First International Workshop on Biogasification and Biorefining of Texas Lignite

First International Workshop on Biogasification and Biorefining of Texas Lignite

Resources and Conservation, 15 (1987) 229-247 Elsevier Science Publishers B.V.. Amsterdam - Printed 229 in The Netherlands Meeting Report First Int...

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Resources and Conservation, 15 (1987) 229-247 Elsevier Science Publishers B.V.. Amsterdam - Printed

229 in The Netherlands

Meeting Report

First International Workshop on Biogasification and Biorefining of Texas Lignite DONALD L. WISE* Dynatech Scientific Inc., Cambridge, MA 02139 (U.S.A.) (Received


15,1986; accepted October 28,1986)

ABSTRACT The First International Workshop on Biogasification and Biorefining of Texas Lignite was held June 10-11, 1986 at the Woodlands, Houston, Texas. The meeting was sponsored by Houston Lighting and Power Company and involved the participation of selected research workers in an overall project dealing with biotechnology applied to fossil fuels. This innovative concept of university participation has the objective of increasing the potential for utilization of Texas lignite through modern biotechnology. Reports on sponsored research at this meeting were primary focused on methane production. Also included were presentations on the broader potential for a biotechnology-based system for profitable utilization of Texas lignite, i.e., extension to a biorefinery.



William M. Menger (Chief Consulting Engineer, Houston Lighting and Power Company) provided background material for the purpose of this meeting. Houston Lighting and Power (HL&P) is the nation’s seventh largest investor-owned electric utility based on kilowatt-hour sales, and the world’s largest user of natural-gas fuel. This was true in 1985 even though 29% of its fuel requirements were met by burning coal and Texas lignite in its five large generating units designed for these coals. HL&P has a vigorous construction program to build new lignite and nuclear generating units as steps toward reducing its dependence on natural gas and a vigorous research program to find ways to substitute coal and lignite for other of its generating needs. Specifically, it is seeking ways to develop the capability’ of burning coal or lignite in gas-fired boilers and conducting other research into ways of converting coal and/or lignite economically into gaseous fuels. Thermal gasification of coal using high-pressure processes was studied early, closely followed by research in burning dry micronized coal and mixtures of coal and water in boilers. Biological gasification processes for coal and lignite were considered to be a research opportunity which offered potential advantages of lower cost and higher efficiency than the various thermal processes. * Now with: Cambridge 0166-3097/87/$03,50


Inc., Belmont,

MA 02178, U.S.A.

0 1987 Elsevier Science Publishers


The first investigation HL&P did into biological processing of coal was to seek ways of using microbes to reduce the sulphur content prior to burning. This led to a review of the Department of Energy’s (DOE) study of the biological gasification of peat which led to a system-engineering review of the requirements to extend the DOE study to gasification of lignite. The review indicated several weaknesses, or areas of opportunity for improvement, in the process: (1) about 15% of the capital cost of the project was in compressors to convert the final product from atmospheric to a pressure suitable for pipeline use; (2) about 20% of the energy requirements for the process were to operate the compressors; (3) about 20% of the capital cost of the project was in the tankage required for the process to take place; and (4) no microbe had been identified which had successfully converted lignite or lignite extract to methane gas. Based on this work, a “conceptual design” for a lignite refinery was developed. It was called this because a study indicated the possibility that it would produce carbon dioxide and an array of selected organic chemicals in addition to pipeline quality methane. (A patent was applied for in 1984. ) Immediate experimental work is now focused on Texas lignite, although a wide range of other lignaceous materials, such as subbituminous coal and peat, are suitable. The lignaceous structure is first broken down into simple water-soluble aromatic compounds (Stage I) by aqueous alkali pretreatment at temperatures of less than 300” C, well below that required for conventional coal gasification/ liquefaction. Following this, the resulting simple single-ring aromatic chemiicals are either recovered or processed further (Stage II). Anaerobic digestion (Stage II) of the pretreated lignite to produce pipeline-quality gas is under experimental development. In order to assess the feasibility of this process, the conceptual design for a 20,000-Mg/d facility is being examined. Due to its large scale, underground cavern anaerobic digesters are incorporated in the design. Briefly, the salient features of this lignite refinery process are: (1) operates at substantially lower pressures and temperatures than conventional gasification processes, thus reducing capital costs; ( 2 ) from suitable lignaceous feedstocks, produces pipeline-quality methane; and (3) produces liquid fuels and organic chemicals by utilizing lower-rank coals, especially lignite and subbituminous coal, in which the high moisture content is an advantage (rather than a liability, as with other processes). It is to be expected that lower-ranked coals and lignite, naturally having high moisture content and also having substantial volatile matter and less complex structure, will be most appropriate. Rather than compete with present coal conversion processes, it is anticipated that the lignite refinery process should open up new fossil fuel reserves of vital importance to the United States.


