prodn. of 19.4 ± 3.6, 28.3 ± 1.7, 37.3 ± 7.1 and 45.7 ± 0.8 L kg-1, for raw manure, blended manure, manure and mulched switchgrass, manure and pretreated switchgrass, resp. Two 6-L lab-scale anaerobic digesters were operated for 130 days to assess the benefit of co-digesting switchgrass with bovine manure (digester #2), at a 20% wet mass fraction, compared with a manure-only operation (digester #1) The digesters were operated at an hydraulic retention time of 37 ± 6 days and at loads of 2.4 ± 0.6 and 2.6 ± 0.6 kg total volatile solids (TVS) L-1 day-1 for digesters #1 (D1) and #2 (D2), resp. The TVS degrdn. reached 25 and 39%, which resulted in a methane prodn. of 1.18 ± 0.18 and 2.19 ± 0.31 L day-1 for D1 and D2, resp. The addn. of 20% on a wet mass ratio of switchgrass to a manure digester increased its methane prodn. by 86%. The co-digestion of switchgrass in a 500 m3 manure digester could yield up to 10.2 GJ day-1 of purified methane or 1.1 MWh day-1 of electricity.
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    44. 44
      Chen, Y.; Cheng, J. J.; Creamer, K. S. Inhibition of Anaerobic Digestion Process: A Review. Bioresour. Technol. 2008, 99 (10), 4044– 4064,  DOI: 10.1016/j.biortech.2007.01.057
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      Inhibition of anaerobic digestion process: A review
      Chen, Ye; Cheng, Jay J.; Creamer, Kurt S.
      Bioresource Technology (2008), 99 (10), 4044-4064CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      A review is given. Anaerobic digestion is an attractive waste treatment practice in which both pollution control and energy recovery can be achieved. Many agricultural and industrial wastes are ideal candidates for anaerobic digestion because they contain high levels of easily biodegradable materials. Problems such as low methane yield and process instability are often encountered in anaerobic digestion, preventing this technique from being widely applied. A wide variety of inhibitory substances are the primary cause of anaerobic digester upset or failure since they are present in substantial concns. in wastes. Considerable research efforts have been made to identify the mechanism and the controlling factors of inhibition. This review provides a detailed summary of the research conducted on the inhibition of anaerobic processes. The inhibitors commonly present in anaerobic digesters include ammonia, sulfide, light metal ions, heavy metals, and orgs. Due to the difference in anaerobic inocula, waste compn., and exptl. methods and conditions, literature results on inhibition caused by specific toxicants vary widely. Co-digestion with other waste, adaptation of microorganisms to inhibitory substances, and incorporation of methods to remove or counteract toxicants before anaerobic digestion can significantly improve the waste treatment efficiency.
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    45. 45
      Caporaso, J. G.; Lauber, C. L.; Walters, W. A.; Berg-Lyons, D.; Lozupone, C. A.; Turnbaugh, P. J.; Fierer, N.; Knight, R. Global Patterns of 16S RRNA Diversity at a Depth of Millions of Sequences per Sample. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 4516– 4522,  DOI: 10.1073/pnas.1000080107
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      Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample
      Caporaso, J. Gregory; Lauber, Christian L.; Walters, William A.; Berg-Lyons, Donna; Lozupone, Catherine A.; Turnbaugh, Peter J.; Fierer, Noah; Knight, Rob
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (Suppl. 1), 4516-4522, S4516/1-S4516/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The ongoing revolution in high-throughput sequencing continues to democratize the ability of small groups of investigators to map the microbial component of the biosphere. In particular, the coevolution of new sequencing platforms and new software tools allows data acquisition-and anal. on an unprecedented scale. Here we report the next stage in this coevolutionary arms race, using the Illumina GAIIx platform to sequence a diverse array of 25 environmental samples and three known "mock communities" at a depth averaging 3.1 million reads per sample. We demonstrate excellent consistency in taxonomic recovery and recapture diversity patterns that were previously reported on the basis of meta-anal. of many studies from the literature (notably, the saline/nonsaline split in environmental samples and the split between host-assocd. and free-living communities). We also demonstrate that 2,000 Illumina single-end reads are sufficient to recapture the same relationships among samples that we observe with the full dataset. The results thus open up the possibility of conducting large-scale studies analyzing thousands of samples simultaneously to survey microbial communities at an unprecedented spatial and temporal resoln.
