Black Carbon Impacts on Paraburkholderia xenovorans Strain LB400 Cell Enrichment and Activity: Implications toward Lower-Chlorinated Polychlorinated Biphenyls Biodegradation Potential

Volatilization of lower-chlorinated polychlorinated biphenyls (LC-PCBs) from sediment poses health threats to nearby communities and ecosystems. Biodegradation combined with black carbon (BC) materials is an emerging bioaugmentation approach to remove PCBs from sediment, but development of aerobic biofilms on BC for long-term, sustained LC-PCBs remediation is poorly understood. This work aimed to characterize the cell enrichment and activity of biphenyl- and benzoate-grown Paraburkholderia xenovorans strain LB400 on various BCs. Biphenyl dioxygenase gene (bphA) abundance on four BC types demonstrated corn kernel biochar hosted at least 4 orders of magnitude more attached cells per gram than other feedstocks, and microscopic imaging revealed the attached live cell fraction was >1.5× more on corn kernel biochar than GAC. BC characteristics (i.e., sorption potential, pore size, pH) appear to contribute to cell attachment differences. Reverse transcription qPCR indicated that BC feedstocks significantly influenced bphA expression in attached cells. The bphA transcript-per-gene ratio of attached cells was >10-fold more than suspended cells, confirmed by transcriptomics. RNA-seq also demonstrated significant upregulation of biphenyl and benzoate degradation pathways on attached cells, as well as revealing biofilm formation potential/cell–cell communication pathways. These novel findings demonstrate aerobic PCB-degrading cell abundance and activity could be tuned by adjusting BC feedstocks/attributes to improve LC-PCBs biodegradation potential.


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S1. Supplemental Methods and Materials S1.1 K1 Medium Preparation and Carbon Source Addition K1 medium was made by adding 100 mL K1 10× stock solution and 20 mL Hutner mix into 880 mL sterile deionized (DI) water.K1 10× stock solution was made by adding 15.9 g K 2 HPO 4 , 2.5 g (NH 4 ) 2 SO 4 , and 1.77 g NaH 2 PO 4 •H 2 O into 1000 mL DI water.After autoclaving K1 10× stock solution for 30 minutes at 121°C, sterile vitamins were added, including 250 µL 10 mg/mL of thiamine, nicotinic acid, nyo-inositol, and riboflavin in phosphate buffer and 25 µL 2 mg/mL of D-biotin in methanol.Hutner mix contains various salts and "Metals 44" solution as described below.First, nitrilotriacetic acid (NTA) (10 g) was added into 600 mL DI water with a magnetic stir bar on a stir plate.The cloudy NTA solution dissolved after adding KOH pellets.Afterwards, 14.5 g MgSO 4 •7H 2 O, 3.33 g CaNO 3 , 9.25 mg (NH 4 ) 6 Mo 7 O 24 •24H 2 O, 99 mg FeSO 4 •7H 2 O, and 50 mL Metals 44 were added sequentially.After all chemicals were completely mixed, an additional 400 mL DI water was added into the solution, followed by pH adjustment to 6.6-6.8.Solution was filtered through 0.2 µm filters and stored at room temperature.Metals 44 was made by adding the following chemicals to 400 mL DI water: 1 g EDTA, 4. A few drops of H 2 SO 4 were added to dissolve metals, and the solution was filtered through 0.2 µm filters and stored at room temperature.
Additional carbon sources were added to the K1 medium for cell growth.Based on their theoretical oxygen demand, 5 mM biphenyl crystal or 10 mM sodium benzoate was needed.Depending on the final volume of K1 medium, different mass of carbon sources were added.For example, when the volume of K1 medium is 250 mL, 0.19 g biphenyl crystal or 0.36 g sodium benzoate is needed.The abundance of bphA in attached and suspended LB400 cells after different incubation periods.The impacts of incubation time on bphA abundance in attached cells and suspended cells were tested.The bphA abundance of attached cells was lower after a 20-day incubation than after a 10day incubation (Figure S1a).The bphA abundance of suspended cells (Figure S1b, c) was not significantly different (p>0.05) after cultures entered stationary phase (~6d, Figure S11).