Following this overview, technical papers were presented on the projects sponsored by HL&P. A brief summary of the papers is as follows, beginning with the major theme paper. Biological methane production from Texas lignite The major-theme paper was “Biological Methane Production from Texas Lignite”, presented by Alfred P. Leuschner, along with Mark J. Laquidara and Annette S. Martel. (An appendix lists the presentors and their affilitations.) The process involves the microbial conversion of Texas lignite to methane gas [l] by physical/chemical pretreatment followed by microbial fermentation. The process consists of crushing and grinding lignite, chemical pretreatment by additions of alkali and a free-radical initiator at 250°C to solubilize the lignite and reduce the molecular weight of the chemical constituents formed, and subsequent anaerobic fermentation to produce methane. Two types of fermentations are presently being examined. The first is traditional digestion using anaerobes from sewage treatment plants (and other sources). The second is digestion at high salt concentrations using anaerobic organisms from the Gulf of Mexico, the Great Salt Lake, and the Dead Sea. This process is also being examined for a flow rate of about 20 Mg of lignite per day. Evaluation of process economics, for capital and operation and maintenance costs and unit gas cost were presented. As described by Mr. Leuschner, the first step in this process requires that the mined lignite be crushed and ground to a powder. For this experimental program, a particle size of 0.7 mm (or 150 mesh) was selected. Water is then added to produce a 10% total-volatile-solids slurry (wet weight basis) which is then pretreated in a plug-flow reactor operated at short retention times 250°C for 10 to 100 minutes. Alkali and hydrogen peroxide are added. After this treatment, unreacted solids are removed as a low-grade fuel while the solubilized lignite solution undergoes fermentation. Mr. Leuschner noted that this process is being evaluated on the basis of converting 20,000 Mg/d. At a 10% solids slurry this represents a flow of approximately 100 million liters of slurry. Due to the relatively long retention times for the anaerobic fermentation, and the high flow rate, above-ground, steel tanks were ruled out as economically infeasible. Thus, a more economic method of producing large-volume reactors is required. In a report to HL&P, by Fenix and Scisson, Inc. (Tulsa, Oklahoma) [ 21, two underground cavern designs were identified. The first consisted of traditional room and pillar mining to produce a series of packed-bed digesters. The mined rock would be crushed to 2.5-5 cm and replaced in the digester to form a support surface for the anaerobic microorganisms.


The second reactor construction method described would utilize solution mining of a salt dome to create a large volume in what is termed an “inverted morning glory” shape. The anaerobic digester would be operated as a completely mixed reactor. The most severe limitation in operating would be to prevent dissolution of the cavern walls, which if allowed to occur, would cause the cavern to grow and eventually collapse. Fenix and Scisson estimated that if the reactor were operated at 75% of salt saturation, growth of the reactor would be minimized and provide a twenty-year life. However, this restriction means that the anaerobic digestion process would have to be performed at salt concentrations far above known toxicity levels. In order to circumvent this problem, the possibilities of using anaerobic halophiles to perform the fermentation were explored. After digestion the liquid stream is subjected to liquid/solid separation. The solids resulting from this step would be used as a low-grade fuel; the liquid requires disposal. The evolved gas will contain methane and carbon dioxide. Gas purification is used to split the gas into its two components. The methane produced will be of sufficient quality to be injected into a gas pipeline. The carbon dioxide side stream will be sold for use in tertiary oil recovery. Chemical pretreatment of lignite Closely coupled with the above major-theme paper was one on “Chemical Pretreatment of Lignite” by Prof. Hans E. Grethlein, Dartmouth College. He noted that in order to convert a heterogeneous, insoluble material by microbiological means, it is necessary to first solubilize the fuel so that it can pass through the microbial cell walls where it serves as an energy source or nutrient for the cell. For biogasification, a series of synergistic groups of organisms coexist in carrying out hydrolysis, acetogenesis, and methanogenesis. The first step, hydrolysis, is common to many biodegradations and has been studied in some detail for lignocellulose conversion to sugars by cellulase enzymes [ 3-51. To some extent it is a useful model for the study of lignite biogasification. Often the solubilization is the rate-limiting step. The breakdown is facilitated by the presence of the proper extracellular enzyme. When the enzyme has access to a labile bond, and the substrate has a reasonable number of such bonds, then the hydrolysis will result in producing sufficiently small, water-soluble molecules. Naturally, the breakdown products cannot be toxic to any of the many organisms that form this synergistic microbial system. To overcome the enzyme inaccessibility problem, Prof. Grethlein noted that some type of pretreatment of the substrate is needed [ 61. In the case of lignocellulose, for which there are many sources of cellulase, it is clear that the major requirement of a practical pretreatment is to increase the pore size of the microporous substrate to accommodate the relatively large macromolecules, the cellulase enzymes [ 7, 81. For example, native wood has about 0.5


