Energy & Fuels 1993, 7, 380-383
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Microbiological Analysis of Argonne Premium Coal Samples J. Kevin Polman,’ Cynthia R. Breckenridge, and Karen M. Delezene-Briggs Biotechnology and Environmental Sciences Group, Idaho National Engineering Laboratory, EG&G Idaho, Inc., Idaho Falls, Idaho 83415-2203 Received October 23, 1992. Revised Manuscript Received February 17, 1993
The microbial content of different lots of three Argonne Premium Coals (North Dakota Beulah Zap lignite, Wyodak subbituminous, and Illinois No. 6 bituminous) was analyzed using 28 kinds of growth medium incubated under aerobic, microaerobic, and anaerobic conditions. This work was initiated for the purpose of isolatingmicroorganismsthat naturally occur in coal, with the presumption that they would be valuable for coal bioconversion technology. The types of microorganisms that were targeted for cultivation included aerobes, microaerophiles, facultative and strict anaerobes, fungi, sulfate-reducing bacteria, methanogenic bacteria, and chemoheterotrophs. Media included nutrient agar and broth, yeast/mold agar and broth, lactate medium, Bacto anaerobic agar, triple sugar iron agar, selenite broth, Eugon broth, Brewer modified thioglycollate medium, thioglycollate medium without dextrose,and 11different methanogen enrichment media containingvariousprimary carbon sources. After a lengthy incubation period, growth was determined by turbidity and/or production of methane. Results indicated that no viable microorganisms were derived from the coal samples. Apparently these coals are rather pristine with respect to the presence of either living or dormant aerobic and anaerobic chemoheterotrophs, and methanogens. While the growth media employed for enrichments did not target all possible microorganisms, it is clear that these coals contain little or no viable biomass as part of their physical structure. These observations are interesting and surprising from an ecologicalperspective,and they also increase our knowledgeabout the physical composition of coal. Introduction Various effects of microorganisms and enzymes on coal have been documented.14 These include alkali-induced coal solubilization,u chelator-induced solubilization,g-ll coal desulfurization,12-14utilization of coal mobile phase components as growth substrates,’”17 and depolymeri-
* Corresponding author.
Phone: 208-526-9598. FAX: 208-526-0828. (1) Faison, B. D. Crit. Reu. Biotechnol. 1991, 11, 347-366. (2) Catcheside, D. E. A.; Mallet, K. J. Energy Fuels 1991,5,141-145. (3) Scott, C. D.; Strandberg, G. W.; Lewis, S. N. Biotechnol. Bog. 1986,2, 131-139. (4) Faison, B. D. Bioltechnol. 1991, 9, 951-956. (5) Gupta, R. K.; Spiker, J. K.; Crawford, D. L. Can. J. Microbiol. 1988, 34, 667-674. (6) Quigley, D. R.; Ward, B.; Crawford, D. L.; Hatcher, H. J.; Dugan, P. R. Appl. Biochem. Biotechnol. 1989,20121,735-763. (7) Runnion, K.; Combie, J. D. Appl. Biochem. Biotechnol. 1990,241 25, 817-829. (8) Quigley, D. R.; Wey, J. E.; Breckenridge, C. R.; Stoner, D. L. Res. Conseru. Recycl. 1988, I , 163-174. (9) Cohen, M. S.; Feldman, K. A,; Brown, C. S.; Gray, E. T., Jr. Appl Enuiron. Microbiol. 1990,56, 3285-3291. (10) Fredrickson, J. K.; Stewart, D. L.; Campbell, J. A.; Powell, M. A.; McMulloch, M.; Pyne, J. W.; Bean, R. M. J. Ind. Microbiol. 1990, 5, 401-406. (11) Quigley, D. R.; Breckenridge, C. R.; Dugan, P. R. Energy Fuels 1989,3,57i-574. (12) Stoner, D. L.; Wey, J. E.; Barrett, K. B.; Jolley, J. G.; Wright, R. B.: Dugan, P. R. Appl. Enuiron. Microbiol. 1990,56, 2667-2676. (13)-Johnson, D:B. Processing and Utilization of High-Sulfur Coals IV; Elsevier Science Publishing Co.: New York, 1991; pp 567-577. (14) Eligwe, C. A. Fuel 1988,67,451-458. (15) Polman, J. K.; Breckenridge, C. R.; Dugan, P. R.; Quigley, D. R. Appl. Biochem. Biotechnol. 1991,28129,487-494. (16) Ralph, J. P.; Catcheside, D. E. A. Proceedings: 1991 Second International Symposium on the Biological Processing of Coal; Electric Power Research Institute: Palo Alto, CA, 1991; pp 4/57-4/71. (17) Polman,J. K.; Breckenridge, C. R.; Dugan, P. R.; Quigley, D. R. Proceedings: 1990 First International Symposium on the Biological Processing of Coal; Electric Power Research Institute: Palo Alto, CA, 1990; pp 4/62-4172.