S1.2 Cell Attachment Visualization
Confocal Laser Scanning Microscopy (CLSM).Cell attached to BC surfaces were stained with 10 µM SYTO9 and 60 µM propidium iodide (PI) in phosphate-buffered saline (PBS) for 15 minutes at room temperature in the dark.After rinsing residual dyes with 1 mL PBS, samples were covered in 90% glycerol with 10 mM Tris-HCl (pH 8) prior to CLSM imaging.Reflectance and fluorescence imaging were observed under 488 nm and 499-600 nm, respectively.PBS was made by adding 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , and 0.24 g KH 2 PO 4 into 1 L DI water, which pH was adjusted to 7.4.Scanning Electron Microscopy (SEM).Before inserting samples into SEM chamber, all samples were air dried, coated with silver solution (60% Ag in 1-methoxy-2-propanol, Electron Microscopy Sciences, Hatfield, PA) on an aluminum holder, and mounted with a conductive layer of gold or iridium sputter (EMS 150T ES, Quorum Technologies, Lewes, United Kingdom).

S1.3 Modified Procedures for DNA Extraction
Instead of using a Sterivex filter unit, two mL of liquid samples (diluted as appropriate) were directly added to a glass PowerBead tube with 0.9 mL warm MBL solution and then incubated at 90°C for 5 mins.After cooling for 2 mins, the solution was vortexed at maximum speed for 5 mins and centrifuged at 4000×g for 1 min.The remaining procedures followed the kit.S3).An ABI 7000 Sequence Detection System (Applied Biosystems, Grand Island, NY) was used with the following thermocycling conditions: 10 min at 95℃, 40 cycles at 95℃ (15 s) and 60℃ (1 min), and a dissociation step.A bphA standard curve was prepared in triplicate (Table S3); for luciferase mRNA, standard DNA templates were gBlock fragments (Integrated DNA Technologies, Coralville, IA).Melt curve analysis indicated single peaks in standards and samples for bphA (85°C) and luciferase (81°C).The entire qPCR procedure was conducted in a biosafety cabinet (BSC).All supplies such as 8channel pipettes and repeater were wiped with 70% ethanol before being brought into the BSC.All supplies were irradiated under UV light in the BSC for 15 minutes.All reagents were thawed in the ice basket, including Power SYBR green master mix, primers, DNA samples, and DNA standards, to keep the reagent activity from decreasing.Known amounts of bphA, cloned into the 2.1-TOPO vector as described below, were used to prepare the standard DNA.Concentrated samples were diluted to 5-10 ng/µL using qPCR-grade water prior to analysis on instrument.Luciferase mRNA recovery efficiency was determined as the ratio of luciferase gene copies from qPCR analysis over spiked luciferase mRNA gene copies (6.08×10 9 cDNA per ng luciferase mRNA, based on dsDNA).The residual bphA in RNA samples after DNA digestion was measured in control qPCRs using RNA as the template.Although incomplete DNA digestion could contribute to the low level of DNA contamination, the difference between bphA transcript abundance in samples and RNA controls was at least four orders of magnitude (Figure S2).
Transcript levels for bphA in samples are shown in Figure 3. Liquid was autoclaved for 30 minutes at 15 psi, and kanamycin was added after the solution cooled to about 55°C.For LB plate containing 50 µg/mL kanamycin, additional 15g/L agar was added into LB medium before autoclaving.
RNA-seq data analysis.The first base of every read was trimmed by fastp program since the per base sequence quality was low (<20).For HISAT2, paired-end data from single interleaved dataset was selected for the library setting, and reverse (RF) was specified for strand information.For FeatureCount, GFF feature types filter was set as "gene" instead of "exon".

S1.6 Biphenyl, Benzoate and Acetate Analysis
Biphenyl extraction.Biphenyl was extracted from liquid samples (2 mL) via liquid-liquid extraction.Liquid samples were collected by glass analytical syringe into glass vials.Hexane was added at a 1:1 ratio (hexane:sample, v/v), followed by vortexing (10 seconds) and centrifugation (3 min at 1550×g).Supernatant was transferred to new glass vials for silica column filtration (Figure S8).Each hexane extract was filtered through a silica column and rinsed three times with hexane, with all filtrates collected in TurboVap tubes.The sample extract was concentrated to ~0.5mL using TurboVap II Concentration Workstation (Biotage, Uppsala, Sweden).The concentrated extract was transferred to crimp-top glass autosampler vials for gas chromatography.