mL of pore volume/g of dry matter, which corresponds to about 500 m’/g. Over 90% of these pores are less than 50 8. Since the enzyme is estimated to be 51 A in diameter, it is clear that the hydrolysis is limited to the external bulk surface area of the lignocellulose. After grinding to 400 mesh, the particles still have less than 1 m2/g external area. Grinding to achieve significant surface area, comparable to 500 m2/g, would mean a reduction to a particle size of 80 A.Fortunately, more practical ways are available to increase the enzyme accessibility. A thermal chemical pretreatment of lignocellulose can remove the easily hydrolyzable hemicellulose, which constitutes about 25% of the dry weight of wood, and greatly increases the porosity of the solid residue. It is important that the removal of hemicellulose is done quickly to avoid subsequent humicsubstance productions from the sugar formed by the thermal chemical hydrolysis of the hemicellulose. The Dartmouth group carries out this pretreatment as follows. In a continuous plug-flow reactor an aqueous slurry of substrate is suspended, a catalyst (such as H,SO, or NaOH) is added which is above the inlet of a Moyno Pump (a moving-cavity positive-displacement type) to pressurize the slurry above saturation for the desired operating temperature. Rapid heating to 180” to 200’ C is achieved by live steam injection from a self-contained electric steam generator. The flow is controlled by a temperature controller proportional to the slurry flow. The reactor is a tube, 12 mm outside diameter, approximately 2 m in length, well insulated. The outside wall temperature is monitored at several points along the length to insure that the reactor is essentially isothermal. The residence time can be varied by adjusting the flow rate and the length of the tube. The current design can operate from 4 to 20 s. A modified design, with larger-diameter reactor, was carried out with 1% H,SO, for 8 s, and the porosity of the residue was progressively increased with a significant increase in the 50 to 100 A region. From the pore-volume distribution, it is possible to estimate the surface area accessible to a 51 A enzyme. The initial rate of enzymatic hydrolysis under a standard test condition of excess enzyme to substrate should be expected to be in proportion to the surface area available to the enzyme. In the case of lignite, Prof. Grethlein indicated that the structure can also be hydrolyzed at carbon-oxygen-carbon bonds with alkali. Since there is not the equivalent of an oxygen bond between every monomer (as in a polysaccharide) , the number of bonds that can be hydrolyzed in lignite are fewer than in cellulose. Thus, a secondary reaction, such as an oxidation of carbon-carbon bonds, is necessary. While the composition of the soluble and residual products from pretreatment of lignite with alkali are presently unknown, it is apparent that the batch reactor, with its long heat-up and cool-down cycle, is not as effective as the flow reactor in producing soluble organic matter. For example, the total dissolved volatile solids (TDVS) changes from 5.9% to 3.9% as the


batch reaction time with alkali increases from 120 to 180 s; the TDVS is as high as 20% for the flow reactor for 60 s. In the case of lignite, with so few oxygen bonds to hydrolyze, it is believed by the Dartmouth group that the best pretreatment would be the one that produces the most soluble, bioconvertable organic matter. However, it remains to be shown what the reactivity of the residual lignite solids is to biogasification, once suitable lignite consuming cultures are established. In the meanwhile, the evaluation of the pretreatment for lignite has proceeded to increase the soluble fraction. Although alkaline hydrolysis gives only a modest increase in soluble matter, the combination of hydrogen peroxide and alkali is potent. It is clear that the flow reactor gives more soluble material than the batch reactor when peroxide is used with alkali. For example, in one run the increase in soluble volatile matter as a percent of the original total volatile matter in the lignite for various combinations of alkali and alkali with H,O, gave more than 77% solubilization. Defining the optimum pretreatment conditions is dependent on the biogasification culture used. To date, the limited testing of biogasification of the pretreated lignite shows that a greater fraction of organic matter is converted to methane for the ultrafiltered soluble matter than for the plain filtered matter. The ultrafiltered matter is all less than 500 molecular weight. Identification and quantitation of lignin-degrading enzymes in fungi As an alternative to chemical pretreatment, the use of enzymes to breakdown lignites was described. Prof. J.K. Marquis and Dr. B. Gallo of Boston University discussed the “Identification and Quantitation of Lignin Degrading Enzymes in Fungi”. Prof. Marquis noted that Cohen and Gabriele in 1982 [ 91 first reported that the fungus Polyporus uersicolor (or Trametes uersicolor) grew on pieces of lignite, but they did not establish that the degradation of lignite occurred enzymatically or that specific digestive enzymes were produced. They concluded that white-rot decay was based on the production of peroxidase enzymes, but the mechanism and biochemical pathways of lignin biodegradation were not specifically defined. Prof. Marquis also noted that in 1983, Tien and Kirk [lo] reported discovery of a lignin-degrading enzyme from the basidiomycete Phanerochaete chrysosporium. Their ligninase is extracellular and requires HzOz for activity. Subsequently, Michael Gold and his colleagues in Oregon [ 111 purified a ligninase or, more accurately, a family of ligninases, and began to characterize the enzyme proteins from the white-rot basidiomycete P. chrysosporium. They partially purified a peroxide-requiring diarylpropane oxygenase with a molecular weight of about 41,000 and containing a heme moiety. Synthetic substrates, such as veratryl alcohol, are oxidized to aldehydes which are readily identified by thin-layer chromatography (including anisaldehyde, benzalde-


