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DEVELOPING TECHNOLOGY Riding the fossil fuel biodesulfurization wave
Daniel J. MonticelloB iodesulfurization is a process that removes sulfur from fossil fuels using a series of enzyme-catalyzed reactions. In 1993, Iain Campbell, a professor of microbiology at the University of Pittsburgh and long-time participant in biodesulfurization research, wrote an article for CHEMTECH entitled "Catching the biodesulfurization wave" (1). It outlined the science and politics of biodesulfurization research in the United States, focusing on the "wave" of government support of this technology in the late 1980s and the results of that support. Since then several industrial, government, and university laboratories have caught the wave and have been riding it for almost five years. In this article, I describe the issues that drive the continued interest in the biodesulfurization of fossil fuels, look at the agencies (governmental, academic, and industrial) that have sponsored the work, identify the major players in this field, and outline the scientific and technological progress that has been made as we have been riding the wave of this development effort.
The nature of the problem The easiest way to limit the amount of sulfur dioxide emitted into the
air is to limit the amount of sulfur in fuel. Another (but less
practical) way would be to make more efficient scrubbers on stationary
emitters and install them on mobile emitters (such as trains, planes,
and automobiles). Understandably, government agencies have opted for
regulations that mandate the level of sulfur in the fuel. Gasoline and
diesel fuel have been particularly attractive targets. "Straight-run
diesel" (taken directly from the crude distillation tower) can have
sulfur ranging from <500 ppm to >5000 ppm, depending on which crude
oil is used and whether it is desulfurized already in refineries.
Currently, U.S. on-highway diesel fuel must be <500 ppm sulfur, but
even lower sulfur levels have been mandated. Some of these levels are
shown in Table 1.
Fuel desulfurization seems like a reasonable approach to reducing acid
rain except for one inevitable problem: It costs money, and as the
extent of desulfurization increases, the costs escalate rapidly. This
is a direct result of the process chemistry, hydrodesulfurization, used
in refineries to remove sulfur. Hydrodesulfurization is a high-pressure
(150-250 psig) and high-temperature (200-425 °C) process that
uses hydrogen gas to reduce the sulfur in petroleum fractions
(particularly diesel) to hydrogen sulfide, which is then readily
separated from the fuel. Hydrodesulfurization units are expensive to
build and operate. In addition, this chemistry does not work well on
certain sulfur molecules in oil, particularly the polyaromatic sulfur
heterocycles (PASHs) found in heavier fractions (Table 2)
(2). It is this limitation in the conventional technology
that has tempted researchers over the past 50 years to venture into the
inviting waters of biotechnology to catch the wave of alternative
sulfur removal processes.
Our understanding of the impact of sulfur emissions on the environment
has grown increasingly sophisticated over the last 20 years, but the
desire to desulfurize oil is not new, and the interest in this issue
has indeed come in waves, as Campbell described. The first recorded
wave was in the early 1950s, when a series of U.S. patents were issued
on "microbial desulfurization" processes (3). They never
actually worked, but that's another story.
The next big wave was in the late 1970s and early 1980s. The U.S.
Department of Energy (DOE) and other organizations sponsored work
around the country to try once again to crack this scientific and
technological nut. Although significant progress was made
(4), the biggest development was the clear insight into what
didn't work. The bacteria that had been isolated at the time were not
appropriate for commercial desulfurization technologies because they
attacked the hydrocarbon portion of PASHs and only coincidentally
solubilized the sulfur molecules to water, thus removing them from oil.
Many of the polyaromatic molecules (naphthalene, phenanthrene, etc.)
were also attacked. This was successful desulfurization, but the cost
was too much of the fuel value of the oil. It was clear then that
further development work was pointless until an enzymatic system that
specifically attacked the sulfur atom in the PASHs in oil could be
identified.
Campbell's article also described the next wave of activity, in the
later 1980s, directed (successfully) at identifying bacteria that could
liberate sulfur from the model PASH dibenzothiophene without attacking
the hydrocarbon. This development and the subsequent characterization
of the system lead to the latest and, by far, the largest and most
sustained wave of development. It looks as if this effort has been
sufficient to create a nascent biodesulfurization technology. It
remains to be seen whether this effort will be sufficient to complete
the journey or whether more breakthroughs will be needed for the
widespread application of this technology.