zation and polymerization of coal macromolecules.la21 Overall, our research is directed toward the biological conversion of coal to liquid fuels that have less tendency to produce pollution upon combustion. One of our primary goals is to bring about the biologically-catalyzeddepolymerization of macromolecular coal to the extent that low molecular weight fuels, such as ethanol, are produced. This bioprocesswould be analogousto the biologicalconversions of such macromolecular substrates as lignocellulose and starch to fermentation products, e.g., ethanol and acetic acid.22An approach to discoveringmicroorganismswhich are capable of extensive depolymerization of coal is to isolate microbes that are associated with coal seams in natural ecological settings. Such microorganisms, probably having been associated with coal for a considerable time, would likely be utilizing some portion of the coal organic carbon for survival; they might also be resistant to any toxic effects of coal. The Argonne Premium Coal Sample collection is a good source of coal seam samples for which the integrity of the original coal samples has been preserved.23 The samples are derived from subsurface coal seams, are collected and (18) Wondrack, L.;Smnto,M.; Wood, W. A. Appl.Biochem.Biotechnol. 1989,20121,765-780. (19) Gupta, R. K.; Deobald, L. A,; Crawford, D. L. Appl. Biochem. Biotechnol. 1990,24/25,1-6. (20) Polman, J. K.; Breckenridge, C. R.; Quigley, D. R. Proceedings: 1991 Second International Symposium on the Biological Processing of Coal; Electric Power Research Institute: Palo Alto, CA, 1991; pp P/63P177. (21) Wyza, R. E.; Desouza, A. E.; Isbister, J. D. Proceedings, Biological Treatment of Coals Workshop;Idaho National Engineering Laboratory: Idaho Falls,ID, 1987; pp 119-132. (22) Leeper, S. A.; Andrews, G. F. Appl. Biochem. Biotechnol. 1991, 28129,499-511. (23) Vorres, K. S. Energy Fuels 1990,4, 420-426.
0887-0624/93/2507-0380$04.oo/o 0 1993 American Chemical Society
Energy & Fuels, Vol. 7, No.3, 1993 381
Microbiological Analysis of Argonne Premium Coal
transported with the objective of preserving the anaerobic nature of the original samples, and are ground, homogenized, and packaged with the intention of preserving the original state of the coal in ita natural seam. Accordingly, these samples should contain good indicators of the natural microbial communities of subsurface coal seams. Claimshave been made that Argonnecoals contain viable organisms.23 If such is the case, the presence of microorganisms, and consequently metabolic activity, might contribute to instability of the coal samples. This would pose a problem for the Argonne Premium Coal Program since stability of these samples for the purposes of repeatable coal chemistry research is the cornerstone of the program. However, even if these coal samples do contain microorganisms, one cannot conclude that this resulta in excessiveinstability of the samples in the absence of data concerning the frequency of occurrence of these microorganisms. Infrequent occurrence of microbes in coal would imply that viable microbes make up very little of the physical composition of coal and thus would be an insignificantfactor in terms of coal stability. We examined three representative Argonne coals for the presence of microbes using a multitude of different media. We carefully employed stringent aseptic technique to be certain that any microorganisms arising in cultures were actually derived from the coal samples.