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    46. 46
      Kozich, J. J.; Westcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Appl. Environ. Microbiol. 2013, 79 (17), 5112– 5120,  DOI: 10.1128/AEM.01043-13
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      Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq illumina sequencing platform
      Kozich, James J.; Westcott, Sarah L.; Baxter, Nielson T.; Highlander, Sarah K.; Schloss, Patrick D.
      Applied and Environmental Microbiology (2013), 79 (17), 5112-5120CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
      Rapid advances in sequencing technol. have changed the exptl. landscape of microbial ecol. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is crit. to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concn. of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large nos. of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
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    47. 47
      Oksanen, J. Multivariate Analysis of Ecological Communities in R: Vegan Tutorial. http://phylodiversity.net/azanne/csfar/images/8/85/Vegan.pdf (accessed 2018-02-07).
      There is no corresponding record for this reference.
    48. 48
      ter Braak, C. J. F.; Verdonschot, P. F. M. Canonical Correspondence Analysis and Related Multivariate Methods in Aquatic Ecology. Aquat. Sci. 1995, 57 (3), 255– 289,  DOI: 10.1007/BF00877430
      There is no corresponding record for this reference.
    49. 49
      Werner, J. J.; Garcia, M. L.; Perkins, S. D.; Yarasheski, K. E.; Smith, S. R.; Muegge, B. D.; Stadermann, F. J.; Derito, C. M.; Floss, C.; Madsen, E. L. Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation. Appl. Environ. Microbiol. 2014, 80 (11), 3375– 3383,  DOI: 10.1128/AEM.00166-14
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      Microbial community dynamics and stability during an ammonia-induced shift to syntrophic acetate oxidation
      Werner, Jeffrey J.; Garcia, Marcelo L.; Perkins, Sarah D.; Yarasheski, Kevin E.; Smith, Samuel R.; Muegge, Brian D.; Stadermann, Frank J.; DeRito, Christopher M.; Floss, Christine; Madsen, Eugene L.; Gordon, Jeffrey I.; Angenent, Largus T.
      Applied and Environmental Microbiology (2014), 80 (11), 3375-3383, 9 pp.CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
      Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, the authors experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidn. In comparison with control digesters, the authors obsd. shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the digesters' 3-yr operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When the authors perturbed the digesters by increasing the ammonia concn. or temp., the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidn. had allowed the exptl. bioreactors to regain stable operation. The emergence of syntrophic acetate oxidn. coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. The authors' 16S rRNA gene anal. also revealed that acetate-fed enrichment expts. resulted in communities that did not represent the bioreactor community. Anal. of shotgun sequencing of community DNA suggests that syntrophic acetate oxidn. was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.
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    50. 50
      Vanwonterghem, I.; Jensen, P. D.; Dennis, P. G.; Hugenholtz, P.; Rabaey, K.; Tyson, G. W. Deterministic Processes Guide Long-Term Synchronised Population Dynamics in Replicate Anaerobic Digesters. ISME J. 2014, 8 (10), 2015– 2028,  DOI: 10.1038/ismej.2014.50
      50
      Deterministic processes guide long-term synchronised population dynamics in replicate anaerobic digesters
      Vanwonterghem, Inka; Jensen, Paul D.; Dennis, Paul G.; Hugenholtz, Philip; Rabaey, Korneel; Tyson, Gene W.