Industry experts suggest that the refining industry will have to spend about $37 billion on new desulfurization equipment and an additional $10 billion on annual operating expenses over the next 10 years to meet the new sulfur regulations. In addition to this opportunity in the refinery, there is also a large potential in the desulfurization of crude oil itself. Approximately half of the 60 million barrels of crude produced each day is considered "high sulfur" (>1%). The partial desulfurization of this material represents a significant chance to "upgrade" the crude and its value. My employer, Energy BioSystems Corporation (EBC), has spent the most money worldwide on microbial desulfurization research since 1992. The other major spender has been the Petroleum Energy Center (PEC) in Japan. EBC has spent about $50 million isolating, characterizing, and manipulating the desulfurization genes from a variety of microorganisms and developing and testing the reactor, separations, and recovery technology that is required to commercialize biodesulfurization. As a publicly traded company in the United States, these numbers are fairly easily calculated based on annual reports. PEC has also been active in this area and, in 1994, pledged $50 million of its own for development of the technology. PEC is a Japanese government-industry consortium, and therefore, it is difficult to determine the actual spending. The number of papers that have appeared over the past three years indicates a substantial, if fragmented, program. The U.S. government, through the ongoing support of several academic and government labs, has spent about $10 million since 1990, including a $3-million Advanced Technology Program (ATP) grant to EBC for crude oil desulfurization, and the steady participation of DOE in cooperative R&D arrangements at the Oak Ridge and Brookhaven National labs and small sponsored programs at the Institute of Gas Technology and other academic labs. DOE recently awarded EBC a $2.4-million grant to pursue gasoline biodesulfurization. The oil companies have spent only small amounts on this wave of technology development, mostly being content to sit on the sidelines and watch. The exceptions to this have been
To manipulate the bacteria that are responsible for these important reactions, we must understand their underlying genetics, biochemistry, enzymology, and microbiology. As a result, the big winner of this research wave thus far has been the scientific community. The metabolic pathway for the sulfur-specific oxidation of dibenzothiophene (DBT) was completely unknown 10 years ago. Today, the details of the pathway are published and are on the University of Minnesota biocatalysis/biodegration database Web site ( http://www.labmed.umn.edu/umbbd/index.html). Model systems for desulfurization. The usual model molecule for studies of desulfurization is DBT. It has been used for many years in the development of conventional catalysts for hydrodesulfurization, and it is representative (to a degree) of the more troublesome molecules in the diesel fractions of crude oil (2). There is actually not much DBT in hydrotreated diesel fuel, but this simple PASH is still a reasonable choice. It is one of many tens of thousands of PASHs found in a hydrotreated diesel sample, and the alkyl side chains that generate all these isomers have been shown to significantly affect the relative reactivity of the molecules with inorganic (cobalt-molybdenum) and organic (enzyme) catalysts (5). The alkylated DBTs (Cx-DBTs) are the actual targets for the technology and should be evaluated as a whole whenever possible. However, most of the basic scientific work that has been reported has been done with DBT. Sulfur-specific Cx-DBT metabolism. In the following sections, I outline the latest work on the metabolism of Cx-DBTs via the hydrocarbon-conserving (4S) pathway first proposed by Iain Campbell and Kee Rhee at DOE's Pittsburgh Energy Technology Center. The activity has been observed in many species of bacteria since the first confirmed isolation by Kilbane in 1988 (6). In most cases, the bacteria have been closely related and catalyze the same reaction. The general steps in this biodesulfurization system as we understand it from studies on various bacterial species are illustrated in Figure 2.
The first step in desulfurization of these molecules is the transfer of the molecules from the oil into the cells. It appears that in Rhodococcus spp., these molecules are transferred directly from the oil to the cells. This is likely, because Rhodococcus spp. and other bacteria have been shown to metabolize many "insoluble" molecules in this fashion. The desulfurization (dsz) genes have been transferred to other organisms, such as Escherichia coli and Pseudomonas putida (7). In these cells, the PASHs appear to partition to the water before being brought into the cell. Conversion to the sulfone. The enzyme directly responsible for the first two oxidations has been isolated and characterized in some detail (8). The gene for this enzyme (dszC) has been cloned and sequenced. The enzyme has been named DBT monooxygenase (FMNH2:DBT oxidoreductase) to reflect the reaction it catalyzes: the transfer of an electron from flavin mononucleotide (FMNH2) to DBT to produce oxidized FMN (FMNH2), DBTO, and DBTO2. DBT monooxygenase catalyzes the oxidation of DBT to the sulfoxide and also the oxidation of the sulfoxide to the sulfone. The enzyme appears to operate as a tetramer in the cell. The genes for this enzyme have been cloned and described (9, 10). Cleavage of the first C-S linkage. The first cleavage of the carbon sulfur bonds is catalyzed by DBT sulfone monooxygenase (FMNH2:DBTO2 oxidoreductase, which transfers another electron from FMNH2 to DBTO2). This enzyme and its gene, dszA, have also been characterized (5, 8, 9). It appears to operate in the cell as a dimer. Liberation of inorganic sulfur. Production of sulfite and an intact hydrocarbon molecule is the last reaction in the pathway. This is catalyzed by a "desulfinase" (coded by the dszB gene) and leads to the release of the sulfur as sulfite and the production of the oil-soluble product, hydroxy biphenyl (HBP). In nature, the cell has achieved its goal at this point--it has the sulfur it needs to grow. The sulfite can be reduced to sulfide and incorporated into sulfur-containing amino acids and vitamins necessary for growth. Technologically, this is just the beginning. The flux through this pathway must be amplified many hundredfold over normal levels to turn this biochemical pathway into a industrially useful technology. This genetic and metabolic engineering effort is underway currently. The supply of reducing equivalents. Another important enzyme in this system is the reduced nicotinamide adenosine dinucleotide (NADH):FMN oxidoreductase, which keeps the supply of reduced FMN in balance (1). The primary enzyme for this task in Rhodococcus spp. is a dedicated flavin reductase (11). (Details of the genetics for this enzyme have been submitted for publication by Charles Squires and colleagues at EBC.) Regulation of the activity in the cell. Expression of the dsz genes in Rhodococcus spp. is carefully regulated in "wild-type" cells. Presumably, this is because the cells are rarely in a carbon-rich, sulfur-poor environment that would favor cells expressing these genes. When the cells are grown in a medium with even small amounts of sulfur, they stop expressing the DSZ phenotype, presumably by repressing enzyme synthesis at the level of transcription. My colleagues and I have characterized the nature of this regulation in some detail (12) and are in the process of modifying cells to effect this regulation so that the gene will work continuously under process conditions.