Silica column filtration setup for biphenyl extraction.
A silica column was assembled using glass wool, silica gel, and acidic silica gel, respectively, in a 23 cm disposable Pasteur pipette placed in a pipette holder.Glass wool was packed into the funnel position, where the edge and the center of cotton were sturdy and flat.Less than 1 cm silica gel was poured through the funnel, followed by acidic silica gel until there was about 2 cm away from the top of the pipette.All columns were conditioned by running through hexane for two times.guard column. 3The HPLC operating mode was isocratic and an injection volume of 10 µL was loaded onto the column at ambient temperature.The mobile phase contained ammonium acetate buffer (90%) and acetonitrile (10%) at a flow rate of 0.8 mL/min.Sample data collection time was 8 mins per sample with UV detection at 225 nm.Ammonium acetate buffer was prepared with 0.3 g ammonium acetate adding in 900 mL DI water, followed by the addition of glacial acetic acid adjusting pH to 3.9.The buffer solution was then transferred to 1 L volumetric flask and filtered through 0.22 µm nylon filter.Method detection level (MDL) was 0.48 mM.Desired MDL was 3.14 times standard deviation of seven replicates of benzoate solution.The recovery efficiency was within the range of 50-150%, and replicate measurements were in the range of one to five times the MDL.MDL determination followed the instructions of Standard Methods for the Examination of Water and Wastewater. 4 Acetate measurement.Acetate concentrations in liquid samples were measured by Ion Chromatography (Dionex ICS-2100) equipped with AS11-HC (250×4 mm) anion exchange column and an AG11 guard column.The column temperature was 30℃, the cell temperature was 35℃, and the suppressor was set at 41 mA.The effluent flow rate was 1.1 mL/min.The eluent gradient was KOH ramp from 1 to 60 mM with a total run time of 20 mins.Method detection level was 0.017 mM.Desired MDL was 3.14 times standard deviation of seven replicates of acetate solution.The recovery efficiency was within the range of 50-150%, and replicate measurements were in the range of one to eight times the MDL.Determination also followed the instructions of Standard Methods for the Examination of Water and Wastewater.

Figure S1 .
Figure S1.The bphA abundance in suspended and attached LB400 cells when 22.8 mg of biphenyl was added as sole carbon and energy source.(a) BphA abundance on the BC surface (attached) after 10-day and 20-day incubations.(b) BphA abundance in suspended cells after 10-day and 20day incubation.(c) BphA abundance in suspended cells after 3-day, 7-day, and 10-day incubation.The unit in (a) was based on wet weight.LB: large corn kernel biochar, SB: small corn kernel biochar, GAC: granular activated carbon, and LCT: live cell treatment without BC addition.The error bars represent the standard deviation of biological duplicates and qPCR technical replicates.Note the log-scale on the vertical axis.

Plasmid bphA standards transformation to 2 . 1 -
TOPO vector.Two microliters of plasmid bphA standards were gently mixed with a vial of Chemically Competent E. coli cells provided in TOPO TA Cloning kit (Life Technologies, Warrington, UK).After incubating on ice for 10 minutes, cells were provided with heat-shock at 42°C water bath for exactly 30 seconds and then immediately transferred to ice.Afterwards, 250 µL of room temperature S.O.C. medium (provided by TOPO TA Cloning kit) was added into the cells and shaken horizontally (200rpm) at 37°C for 1 hour.20 µL of E. coli culture were spread on a prewarmed LB plate containing 50 µg/mL kanamycin and incubated overnight at 37°C. 10 single colonies were picked from the plate and cultured in LB medium containing 50 µg/mL kanamycin overnight to grow enough E. coli cells for further plasmid DNA extraction which was performed based on PureLink Quick Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA) instructions.To verify the transformed plasmid, polymerase chain reaction (PCR) and DNA sequencing were performed to obtain the whole plasmid DNA sequences.LB medium containing 50 µg/mL kanamycin was prepared by adding 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1L DI water, and then pH was adjusted to 7.