hyde, and veratraldehyde) . Furthermore, they demonstrated that the ligninase per se is really a family of enzymes, i.e., an enzyme that exists in multiple molecular forms, and that at least three of these forms are glycoproteins. Also, not all of these enzyme forms are peroxide-dependent. It is particularly important to note that these multiple glycoproteins differ significantly in the kinetics of binding to lectins such as concanavalin A (carbohydrate-binding compound) and may represent an unusual case where the carbohydrate portion of a glycoprotein has some modifying effect on the enzyme function. All of this work is preliminary enzymology and has been focused on the single fungal organism (P. chrysosporium) . Prof. Marquis pointed out that recently, Kirk and his colleagues at Repligen Corporation (Cambridge, Massachusetts) identified two major research needs in the study of ligninase: (1) To identify the full complement of enzymes needed to degrade lignin to assimilable fragments; the ligninases alone are insufficient. The complexity of the system, particularly the interaction and interdependence of multiple enzymes, is of great interest. At the moment this Boston University group is working with several culture filtrates. The primary enzyme assay is the measurement of veratryl alcohol oxidation to veratraldehyde which is readily quantitated at 310 nm. Furthermore, these workers achieved a lOO-fold purification of ligninase activity by concanavalin A affinity chromatography, a technique which is not selective for the various molecular forms of the enzyme. (2 ) To maximize the production of ligninase so that their practical potentials can be assessed, growth of four fungal strains will be studied; Pork placenta was chosen as a non-ligninase producing control. In the next year, the Boston University group will continue to work toward both these goals, i.e., production of ligninase and characterization of the enzyme complex required for degradation of lignin and lignite. Anaerobic metabolism of aromatic compounds The anaerobic metabolism of aromatic compounds was discussed by J.D. Haddock and J.G. Ferry (Virginia Polytechnic). This research relates to the anaerobic microbial degradation of aromatic compounds found in pretreated Texas lignite to products that support the production of methane gas. The goals of the research are: (1) to determine the properties and mechanisms of action of key enzymes involved in the anaerobic degradation of monoaromatic substrates; and ( 2 ) to relate this information to the physiology of the organism in order to gain insight into the interaction between the organism and its environment. In order to achieve the overall objectives of this study, it was necessary to obtain a microorganism capable of the degradation of aromatic compounds under anaerobic conditions. Even though anaerobic aromatic degradation had been demonstrated as early as the early 1930’s [ 121, few organisms have been described in pure culture with that capacity. The described


organisms span a wide range of anaerobic physiological types including photosynthetic nonsulfur purple bacteria [ 13-151. The latter group is considered most appropriate for this project, since only fermentation of aromatic substrates would be expected to produce degradation products that might be subsequently converted to methane. Fermentative end products known to support methanogenesis include acetate, formate, COZ, and H,. Photosynthetic nonsulfur purple bacteria utilize organic compounds as a carbon source for growth [ 161 while the nitrate- and sulfate-respiring organisms would be expected to mineralize aromatic compounds to COP, and to compete with methanogens for substrates such as H,. Mr. Haddock noted that few anaerobic bacteria have been described in pure culture that utilize fermentative pathways to degrade aromatic compounds. Tsai and Jones [ 171 isolated and described a Coprococcus sp. and a strain of Streptococcus bouis from the rumen that degraded phloroglucinol (1,3,5-trihydroxybenzene) anaerobically. An isolate of the former species, strain Pe151q2, was shown to ferment phloroglucinol to acetate and CO, [ 181. Prior to those reports, fermentation of aromatic compounds had only been demonstrated in mixed microbial populations derived from sources such as anaerobic sewagesludge digestor [ 191. Recently, additional aromatic fermenting organisms have been isolated from the rumen [ 201 and freshwater and marine sediments [ 211, but the list is still small. With the above considerations in mind, this group developed a two-pronged approach to the research problem. The most immediate goal was to obtain a pure culture of a fermentative organism that could easily be maintained and mass-cultured in order that sufficient cell material would be available for enzymatic studies. Therefore, pure cultures of two of the described aromatic-ferPelobacter acidigallici strain MaGal[ 211 and menting organisms, Eubacterium oxidoreducens strain G-41 [ 201 were obtained from the authors. The P. acidigallici strain was chosen because of its reported short lag phase and lack of clumping during growth. In addition, strain MaGalwas a marine organism which might be adapted to the high salt environment being considered. The second line of research undertaken involved attempts to isolate new organisms capable of fermenting aromatic compounds to methanogenic substrates. Anaerobic enrichment cultures were established to provide a source from which isolation could be attempted. Besides pretreated Texas lignite as an enrichment substrate, benzoic acid and gallic acid have been shown to be a substrate suitable for the isolation of aromatic fermenters. Isolation and characterization sediments of the Dead Sea

of novel anaerobic halophilic bacteria from

A paper on the “Isolation and Characterization of Novel Anaerobic Halophilic Bacteria from Sediments of the Dead Sea”, was given by Dr. Aharon