Table 1. Microbiological Media Utilieed for Coal Analyses medium acronym medium components NA nutrient ag@ nutrient broth" NB 0.5 X NB 0.5X strength nutrient broth 0.1x strength nutrient broth, basal saltsb 0.1 X NB/BS 0.1% glucose (dextrose),basal saltsb GM BAA Bacto anaerobic a g e BrAA Brewer modified anaerobic a g e BMT Brewer modified thioglycollatebrothC TGw/oG thioglycollatemedium without dextrosea 0.5 X TGw/oG 0.5X strength TGw/oG 0.1 X TGw/oG 0.M strength TGw/oG triple sugar iron a g e TSIA SB selenite brotha LM sodium lactate, basal salts, yeast extractd yeasts/molds a g e YMA yeasts/molds brotha YMB Eugon broth" EB basal salts: 1% SATe AMM basal salts: 0.15% NazC03.10H20 CMM basal salts: 1% methanol MMM AMM, vitamind AMMv CMM, vitamind CMMv MMM, vitamind MMMv 1 X NB/AMM 0.8% nutrient broth, 1%S A P 0.6 X NB/AMM 0.6% nutrient broth, 1%SATe 0.3 X NB/AMM 0.3% nutrient broth, 1%SATe 0.1 X NB/AMM 0.1% nutrient broth, 1%SATe NB/A/BSE/MM basal salts! 0.2% nutrient broth, 1%SATe a
Experimental Section Microbiological Media and Coals. All media utilized are described in Table I. Many of these growth media are standard for microbial cultivation, were obtained from commercial sources (Table I), and have been shown to foster excellent growth of appropriate microorganisms. Several different lots of Argonne coals were tested for the presence of microorganisms, and these coals were procured from the Argonne Premium Coal Sample Program.23 In all cases, one enrichment culture was prepared per coal (or coal mixture) per medium type. Strict Anaerobic Enrichments. Argonne Premium Coal Sample bottles of Illinois No. 6 bituminous, Wyodak subbituminous, and North Dakota Beulah Zap lignite (5 g each, -100 mesh) were surface sterilized with 95% ethanol. The bottles were broken open in air with a flame-sterilized spatula, and coal was transferred to a sterile vial. The vials were immediately transferred to an anaerobic chamber (atmosphere 90% nitrogen, 5 % hydrogen, 5% carbon dioxide; chamber was equipped with an oxygen-scavengingpalladium catalyst system) and mixed with 100 mL of sterile anaerobic demineralized water. Strict anaerobes, such as Clostridium acetobutylicum, that require media with low redox potentials, are routinely cultured in this chamber. A drop (50-100 pL) of coal suspension was added to each culture vessel. Coal was visibly present in the drop of suspension and, assuming uniform suspension of coal particles, 2.5-5 mg of coal went into each culture. Cultures were incubated at 24 OC, stationary. Growth was monitored by repeatedly checking for changes in turbidity of broth cultures, or presence of colonies on plates, over a month-long incubation. AerobicIMicroaerobic Enrichments. Argonne coal samples were removed from vials as previously described, and mixed with 10 mL of water. Coal mixes were used to inoculate media as described for strict anaerobic enrichments. Broth tubes were incubated a t 37 "C, stationary (microaerobic conditions), and plates were incubated a t 22 OC (aerobic conditions). Growth was monitored as previously described. Anaerobic Enrichments for Methanogens. Methanogen media (2 mL) were dispensed to serum bottles (18 mL). Culture vessels were transferred to an anaerobic chamber (as described above). Equal amounts (10g) of Wyodak, Beulah Zap, and Illinois
DZFCO Manual of Dehydrated Culture Media and Reagents
for Microbiology, 10th ed.; DIFCO Laboratories: Detroit, MI. Reference 15. BBL, Becton Dickinson Co., Cockeysville, MD. Krieg, N. R. Manual of Methods for General Bacteriology; American Society for Microbiology: Washington, DC, 1981;pp112-142. e SAT, sodium acetate trihydrate. f Vitamin supplements included the following (final concentrations, % w/v, in parentheses): thiamine (0.OOO 05); biotin (0.OOO 02); B-12(0.OOO 02); yeast extract, DIFCO (0.001);yeast nitrogen base, DIFCO (0.001);nutrient broth (0.001); heart infusion broth, DIFCO (0.001); peptone, DIFCO (0.001).