      ISME Journal (2014), 8 (10), 2015-2028CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)
      A replicate long-term expt. was conducted using anaerobic digestion (AD) as a model process to det. the relative role of niche and neutral theory on microbial community assembly, and to link community dynamics to system performance. AD is performed by a complex network of microorganisms and process stability relies entirely on the synergistic interactions between populations belonging to different functional guilds. In this study, three independent replicate anaerobic digesters were seeded with the same diverse inoculum, supplied with a model substrate, α-cellulose, and operated for 362 days at a 10-day hydraulic residence time under mesophilic conditions. Selective pressure imposed by the operational conditions and model substrate caused large reproducible changes in community compn. including an overall decrease in richness in the first month of operation, followed by synchronised population dynamics that correlated with changes in reactor performance. This included the synchronised emergence and decline of distinct Ruminococcus phylotypes at day 148, and emergence of a Clostridium and Methanosaeta phylotype at day 178, when performance became stable in all reactors. These data suggest that many dynamic functional niches are predictably filled by phylogenetically coherent populations over long time scales. Neutral theory would predict that a complex community with a high degree of recognized functional redundancy would lead to stochastic changes in populations and community divergence over time. We conclude that deterministic processes may play a larger role in microbial community dynamics than currently appreciated, and under controlled conditions it may be possible to reliably predict community structural and functional changes over time.
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    51. 51
      Sträuber, H.; Schröder, M.; Kleinsteuber, S. Metabolic and Microbial Community Dynamics during the Hydrolytic and Acidogenic Fermentation in a Leach-Bed Process. Energy. Sustain. Soc. 2012, 2 (1), 13,  DOI: 10.1186/2192-0567-2-13
      There is no corresponding record for this reference.
    52. 52
      Meng, Y.; Mumme, J.; Xu, H.; Wang, K. A Biologically Inspired Variable-PH Strategy for Enhancing Short-Chain Fatty Acids (SCFAs) Accumulation in Maize Straw Fermentation. Bioresour. Technol. 2016, 201, 329– 336,  DOI: 10.1016/j.biortech.2015.11.064
      52
      A biologically inspired variable-pH strategy for enhancing short-chain fatty acids (SCFAs) accumulation in maize straw fermentation
      Meng, Yao; Mumme, Jan; Xu, Heng; Wang, Kaijun
      Bioresource Technology (2016), 201 (), 329-336CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      This study investigates the feasibility of varying the pH to enhance the accumulation of short-chain fatty acids (SCFAs) in the in vitro fermn. of maize straw. The corresponding hydrolysis rate and the net SCFA yield increased as inoculum ratio (VSinoculum/VSsubstrate) increased from 0.09 to 0.79. The pH were maintained at 5.3, 5.8, 6.3, 6.8, 7.3, and 7.8, resp. A neutral pH of approx. 6.8 was optimal for hydrolysis. The net SCFA yield decreased by 34.9% for a pH of less than 5.8, but remained const. at approx. 721 ± 5 mg/gvs for a pH between 5.8 and 7.8. In addn., results were obtained for variable and const. pH levels at initial substrate concns. of 10, 30 and 50 g/L. A variable pH increased the net SCFA yield by 23.6%, 29.0%, and 36.6% for concns. of 10, 30 and 50 g/L. Therefore, a variable pH enhanced SCFA accumulation in maize straw fermn.
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    53. 53
      Zhang, M.; Zhang, G.; Zhang, P.; Fan, S.; Jin, S.; Wu, D.; Fang, W. Anaerobic Digestion of Corn Stovers for Methane Production in a Novel Bionic Reactor. Bioresour. Technol. 2014, 166, 606– 609,  DOI: 10.1016/j.biortech.2014.05.067
      53
      Anaerobic digestion of corn stovers for methane production in a novel bionic reactor
      Zhang, Meixia; Zhang, Guangming; Zhang, Panyue; Fan, Shiyang; Jin, Shuguang; Wu, Dan; Fang, Wei
      Bioresource Technology (2014), 166 (), 606-609CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      To improve the biogas prodn. from corn stovers, a new bionic reactor was designed and constructed. The bionic reactor simulated the rumen digestion of ruminants. The liq. was sepd. from corn stovers and refluxed into corn stovers again, which simulated the undigested particles sepd. from completely digested materials and fed back again for further degrdn. in ruminant stomach. Results showed that the bionic reactor was effective for anaerobic digestion of corn stovers. The liq. amt. and its reflux showed an obvious pos. correlation with biogas prodn. The highest biogas prodn. rate was 21.6 mL/gVS-added d, and the total cumulative biogas prodn. was 256.5 mL/gVS-added. The methane content in biogas ranged from 52.2% to 63.3%. The degrdn. of corn stovers were greatly enhanced through simulating the animal digestion mechanisms in this bionic reactor.