Oren (Hebrew University of Jerusalem). Dr. Oren indicated that he was studying the microbiology of anaerobic sediments of the Dead Sea by isolating and characterizing the bacteria present. The ultimate goal is to examine whether there exists a potential for breakdown of lignite by anaerobic halophilic microorganisms. Dr. Oren noted that two obligatory anaerobic chemoorganotrophic moderately halophilic bacteria have been isolated in the past from the bottom sediments of the Dead Sea: Hulobacteroides halobius, a long, motile rod, and Clostridium lortetii, a rod-shaped bacterium producing endospores with attached gas vacuoles [ 22, 231. He also included in his studies Haloanaerobium prae&ens, a rod-shaped halophilic anaerobic isolated by Zeikus and coworkers from sediments of the Great Salt Lake, Utah. Dr. Oren has set up enrichment cultures for anaerobic halophilic bacteria, using a variety of substrates, and using anaerobic hypersaline sediments from two sites on the shore of the Dead Sea: black mud from the shore near Massada, and mud from a sulfur spring near Ein Gedi. Enrichments inoculated with the latter yielded moderately halophilic facultatively anaerobic bacteria such as Vibrio costicola, while from the first source a new spore-forming motile rodshaped bacterium was isolated, designated strain DY-1. Hulobacteroides halobius and strain DY-1 ferment carbohydrates such as glucose, fructose, sucrose, and starch to products such as ethanol, acetate, butyrate, HP, and C02. The growth requirements of Clostridium lortetii have not been well characterized; it grows in a rich medium containing amino acids and yeast extract, and produces acetate and other acids [ 24, 251. The Great Salt Lake isolate Huloanaerobium praevalens ferments carbohydrates, peptides, amino acids, and pectin to acetate, propionate, butyrate, C02, and HP. Attempts by Dr. Oren to isolate sulfate-reducing bacteria from sediments of the Dead Sea remained without success, though indications of biological sulfate reduction in Dead Sea sediments exist; halophilic methanogens, though not yet found in the Dead Sea, have been described from other hypersaline environments. All obligately anaerobic halophiles isolated thus far are moderately halophilic, with optimal NaCl concentrations between 1 and 4-M, and all are mesophiles, growing best at temperatures between 35 and 45 ‘C. The four obligately anaerobic halophilic bacteria examined (Hulobacteroides halobius, Clostridium lortetii, the spore-forming strain DY-1, and HuZoanaerobium praevalens) are closely related; they are gram-negative, share a low percentage of guanine and cytosine in their DNA, and analysis of their 16s rRNA shows they are related to each other, but unrelated to any of the other subgroups of the eubacterial kingdom, to which they belong. Physiology and biotechnology of halophilic anaerobes for application to Texas lignite A paper closely related to Dr. Oren’s, entitled Physiology and Biotechnology of Halophilic Anaerobes for Application to Texas Lignite”, was given by Sirirat


Rengpipat and Prof. J. Gregory Zeikus (Michigan State University). Dr. Zeikus noted that a variety of halophilic anaerobes were enriched from hypersaline environments by selecting for bacterial strains that grew readily on inexpensive nutrients and which made either exopolysaccharides, or made organic acid salts and alcohols as catabolic end products during the hydrolysis of complex organic matter. One prolific species, Hulobacteroides acetoethylicus sp. nov., was isolated from deep subsurface brine waters associated with an injection water filter on an offshore oil rig in the Gulf of Mexico. This species is used as a model biocatalyst to understand how chemoorganotrophic anaerobes adapt to extreme salt stress and to understand how to bioengineer haloanaerobes for technology applications. Studies to date imply that chemotrophic haloanaerobes, unlike aerobic Halobacterium species, do not actively expend ATP to prevent the internal accumulation of sodium or to synthesize an anabolic osmoregulant. Rather, species proton motive force and catabolism are metabolically controlled in relation to an optimal internal sodium concentration that is in dynamic equilibrium with environmental hypersalinity. The impact of this finding and the physiological properties of H. acetoethylicus were discussed in relation to strain improvements needed to develop an industrial fermentation of complex organic matter such as pretreated Texas lignite in an anoxic, hypersaline bioreactor system. Extremely thermophilic bacteria from deep-sea geothermal sediments with emphasis on fermentative gasification of lignaceous compounds Continuing with the interest in microorganisms for bioconversion of lignaceous compounds, Dr. Holger W. Jannasch (Woods Hole) discussed, “Extremely Thermophilic Bacteria from Deep-Sea Geothermal Sediments with Emphasis on Fermentative Gasification of Lignaceous Compounds”. Dr. Jannasch noted that at the tectonic ocean spreading center of the Guaymas Basin (Gulf of California) at a depth of about 2,000 m, active hot vents are overlayed by several hundred meters of terrigenous, rapidly depositing sediments containing high concentrations of thermogenic organic matter of diatomacous origin. At certain spots, these sediments reach temperatures of 160°C in a depth of only 60 cm below the 3 to 4°C sediment-surface/bottom-water interface. The thermally altered organic materials are represented by a wide range of bituminous and sub-bituminous materials which, in the upper sediment layers, are mixed with young photosynthetically produced organic deposits. The presence of metalliferous brines of the structurally continuous Salton Sea Trough has been speculated. This general geological-geochemical situation appears to be highly promising for the presence of a variety of physiological types of thermophilic and extremely thermophilic, archaebacterial or eubacterial microorganisms. From Dr. Jannasch’s last deep-ocean diving cruise, core material (stored