No. 6 coals were mixed together in one vessel and also transferred to the anaerobic chamber. NazS.9H20 (final concentration 0.03 %) and coal (0.1 g) were added to each culture vessel, which were then capped with serum seals. The cultures were incubated for a month a t 25 "C, stationary. Growth was determined by changes in turbidity, and methane content was analyzed by performing gas chromatographic analyses of 1-mL headspace samples using a CTR I column (Alltech, Deerfield, IL) at 30 OC (carrier gas helium, 10 mL/min; thermal conductivity detector). Positive controls, in which methanogen-containing mud was used as an inoculum,showed substantial methane peaks and detectable turbidity. The absence of a methane peak in experimental cultures was taken to indicate that no methane was present within the limits of our detection.
Results and Discussion After considerable incubation time, there were no indications of any microorganisms having arisen from the Argonne coal samples we tested (Table 11). These data do not support a previous report that Argonne coals contain cultivable bacteria, specificallya Clostridium spaz3Many of the media we utilized in this study are capable of supporting the growth of many species of Clostridium, yet no such organismswere derived from the coal samples. One possible explanation for this is that there may be some clostridia in the coal, but they are present at such a low concentration that they are infrequently discovered in coal samples (see further discussion below). Another possibility is that the clostridia which were discovered were simply contaminants that did not originate from the coal.
382 Energy 13Fuels, Vol. 7, No.3,1993 Table 11. Microbiological Analysis of Argonne Premium Coal Samdes target oxygenCH4 mediaa organismb ationc tempd growthe producd A 22 nm NA GCH AN nm 24 nm MA 37 NB GCH AN nm 24 nm MA 37 0.5 X NB GCH, RS nm MA 37 0.1 X NB/BS GCH, RS MA GM GCH, RS 37 nm AN 24 nm A nm 22 BAA GCH nm 24 AN AN nm 24 BrAA GCH MA nm 37 BMT GCH AN nm 24 nm MA 31 TGw/oG GCH nm 24 AN AN nm 24 0.5 X TGw/oG GCH, RS AN nm 24 0.1 X TGw/oG GCH, RS nm A TSIA GCH, GE 22 nm 24 AN MA nm SB GCH, S 37 nm 24 AN nm MA LM GCH, SRB 37 AN 24 nm MA nm 37 YMB GCH, YM 22 A nm YMA GCH, YM AN 24 nm MA nm 37 EB GCH 24 AN nm 24 AN AMM GCH, AM AN 24 CMM CM AN 24 MMM GCH, MM 24 AMMv GCH, FAM AN AN 24 CMMv FCM 24 MMMv GCH, FMM AN 24 1 X NB/AMM GCH, FAM AN 24 0.6 x NB/AMM GCH, FAM AN 24 0.3 X NB/AMM GCH, FAM AN 24 0.1 x NBIAMM GCH, FAM AN 24 NB/A/BSE/MM GCH, FAM AN
~
~~
a Media formulations are described in Table I. GCH, general chemoheterotroph; RS,revivable sporulating microbes; GE, gram negative enteric bacilli, S,Salmonella;SRB,sulfate reducing bacteria; YM,yeaste, molds, fungi;AM, aceticacid metabolizingmethanogens; CM, carbonate metabolizing methanogens; MM, methanol metabolizing methanogens; FAM, fastidious acetic acid metabolizing methanogens;FCM, fastidious carbonate metabolizing methanogens; FMM, fastidious methanol metabolizing methanogem. A, Aerobic; MA, microaerobic; AN, strict anaerobic. Degrees Celsius. e Minus sign (-) = no turbidity. f Nm = not measured, (-) = no methane detected.