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    54. 54
      Ziemer, C. J.; Sharp, R.; Stern, M. D.; Cotta, M. A.; Whitehead, T. R.; Stahl, D. A. Comparison of Microbial Populations in Model and Natural Rumens Using 16S Ribosomal RNA-Targeted Probes. Environ. Microbiol. 2000, 2 (6), 632– 643,  DOI: 10.1046/j.1462-2920.2000.00146.x
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      Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes
      Ziemer C J; Sharp R; Stern M D; Cotta M A; Whitehead T R; Stahl D A
      Environmental microbiology (2000), 2 (6), 632-43 ISSN:1462-2912.
      A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.
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    55. 55
      Yenigün, O.; Demirel, B. Ammonia Inhibition in Anaerobic Digestion: A Review. Process Biochem. 2013, 48 (5–6), 901– 911,  DOI: 10.1016/j.procbio.2013.04.012
      55
      Ammonia inhibition in anaerobic digestion: A review
      Yenigun, Orhan; Demirel, Burak
      Process Biochemistry (Oxford, United Kingdom) (2013), 48 (5-6), 901-911CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)
      A review. Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis during anaerobic digestion process if it is available at high concns. Therefore, ammonia is regarded as a potential inhibitor during anaerobic digestion, particularly when dealing with complex type of substrates such as manure or the org. fraction of municipal solid waste. Ammonia is produced through biol. degrdn. of nitrogenous matter. NH4+ and free NH3 are the 2 principal forms of inorg. ammonia-N. Both forms can directly and indirectly cause inhibition in an anaerobic digestion system. Particularly, free NH3 is a powerful inhibitor in an anaerobic digester above threshold concns. Process inhibition is related to the particular characteristics of the substrate to be anaerobically digested, pH, process temp. (mesophilic or thermophilic), type of the seed sludge (inoculum), the reactor configuration, and to the concns. of NH4+ and NH3. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts after inhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial population available in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.
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    56. 56
      Ransom-Jones, E.; Jones, D. L.; McCarthy, A. J.; McDonald, J. E. The Fibrobacteres: An Important Phylum of Cellulose-Degrading Bacteria. Microb. Ecol. 2012, 63 (2), 267– 281,  DOI: 10.1007/s00248-011-9998-1
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      The Fibrobacteres: an Important Phylum of Cellulose-Degrading Bacteria
      Ransom-Jones, Emma; Jones, David L.; McCarthy, Alan J.; McDonald, James E.
      Microbial Ecology (2012), 63 (2), 267-281CODEN: MCBEBU; ISSN:0095-3628. (Springer)
      A review. The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognized as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted mol. approaches have demonstrated that novel centers of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degrdn. beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a "third" way of attacking the most abundant form of org. carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilize membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecol. and potential as a source of novel glycosyl hydrolases of biotechnol. importance.
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    57. 57
      Arntzen, M.; Várnai, A.; Mackie, R. I.; Eijsink, V. G. H.; Pope, P. B. Outer Membrane Vesicles from Fibrobacter Succinogenes S85 Contain an Array of Carbohydrate-Active Enzymes with Versatile Polysaccharide-Degrading Capacity. Environ. Microbiol. 2017, 19 (7), 2701– 2714,  DOI: 10.1111/1462-2920.13770
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      Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity
      Arntzen, Magnus O.; Varnai, Aniko; Mackie, Roderick I.; Eijsink, Vincent G. H.; Pope, Phillip B.
      Environmental Microbiology (2017), 19 (7), 2701-2714CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
      Summary: Fibrobacter succinogenes is an anaerobic bacterium naturally colonizing the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilizing the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a com. cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to cryst. cellulose. We hypothesize that this complex has a function in plant cell wall degrdn., either by catalyzing polysaccharide degrdn. itself, or by targeting the vesicles to plant biomass.