frozen, refrigerated, and at room temperature) is available for enrichment experiments to be set up for the ultimate isolation of pure cultures. Three sets of variables will give rise to several hundred combinations of culture conditions: (1) subsamples from different core locations and in situ temperatures; (2) different composition of enrichment media; and (3) enrichment temperatures from ca. 50 to 140°C. Incubations at temperatures beyond 100°C will be done in pressurizable culture tubes. They will be suspended in silicone-oilfilled reactor vessels which can be pressure and temperature controlled. A variety of fermentation substrates (e.g., aromatic compounds, cellulose, lignites, etc.) and salinities will be chosen in consultation with other colleagues of this University Participation Program. Ultimate classification and complete taxonomic description of isolates will be done in cooperative efforts. Biotreatment for organic sulfur removal Another important research topic being explored on the HL&P project is that of “Biotreatment for Organic Sulfur Removal”. This work was described by Dr. Oliver Peoples and Prof. Anthony J. Sinskey (Massachusetts Institute of Technology). Prof. Sinskey pointed out that this project is designed to examine the biochemistry of the removal of sulfate groups from biopolymers, in this case model polysaccharides, by using recombinant DNA technology. Flavobacterium heparinum, a gram-negative bacteria, which can grow on heparin, produces several sulfatases as components of the system for degrading heparin, heparatin sulfate, and chondroitin sulfate. Sulfatases with varied specificities have been successfully purified and characterized from this organism. Prof. Sinskey proposes to isolate the genes encoding these sulfatases by clonand their subsequent use to design overing them in E. coli. Characterization production systems for sulfatase enzymes will follow. An abundant supply of the enzymes, previously only purified in analytical quantities, will enable Prof. Sinskey to carry out broader studies on their enzymatic mechanisms and applications to other sulfated polymers. Prof. Sinskey noted that an important target for biotechnology is the manipulation of microorganisms to facilitate the removal of sulfur groups from organic matter in coals, lignites, petroleums, and biopolymers such as heparin, chondroitin sulfate, and carrageneen. The enzymatic removal of sulfur from polymers in general is a poorly understood phenomenon. However, a number of bacterial carbohydrate sulfatases have been investigated at the biochemical level (for a review, see Ref. [ 261) . Prof. Sinskey believes that the time is ripe to expand this area of research to the molecular level, a process which should be greatly facilitated by the use of recombinant DNA technology. As a model system, the sulfatases from Flavobacterium heparinum have been chosen. There are two main reasons for this choice: first, the organism is a gram-negative facultative anaerobe and produces several different sulfatases;


second, there is a certain amount of biochemical information on these enzymes and three of them have been purified to homogeneity. F. heparinum produces enzymes which degrade the sulfated polysaccharides heparin, heparan sulfate, and chondroitin sulfate. The degradative enzymology for heparin, a poly-dispersed, sulfated polysaccharide of (l-4) linked glucosamine and uranic acid residues, is the best characterized [ 271. First, the enzyme heparinase acts endolytically to cleave a specific subset of glycosidic bonds producing a predominant trisulfated disaccharide and a mixture of tetrasaccharides, hexasaccharides, and higher oligosaccharides. Subsequent steps in the breakdown of the disaccharide involves removal of the 2-O-sulfate from the glucuronic acid moiety followed by cleavage of the glycosidic bond to produce glucuronic acid and glucosamine-2,6-disulfate. Finally, the enzymes sulfamidase and 6-0sulfatase remove the 2-N- and 6-O-sulfate groups to form glucosamine and inorganic sulfate. These three sulfatases are also probably involved in the removal of sulfate groups from the breakdown products of heparan sulfate. Heparin also contains 3-O-sulfate groups [ 281 and the corresponding sulfatase has been identified [ 291. Both heparinase and the sulfatases are regulated at the genetic level, being induced by heparin or the products of heparinase, and repressed by inorganic sulfate [ 301. The 2-0-sulfatase, 6-0-sulfatase, and 30-sulfatase have been purified to homogeneity [ 29,31,32]. Limited data are available for the sulfamidase [ 331. Mechanism of anaerobic degradation of aromatic compounds in Texas lignite by bacteria Work on the “Mechanism of Anaerobic Degradation of Aromatic Compounds in Texas Lignite by Bacteria” was discussed by G. Fuchs, A. Kroger, and R.K. Thauer (all from F.R.G. ) . Briefly, this work deals with: (1) aromatic compounds in Texas lignite as substrates for anaerobic bacteria; (2) isolation of bacteria capable of anaerobic growth on aromatic compounds in Texas lignite; and (3) studies on the biochemistry of degradation of the aromatic compounds by the isolated bacteria. It was noted by Dr. Fuchs that aromatic compounds can be degraded biologically under aerobic or anaerobic conditions. While the mechanisms of aerobic degradation have been thoroughly studied [ 341, there are only few reports on the anaerobic pathways, which differ from the aerobic processes in that oxygenase reactions are not involved [ 14,15,35-441. Anaerobic bacteria are known that oxidize aromatic compounds with protons ( H2 production), sulfate ( H$ production), or nitrate ( N, production). It is generally assumed these bacteria use a common pathway of oxidation, irrespective of the electron acceptor. With protons as electron acceptor, the anaerobic oxidation of aromatic compounds is exergonic only, if the partial pressure of H, is kept low. Therefore, these bacteria require the presence of HZ-utilizing anaerobes for growth (methano-