The previous report also mentions that some time after the vials were sealed, methane was detectable in the coal sample vials.23 This implies that either indigenous methane slowly diffuses out of the coal into the vial headspace or that methanogenic bacteria in the coal samples may be creating methane as part of their metabolic activities. Several methanogenic media were used to cultivate methanogenic bacteria in the coal samples (Tables I and 11). All of these media should support methanogenic growth, and most of the media were demonstrated to be adequate for the enrichment of methanogens from environmentalsamples(see ExperimentalSection). However, none of the incubation vessels containing coal gave rise to the production of methane, even after a month-long incubation (Table 11). This suggests that methanogenic bacteria may not be a common flora of coal seams and that the generation of methane in the coal samples may be abiotic. Another possibility is that the methane was generated by methanogenic bacteria early in the geological history of the coal and was trapped in the coal. The original
Polman et al.
methanogens might then have died off, leaving behind methane that was detected many years later. Since rich media might inhibit the germination of microbial spores preserved within coal, we also used lesser strengths of some media, e.g., nutrient broth and thioglycollate medium (Table 11). In addition, glucose minimal medium was used for some coal enrichments. No growth was observed in coal-containingculturesusing these media. This suggests that the coal contains little or no microbial spores or other dormant forms. Overall, our results with various media and incubation conditions indicate that these three Argonne coals are rather pristine with respect to the presence of living or dormant microorganisms. The observation that no revivable microorganismswere present in the coals is interesting in terms of the microbial ecology of natural coal seams, since one might expect that at least bacterial spores would have been preserved in the coal. The treatment process for sampling and packaging of Argonne coals is unlikely to be inhibitory to microbes that might be found within the coal seams, since the humidity of the samples is maintained, and the anaerobic/microaerobic nature of the originalsubsurface coal seam is preserved in the ~amples.2~ We must note that while we used a broad range of media that were capable of cultivating many types of organisms, the list of media and target microorganisms was by no means inclusive; that is to say, there might have been microbes present for which we simply did not provide sufficientnutrients. For example, no media were included that contained coal substructure model compounds as sole carbon and energy sources (although it is likely that organisms growing on such compounds would grow well in the media we used). Nevertheless, the fact that no organisms were cultivated is surprising. At least part of the reason for this must be the antimicrobial activity that coal has been reported to p0ssess.14*24-27 Another possible reason that microorganisms were not detected in this study may be that they occur with very low frequency in Argonne coals. Both Argonne National Laboratory (ref 23;Karl Vorres, ANL, personal communication) and Oak Ridge National Laboratory (Brendlyn Faison, ORNL, personal communication) have reported the presence of microorganismsin Argonne Premium Coal Samples. At ANL and ORNL, inocula used for microorganism enrichments were 2.5 g of coal per culture and 5 g of coal per culture, respectively. Those levels of inoculation were much higher than the levels used in this study (0.0025-0.1g of coal per culture). In conjunction, all of these observationsindicate that microorganismsare (1)either not present in these samples (2) or present in trace amounts. Our results supply information about the overall physical nature of coal. Apparently, coal contains little or no viable biomass as part of its overall composition. Thus, we must conclude that microorganisms pose little or no threat to the stability of Argonne Premium Coal Samples in terms of their physical and chemical structures. The absence or infrequency of viable biomass that we have demonstrated here implies that, at least in this respect, these coals are reliable for repeatable research results. Our conclusion concerningthis work is that no revivable (24) Olsson, G.; Larseon, L.; Holst, 0.; Karlsson, H.T.Fuel 1989,68, 1270-1274. (25) Rogoff, M. H.; Wender, I. Nature 1961, 192, 378-379. (26) Kosanke, R. M. Science 1954,119,214-216. (27) Schenck, N. C.; Carter, J. C . Science 1954, 119, 213-214.
Microbiological Analysis of Argonne Premium Coal
microorganisms were retrieved from three Argonne Premium Coals, even when exposed to a variety of microbial media. The original intention of the work was to procure microbes that might be beneficial in coal bioprocessing. The experimentation,however, was not unsuccessfulsince the results have broadened our views concerning the overall physical composition of coal,especiallyArgonne Premium
Energy & Fuels, Vol. 7, No. 3, 1993 383
Coal Samples, which are relied upon for repeatable coal research results. Acknowledgment. Funding for the work described herein was provided by the United States Department of Energy (Office of Advanced Research and Technology Development, Office of Fossil Energy, contract DE-AC0776ID01570).