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    58. 58
      Weimer, P. J.; Russell, J. B.; Muck, R. E. Lessons from the Cow: What the Ruminant Animal Can Teach Us about Consolidated Bioprocessing of Cellulosic Biomass. Bioresour. Technol. 2009, 100 (21), 5323– 5331,  DOI: 10.1016/j.biortech.2009.04.075
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      Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass
      Weimer, Paul J.; Russell, James B.; Muck, Richard E.
      Bioresource Technology (2009), 100 (21), 5323-5331CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      A review. Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermn. pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermn. is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective phys. pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermn. of nearly all plant components; efficient recovery of primary fermn. end-products; and prodn. of useful co-products. Ruminal fermn. does not produce significant amts. of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other org. compds., particularly transportation fuels.
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    59. 59
      Stanton, T. B. Glucose Metabolism of Treponema Bryantii, an Anaerobic Rumen Spirochete. Can. J. Microbiol. 1984, 30 (5), 526– 531,  DOI: 10.1139/m84-080
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      Glucose metabolism of Treponema bryantii, an anaerobic rumen spirochete
      Stanton, Thad B.
      Canadian Journal of Microbiology (1984), 30 (5), 526-31CODEN: CJMIAZ; ISSN:0008-4166.
      The pathway of glucose metab. by T. bryantii, an obligately anaerobic spirochete isolated from bovine rumen contents, was studied. Washed cell suspensions of the spirochete consumed glucose and CO2 and produced equimolar amts. of acetate, formate, and succinate. CO2 was essential for glucose metab. Detn. of radioactivity in products formed from 14C-labeled glucose and NaH14CO3 and assays of enzyme activities in cell-free exts. were used to det. the pathway of glucose metab. T. bryantii Catabolized glucose to pyruvate via the Embden-Meyerhof-Parnas pathway. The spirochete used a coliform pyruvate-formate lyase to degrade pyruvate and produce formate and acetate. Succinate was formed by a pathway which involved the condensation of CO2 with pyruvate (or phospho(enol)pyruvate) formed from the breakdown of glucose.
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    60. 60
      Kudo, H.; Cheng, K.-J.; Costerton, J. W. Interactions between Treponema Bryantii and Cellulolytic Bacteria in the in Vitro Degradation of Straw Cellulose. Can. J. Microbiol. 1987, 33 (3), 244– 248,  DOI: 10.1139/m87-041
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      Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose
      Kudo, H.; Cheng, K. J.; Costerton, J. W.
      Canadian Journal of Microbiology (1987), 33 (3), 244-8CODEN: CJMIAZ; ISSN:0008-4166.
      To assess the contribution of individual bacterial species to the overall process of cellulose digestion in the rumen, cellulolytic bacteria (Bacteroides succinogenes and Ruminococcus albus) were tested as pure cultures and as cocultures with noncellulolytic T. bryantii. In studies of in vitro barley straw digestion, Treponema cocultures surpassed pure cultures of the cellulolytic organisms in dry matter disappearance, volatile fatty acid generation, and in the prodn. of succinic acid, lactic acid, and EtOH. Morphol. examn., by electron microscopy, showed that cells of T. bryantii assoc. with the plant cell wall materials in straw, but that cellulose digestion occurs only when these organisms are present with cellulolytic species such as B. succinogenes. These results show that cellulolytic bacteria interact with noncellulolytic Treponema to promote the digestion of cellulosic materials.