241 TABLE 1 Elemnetal


of Texas lignite Typical coal

% by weight C H 0 N residue Atomic ratio C H 0 Chemical summation formula

Texas raw lignite”

Texas pretreated

45.2 3.1 17.1 1.0 32

52.5 3.9 23.1 0.9 19

3.75 3.7 1.07

4.38 3.9 1.44


Average lignite

50.7 4.4 12.9 1.0 32 4.22 4.4 0.81

“Dried at 2 mbar, 25’ C.

gens, acetogens, or sulfate reducers). These mixed cultures are not well suited for studying the biochemistry of the process. In contrast, with nitrate of sulfate, pure cultures growing on aromatic compounds can be obtained. These workers isolated nitrate-reducing bacteria growing with monocyclic aromatic compounds considered as model compounds of the constituents of pretreated Texas lignite. It is also to be noted that raw and pretreated Texas lignite has been analyzed with respect to elemental composition and molecular weight: the chemical summation formula of the treated lignite is approximately C7H702, as shown in Table 1. Process systems for solubilized lignite

Concluding the workshop was a review on process systems available for bioconversion of solubilized lignite by Mr. Don Lapin and Prof. Jack V. Matson (University of Houston). They noted that the effectiveness of anaerobic cultures in large-scale conversion of Texas lignite to methane depends on reactor configuration and biological kinetics. To evaluate design alternatives, a set of six 7 to 8 L pilot reactors are being prepared. The system design provides for continuous pretreated lignite supernatant/filtrate feeding to and gas/effluent collection from the reactors. Of the six reactors, two are upflow packed beds ( anaerobic filters) ; two are upflow anaerobic sludge blanket (UASB ) types; one is an expanded bed partly fluidized by recirculation, and one is baffled. The packing media is sieved lignite, 6.3 to 9.5 mm. Detection time is variable from less than one hour to more than 133 h. Supplemented with substrate


characterization studies, the pilot reactor studies will optimize conditions for lignite bioconversion. The investigation focuses on reactor configuration and conditions for the anaerobic conversion of solubilized lignite feedstock into methane, “Stage II” in the biogasification of Texas lignite proposed by Leuschner [ 11. In Stage I of this process (pretreatment), alkaline hydrolysis cleaves the complex benzene ring structure of the lignite into aqueous aromatic acids, mostly benzene carboxylic acids. These compounds then can be degraded biologically under anaerobic conditions. Several anaerobic reactor (fermenter) types are applicable for the two site locations discussed by Leuschner [ 11, underground rock caverns and underground salt domes. Reactor costs are likely to influence process economics significantly; thus, an evaluation of reactor design for the lignite conversion process is warranted. Mr. Lapin pointed out that unlike aerobic biological processes, methaneproducing anaerobic fermenters require more than one species of bacteria. Of necessity, the anaerobic cultures within a fermenter represent a symbiotic association of at least three different bacterial types in four separate steps [ 411. Hydrolyzing organisms convert complex organics to simple organic compounds (sugars, amino acids, fatty acids) in the first step. For example, cellulose would be converted to sugar. Fermenting organisms, or “acid farmers”, convert the simple organic materials to organic acids, hydrogen, and carbon dioxide. Organic acids formed during fermentation are converted separately to acetate (“acetogenesis”). In the fourth step, methanogenic organisms reduce carbon dioxide to methane and water (“hydrogenotrophic” methanogens) and decarboxylate acetate to methane (“acetoclastic” methanogens) . This association of microorganisms requires that seed material to start an anaerobic reactor be a mixed culture. Mr. Lapin stated that some twenty different species of methanogens are known; fifteen or so reduce carbon dioxide and two or three decarboxylate acetate. One or two species are thought to perform both functions. Methanogens are primitive organisms, obligate anaerobes that rely on the facultative hydrolyzers and fermenters to scavenge toxic oxygen from the reactor system. Further, the slow growth rate of methanogens usually is the rate-limiting step in biomethanation systems. A slug dose of complex organic substrate can accelerate the faster-metabolizing acid formers, resulting in a net accumulation of acid that may “pickle” the reactor. Consequently, pH and volatile-acid monitoring in a reactor are important. Alkalinity must be added to buffer local pH variations. Since the acid formers tend to accumulate near the bottom of an upflow reactor, near the substrate inlet, the pH and volatile-acid profiles dip and bulge (respectively) at this location. For high organic loadings, effluent recycle may be an appropriate measure to spread the conversion process more evenly through the reactor and to dilute the influent. Another effect Mr. Lapin called attention to relates to fermenter hydrogen