Developing the technology The bacteria have solved this problem with the use of a monooxygenase. The oxygen is activated on the surface of the catalyst (with the electron from FMNH2), which is inside the cell and specific for molecules with the physical and chemical properties of the PASHs. This specificity gives the microbial system its advantage over chemical systems for PASH oxidation. Turning this interesting metabolic phenomenon into an industrially useful technology requires the development of a biocatalyst with rates and stability that are amenable to process development. Beyond this, there are numerous problems related to mixing, mass transfer, separations, and byproduct disposition that I will not address here. Genetic engineering. The key to increasing the metabolic flux through the bacteria is to manipulate the basic genetic makeup of the system. Fortunately, this doesn't require much manipulation of the organism as a whole but rather modification of the dsz genes. We achieved a significant increase in the flux simply by amplifying the expression of the four proteins coded by the dsz genes. Initially, the biodesulfurization rate was limited by the actual concentration of these proteins. Further increases in activity may be achieved by modifying the proteins themselves, but this has not been reported. Alternative hosts. Another popular strategy in metabolic engineering is to change the host bacteria strain for the genes entirely, perhaps to take advantage of another strains's growth properties, physical properties (for mixing and separations), or a higher intrinsic metabolic rate. We have successfully done this, as has at least one other lab (14). In general, we prefer to stick as close to the original strain as possible, for technical (e.g., gene expression and codon usage preferences) and regulatory reasons. Biodesulfurization processes. Large-scale use of a biocatalyst presents many challenges. Figure 3 illustrates one manifestation of the technology. The process continues to evolve as we learn more about it (15, 16). In this very simple system, the biocatalyst is supplied to a simple stirred-tank reactor, which is also fed oil, air, and a small amount of water. In the reactor, the PASHs are oxidized to water-soluble products, and the sulfur is segregated into the aqueous phase. After leaving the reactor, the oil-water-biocatalyst-sulfur-byproduct emulsion is separated into two streams: the oil (which is further processed and returned to the refinery) and the water-biocatalyst-sulfur-byproduct stream. A second separation is needed to achieve this and allow most of the water and biocatalyst to return to the reactor for reuse.
In our 5-bbl/day pilot plant, we have evaluated several separation schemes, including simple settling tanks, hydrocyclones, membranes, and low-speed centrifuges. A combination of these technologies works the best; and the final configuration will be determined by conditions in the actual refinery, the precise physical properties of the target fossil fuel stream, and the disposition option chosen for the sulfur byproduct.
The waves ahead The future of oil company and government sponsorship of research related to this metabolic pathway and its application to oil and coal desulfurization is cloudy. Like most oil companies, most international agencies have adopted a wait-and-see attitude. DOE has committed money to gasoline desulfurization, but otherwise the commitment to basic research is small. Insights into the basic molecular mechanisms of these novel enzyme-catalyzed reactions would have an effect on bioremediation research, as well as in the academic and industrial biotransformation community, where similar reactions are used to produce chemicals and pharmaceuticals. Skepticism of a new technology is to be expected. The reluctance of conservative oil companies to encourage the development of new ideas is typical of our risk-averse times. Government agencies have many constituencies and priorities, and helping oil companies solve their problems is not high on their lists. We are making small waves in a very large pool. Still, when the technology can be demonstrated at the commercial level and the technology is implemented in refineries instead of new hydrotreating capacity, the next wave of desulfurization development will not be a wave, but a tsunami! Biodesulfurization of fossil fuels will become a reality by manipulating the bacterial system at the molecular level. Our ability to do this is a result of the many tools that have been developed to work at this level. The waves emanating out from the profound advancements in molecular biology over the past 20 years are not limited to developments in medicine but cover all the areas where biotechnology plays (or might play) a role. In nature, enzymes catalyze millions of unique reactions. Many of these are of great commercial importance already (industrial enzymes are a billion-dollar business), and more will be so in the future. Another area ripe for exploration and exploitation in petroleum biorefining is the use of enzymes and cells to catalyze biotransformations of molecules from petroleum into higher value products. The future may see the use of enzymes to catalyze many of the reactions currently run in a petroleum refinery, such as cracking, viscosity reduction, and demetalization. Developments in science and technology often come in waves. Such is certainly the case for biodesulfurization. Each wave has pushed the technology closer to a commercial reality. With luck, this wave will be the last one needed to get biodesulfurization to the marketplace!
Acknowledgements
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