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    61. 61
      Hahnke, S.; Langer, T.; Koeck, D. E.; Klocke, M. Description of Proteiniphilum Saccharofermentans Sp. Nov., Petrimonas Mucosa Sp. Nov. and Fermentimonas Caenicola Gen. Nov., Sp. Nov., Isolated from Mesophilic Laboratory-Scale Biogas Reactors, and Emended Description of the Genus Proteiniphilum. Int. J. Syst. Evol. Microbiol. 2016, 66 (3), 1466– 1475,  DOI: 10.1099/ijsem.0.000902
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      Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov., isolated from mesophilic laboratory-scale biogas reactors, and emended description of the genus Proteiniphilum
      Hahnke, Sarah; Langer, Thomas; Koeck, Daniela E.; Klocke, Michael
      International Journal of Systematic and Evolutionary Microbiology (2016), 66 (3), 1466-1475CODEN: ISEMF5; ISSN:1466-5026. (Society for General Microbiology)
      Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic lab.-scale biogas reactors. The strains were Gram-neg. rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1-7.8. The main fermn. products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA-DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3-45.6 and 23.8-25.7 % relatedness, resp., which supports the conclusion that both isolates represent novel species. Phylogenetic anal. and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiol., genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addn., an emended description of the genus Proteiniphilum is provided.
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    62. 62
      Azman, S.; Khadem, A. F.; Van Lier, J. B.; Zeeman, G.; Plugge, C. M. Presence and Role of Anaerobic Hydrolytic Microbes in Conversion of Lignocellulosic Biomass for Biogas Production. Crit. Rev. Environ. Sci. Technol. 2015, 45 (23), 2523– 2564,  DOI: 10.1080/10643389.2015.1053727
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      Presence and Role of Anaerobic Hydrolytic Microbes in Conversion of Lignocellulosic Biomass for Biogas Production
      Azman, Samet; Khadem, Ahmad F.; Van lier, Jules B.; Zeeman, Grietje; Plugge, Caroline M.
      Critical Reviews in Environmental Science and Technology (2015), 45 (23), 2523-2564CODEN: CRETEK; ISSN:1064-3389. (Taylor & Francis, Inc.)
      In recent years, biogas prodn. from complex biomass has received great interest. Therefore, many studies have been conducted to understand the anaerobic digestion process and to characterize responsible microbes for the biochem. conversions. Although the knowledge about biogas prodn. in general is rapidly increasing, less information is available about hydrolytic microbes within anaerobic bioreactors. Here, the authors pinpoint the urgent need for solid fundamental knowledge about hydrolytic bacteria within biogas plants. In this review, current knowledge about anaerobic hydrolytic microbes is presented, including their abundance in biogas plants, and the factors impacting their activity.
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    63. 63
      Chow, J. M.; Russell, J. B. Effect of PH and Monensin on Glucose Transport by Fibrobacter Succinogenes, a Cellulolytic Ruminal Bacterium. Appl. Environ. Microbiol. 1992, 58 (4), 1115– 1120,  DOI: 10.1128/AEM.58.4.1115-1120.1992
      63
      Effect of pH and monensin on glucose transport by Fibrobacter succinogenes, a cellulolytic ruminal bacterium
      Chow, Jo May; Russell, James B.
      Applied and Environmental Microbiology (1992), 58 (4), 1115-20CODEN: AEMIDF; ISSN:0099-2240.
      F. succinogenes S85, a cellulolytic ruminal bacterium, required Na for growth and glucose uptake. Cells which were deenergized with iodoacetate (500 μM) could not take up [14C]glucose. However, deenergized cells which were treated with valinomycin, loaded with K, and dild. into Na or Na plus K to create an artificial elec. gradient (Δψ) plus a chem. gradient of Na (ΔpNa) or ΔpNa alone transported glucose at a rapid rate. Cells which were loaded with Na plus K and dild. into Na (Δψ with Na, but no ΔpNa) also took up glucose at a rapid rate. K-loaded cells that were dild. into buffers which did not contain Na (Δψ without Na) could not take up glucose. An artificial ZΔpH which was created by acetate diffusion could not drive glucose transport even if Na was present. The max. rate and affinity of glucose transport (pH 6.7) were 62.5 nmol/mg protein per min and 0.51 mM, resp. S85 was unable to grow at pH <5.5, and there was little glucose transport at this pH. When the extracellular pH was decreased, the glucose carrier was inhibited, intracellular pH declined, the cells were no longer able to metabolite glucose, and Δψ declined. Monensin (1 μM) or lasalocid (5 μM) decreased intracellular ATP and dissipated both the Δψ and ΔpNa. Since there was no driving force for transport, glucose transport was inhibited. These results indicated that F. succinogenes used a pH-sensitive Na symport mechanism to take up glucose and that either a Δψ or a ΔpNa was required for glucose transport.