Figure S3 .Figure S4 .Figure S5 .Figure S6 .S11S1. 5
Figure S2.Residual DNA in RNA samples of attached LB400 cells in different black carbon addition systems after DNA digestion.The unit was based on wet weight of BCs.SB: small corn biochar, LB: large corn biochar, GAC: granular activated carbon, BB: bamboo biochar, and WB: wood biochar.

Figure S8 .
Figure S8.Display of silica column setup used to filter biphenyl extracts.Biphenyl measurement.Extracted samples were run on a Hewlett Packard Model 6890 Gas Chromatograph equipped with 5973 Turbo Pump Mass Selective Detector (GC-MS).A Restek Rtx-VMS capillary column (30 m × 0.25 mm ID, 1.4 µm film thickness) was used with helium as the carrier gas (20 mL/min with split ratio of 10).The initial temperature GC oven temperature was 60°C (hold for 0.5 min), then ramped to 225°C (30°C/min) and hold for 2 mins.MSD electron ionization source was set to 280°C.Benzoate measurement.Benzoate was quantified using high performance liquid chromatography (HPLC, Agilent 1100 series, Hewlett Packard, Palo Alto, CA) with Diode-Array Detection (DAD) and separated on a Sprite Targa reverse-phase C18 column (40 mm × 2.1 mm ×5 µm) with a PEEK

Figure S10 .Figure S11 .
Figure S10.Transcript per gene ratio of suspended and attached LB400 cells in different black carbon addition systems.SB: small corn biochar, LB: large corn biochar, and GAC: granular activated carbon.Liquid phase represents suspended cells, and solid phase represents attached cells.

Figure S13 .
Figure S13.Differential gene expression heatmap of LB400 cells attached to either SB or LB. Green bars represent upregulated genes while yellow bars represent downregulated genes.Both heatmaps were plotted by using Z-scores, standardized based on row, and describe the significant gene expression level of selected genes passing the threshold (Log2 (FC)> |±1|, adjusted p<0.05).The Z-score is the number of standard-deviations that a value was away from the mean of all the values in the same group.Hierarchical clustering was based on the Euclidean distance of each row and demonstrated the similarity of different genes.SB: small corn biochar and LB: large corn biochar.Branches on the left depicted the cluster of selected genes and branches on the top demonstrated the cluster of samples.

Figure S17 .
Figure S17.Benzoate degradation in the liquid phase for both cells attached on SB and GAC and benzoate sorption by plain SB and GAC (n=2).Attached cells were grown under 5 mM biphenyl for 10 days before testing benzoate degradation.The initial sampling time was immediately after BCs (with and without attached cells) and benzoate were added and mixed.The initial benzoate concentration for GAC (without attached cells) was lower than other groups because benzoate was rapidly sorbed by GAC.Attached cells may have affected benzoate sorption efficiency in the GAC_attached cells treatment.SB: small corn biochar and GAC: granular activated carbon.Error bars represent the standard deviation of biological duplicates.MDL represents method detection limit of benzoate.

Figure S18 .
Figure S18.Acetate degradation in the liquid phase for both cells attached on SB and GAC and acetate sorption by plain SB and GAC (n=4).Attached cells were grown under 5 mM biphenyl for 10 days before testing acetate degradation.SB: small corn biochar and GAC: granular activated carbon.Error bars represent the standard deviation of biological duplicates.MDL represents method detection limit of acetate.

Table S4 .
Spearman correlation between BC properties and bphA abundance in attached cells.

Table S5 .
Summary of live to dead cell ratios on corn kernel biochar and GAC surfaces growing under 5 mM biphenyl crystal for 10 days.

Table S6 .
Bph pathway related genes of differential gene expression comparison between attached and suspended LB400 cells.

Table S7 .
Box pathway related genes of differential gene expression comparison between attached

Table S9 .
Genes related to EPS production pathway (EPS pathway II) of differential gene expression comparison between LB and GAC surfaces and SB and GAC surfaces.

Table S10 .
AHL production pathway related genes of differential gene expression comparison356 between SB and GAC surfaces.