concentrations. The standard free energy ( AG"(w ) ) of many hydrogen-producing reactions are positive (for example, ethanol hydrolysis, benzoic acid fermentation, or propionate acidogenesis) . To obtain energy for growth, the organisms effecting these conversions depend on the methanogens to reduce the partial pressure of hydrogen to very low levels. As an example, for propanoic acid conversion, it may be noted that ( lop6 atm < P ( HZ) < 10e4 atm) is necessary for the dG of the acetogenesis/methanogenesis reaction couple to be negative, i.e., so that the reaction will proceed. With hydrogen concentration so low, little energy is available to the hydrogenotrophic methanogens, resulting in very low biomass yields for complete biomethanation of propionate and other organic acids [ 45,461. Experimental studies have verified this observation. Methanogenic associations also feature an inherently low endogenous decay rate; the biomass diminishes very slowly, even in the absence of substrate. Accordingly, a reactor design that retains high biomass concentrations during flow interruptions, and which provides good mixing of substrate and solids when flow resumes, can be expected to perform well under load fluctuations and periods of dormancy. CONCLUSIONS

This First International Workshop on Biogasification and Biorefining of Texas Lignite was useful for several reasons. Foremost, while experimental results are clearly preliminary, every indication is that biotechnology-based processing of selected fossil fuels such as Texas lignite in every respect merits further attention. Pretreatment of the fossil fuel by either chemical (alkaline) or biological (enzymes) means appears to be essential. However, once lowermolecular-weight compounds (especially single-ring aromatic compounds) are obtained, bioconversion to methane appears to be practical. Organic chemicals such as acetic acid may also, it appears, be a commercial product from this overall biorefinery. Due to the bold plans to carry out the bioconversion in large underground salt caverns, the work on halophilic microorganisms appears to have much promise. Further, the potential for organic sulfur removal via biotechnology-based processing appears to have much potential. ACKNOWLEDGEMENT

The author wishes to express his appreciation to William M. Menger, Chief Consulting Engineer, Houston Lighting and Power Company, for his foresight in initiating work in biotechnology applied to fossil fuels and for his sponsorship of this meeting.



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A complete

list of presentors,

in order of presentation,

Mr. William M. Menger Chief Consulting Engineer, Houston Lighting and Power Company Houston, Texas 77001 Mr. Alfred P. Leuschner Dynatech Scientific, Inc. 99 Erie Street Cambridge, Massachusetts


Prof. Hans E. Grethlein Thayer School of Engineering Dartmouth College Hanover, New Hampshire 03755 Prof. J.K. Marquis and Dr. B. Gallo Boston University Boston, Massachusetts 02118 J.D. Haddock and J.G. Ferry Department of Anaerobic Microbiology Virginia Polytechnic and State University Blacksburg, Virginia 24061 Dr. David A. Odelson Department of Microbiology and Immunology Medical College of Virginia Richmond, Virginia 232980001

is as follows.

Dr. Aharon Oren The Division of Microbial and Molecular Ecology The Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem 91904, Israel

Sirirat Rengpipat and Prof. J. Gregory Zeikus Michigan Biotechnology Insitute Department of Microbiology Michigan State University East Lansing, Michigan 48824

Dr. Holger W. Jannasch Woods Hole Oceanographic Institution Woods Hole, Massachusetts

Dr. Oliver Peoples and Prof. Anthony J. Sinskey Department of Applied Biological Sciences Massachusetts Institute of Technology Cambridge, Massachusetts 02139


G. Fuchs Angewandte Mikrobiologie University of Ulm D-7900 Ulm, F.R.G. and A. Krijger Institut fur Mikrobiologie Fachbereich Biologie, J.W. Goethe-University D-6000 Frankfurt/Main, F.R.G. and R.K. Thauer Mikrobiologie, Fachbereich Biologie University of Marburg, Lahnberge, D-3550 Marburg, F.R.G.

Mr. Don Lapin and Prof. Jack V. Matson Environm.ental Engineering Program Department of Civil Engineering University of Houston Houston, Texas 77004 Project coordinator

Donald L. Wise, Ph.D., P.E. President Cambridge Scientific, Inc. Executive Offices 195 Common Street Belmont, MA 02178, U.S.A.