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    64. 64
      Russell, J. B.; Dombrowski, D. B. Effect of PH on the Efficiency of Growth by Pure Cultures of Rumen Bacteria in Continuous Culture. Appl. Environ. Microbiol. 1980, 39 (3), 604– 610,  DOI: 10.1128/AEM.39.3.604-610.1980
      64
      Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture
      Russell J B; Dombrowski D B
      Applied and environmental microbiology (1980), 39 (3), 604-10 ISSN:0099-2240.
      A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.
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    65. 65
      Weimer, P. J. Effects of Dilution Rate and PH on the Ruminal Cellulolytic Bacterium Fibrobacter Succinogenes S85 in Cellulose-Fed Continuous Culture. Arch. Microbiol. 1993, 160, 288– 294,  DOI: 10.1007/BF00292079
      65
      Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture
      Weimer, Paul J.
      Archives of Microbiology (1993), 160 (4), 288-94CODEN: AMICCW; ISSN:0302-8933.
      F. succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of diln. rate (D, 0.014-0.076/h) and extracellular pH (6.11-6.84). Effects of pH and D on the fermn. were detd. by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and sol. sugar concn. to response surface anal. The extent of cellulose conversion decreased with increasing D. First-order rate consts. at rapid growth rates were estd. as 0.07-0.11/h, and decreased with decreasing pH. Apparent decreases in the rate const. with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of sol. sugars (0.04-0.18 g/L, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11-0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to det. that maintenance coeff. (0.04-0.06 g cellulose/g cells-h) and true growth yield (0.23-0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermn. end product, were ≤1.15 mol/mol anhydroglucose fermented and were slightly affected by diln. rate but were not affected by pH. Comparison of the fermn. data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of cryst. cellulose and efficient growth, despite a lower μmax on microcryst. cellulose.
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    66. 66
      Kleerebezem, R.; Joosse, B.; Rozendal, R.; Van Loosdrecht, M. C. M. Anaerobic Digestion without Biogas?. Rev. Environ. Sci. Bio/Technol. 2015, 14, 787,  DOI: 10.1007/s11157-015-9374-6
      66
      Anaerobic digestion without biogas?
      Kleerebezem, Robbert; Joosse, Bart; Rozendal, Rene; Van Loosdrecht, Mark C. M.
      Reviews in Environmental Science and Bio/Technology (2015), 14 (4), 787-801CODEN: RESBC6; ISSN:1569-1705. (Springer)
      Anaerobic digestion for the prodn. of methane contg. biogas is the classic example of a resource recovery process that combines stabilization of particulate org. matter or wastewater treatment with the prodn. of a valuable end-product. Attractive features of the process include the prodn. of a single end-product from a heterogeneous feedstock, and in-situ product sepn. of the gaseous end-product. Despite these intrinsic attractive properties of the process, the economic added value of the biogas produced is limited, enabling the development of alternative processes that yield higher-value end-products. Typically the prodn. of higher value end-products from low value feedstock and industrial wastewater proceeds via intermediate prodn. of org. acids (and carbon dioxide and mol. hydrogen). Optimization of org. acid prodn. from particulate feedstocks and wastewater for development of the org. acid based resource recovery route receives significant research attention. The org. acid stream generated as such, has no economic value, but if org. acids can either be concd. via membrane sepn. or (bio)converted to an end-product that can easily be sepd. from the liq., an attractive biomass processing scheme can be developed. Attractive end-products of org. acid processing include polyhydroxyalkanoates, medium chain length fatty acids, or other org. mols. using bio-electrochem. systems. Overall we suggest that these novel bioprocessing routes for conversion of low value feedstock to higher added value products will contribute to a sustainable future and will change the economic status of org. waste.
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  • Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsestengg.0c00164.

    • Calculations to estimate the rumen content availability in the US, description of chemical and microbial analyses, characterization of inocula and substrates, reactor operating parameters, experimental design, and reactor performance data (PDF)


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