Energy & Fuels 1987, 1 , 80-84
80
Microbial Conversion of Low-Rank Coal: Characterization of Biodegraded Product Bary W. Wilson,* Roger M. Bean, James A. Franz, and Berta L. Thomas Battelle, Pacific Northwest Laboratories, Richland, Washington 99352
Martin S . Cohen, Harold Aronson, and Edward T. Gray, Jr. University of Hartford, West Hartford, Connecticut 0611 7 Received August 26, 1986. Revised Manuscript Received October 29, 1986
We have characterized products obtained from the action of the fungus Polyporus versicolor on oxidized North Dakota (ND) lignite. These analyses showed that, compared to feed coal, the bioconverted materials had higher hydrogen:carbon and 0xygen:carbon ratios, but were proportionately lower in aliphatic hydrogen, as determined by infrared (IR)spectroscopy. The acid-precipitated extract was dissolved in dilute base and analyzed by I3C nuclear magnetic resonance (NMR) spectroscopy. Of the 60% of the carbon accounted for, approximately 51% of the carbon atoms were aromatic, 20% were assigned to carboxylic acid groups, and the remainder were aliphatic carbon. Proton NMR spectra of the acid-precipitated material revealed approximately equal proportions of aromatic and aliphatic hydrogen. The bioconverted materials were highly polar and exhibited a wide range in apparent molecular weight; most material was over 10000 Da a t acidic pH, as determined by ultrafiltration experiments. Freeze-dried product material was soluble in water but was essentially insoluble in other organic solvents. Calorimetric measurements on samples of the freeze-dried extract showed that, on a per-weight basis, it retained 94-97% of the heating value of the feed coal.
Introduction Biological conversion of low-rank coals by bacterial or fungi: or by preparations of the enzymes that they produce: has received increasing attention. Because these processes occur a t ambient temperatures and pressures, they represent a potential cost savings in the processing of certain coals and lignites. Methodologies currently used in thermochemical coal conversion require high temperature and pressure and can result in process streams or products that are more toxic than their petroleum-derived counterparts. Biological reactions studied to date have been mainly those of fungi on lignite, resulting in a water-soluble product that can be precipitated a t acidic pH. Cohen and Gabriele2first reported that fungi could grow directly on, and metabolize, naturally occurring coal. Since that time, Cohen et al.4 reported that a naturally oxidized form of lignite coal, known as Leonardite, is degraded to a black, viscous liquid. Wilson et ale3have shown that the Leonardite degradation product from Polyporus versicolor was water-soluble and contained no detectable polycyclic aromatic hydrocarbons (PAH) having three to six rings. Unlike many thermochemically derived coal products, the bioconverted material was inactive in the microbial histidine reversion assay for mutagenic a ~ t i v i t y . ~ Others have also reported degradation or solubilization of lignites by various fungal strains, including Phanerochaete chrysosporium,5 Penicillium waksmanii,6 and Candida sp. ML13.617 A recent review of coal bioprocessing was made by Olson and Brinkman.s Although conversion has been reported by several species on several lignite substrates, no detailed chemical or spectroscopic characterization of the product materials has appeared in the literature. We report here analyses of the material
* Author to whom correspondence
should be addressed.
produced by action of P . versicolor on Leonardite.
Experimental Section Bioconversion. Stock cultures of P.versicolor are routinely
maintained in both solid Sabouraud maltose agar and Sabouraud maltose broth cultures. All cultures were incubated at 30 O C , relative humidity 84-98%, and pH 5.8. Experimental cultures were also incubated as described above, with the exception of the experiments involving specific additions to the media. Solid cultures were inoculated with a hyphal suspension and allowed to incubate for approximately 12 day to produce a continuous fungal mat. Sterile lignite pieces (approximately5 mm3) were placed directly on the hyphal mat. Although extensive solubilization was evident after 24 h, product was harvested from the cultures after 5 days as described previously2 and freeze-dried, by using a Bellco cold finger at -70 O C , and stored in a desiccator at room temperature. All data reported here were obtained on the freeze-driedbioextract. Solubility Tests. In order to determine solubility characteristics, the bioextact was first lyophilized and then the residue was weighed. The concentrationof solids in the bioextract was (1) Fakausa, R. M. Coal as a Substrate for Microorganisms: Investigation with Microbial Conversion of National Coals, Ph.D. Thesis, Friedrich-Wilhelms Unviersity, Bonn, Federal Republic of Germany,
1981.
(2) Cohem, M. S.; Gabriele, P. D. Appl. Enuiron. Microbiol. 1982,44, 23-27. ( 3 ) Wilson, B. W.; Lewis, E.; Stewart, D.; Li, S.-M.; Bean, R.; Chess, E.; Pyne, J.; Cohen, M.; Aronson, H. in Proceedings of the Direct Liquefaction Contractor’s Meeting; US.Department of Energy: Washington, DC, 1986; pp IV-89-IV-98. (4) Cohen, M. S.; Aronson, H.; Gray, E. T., Jr. In Proceedings of the Direct Liquefaction Contractor’s Meeting; U.S. Department of Energy: Washington, DC, 1986; pp IV-48-IV-64. ( 5 ) Faison, B. D.; Kuster, T. A. In Proceedings, Eighth Symposium on Biotechnology for f i l e s and Chemicah;Scott, C. D., Ed.; Wiley: New York; in press. (6) Ward, B. Syst. Appl. Microbiol. 1985, 6 , 236. (7) Scott, C. D.; Strandberg, G. W. In Proceedings of the Direct Liquefaction Contractor’s Meeting; U.S.Department of Energy: Washington, DC, 1986; pp IV-65-IV-87. (8) Olson, G. J.; Brinkman, F. E. Fuel, in press.
0887-0624/87/2501-0080$01.50/00 1987 American Chemical Society
Microbial Conversion of Low-Rank Coal calculated to be 60 mg/mL. The dry product was a brown, flakelike solid, with the ability to hold a static charge. Solubility of the freeze-dried bioextract was determined by dissolving the particles to saturation (over a period of 2 h) in solvents of varying polarities. Solutions were then evaporated to dryness, and the solutes were weighed to determine solubilities, expressed as grams of solid bioextract per milliliter of solvent. Titration Experiments. A 0.2892-g sample of biodegraded material was dissolved in water and the volume brought to 50 mL. Fifteen-milliliter aliquots were placed in two centrifuge tubes, an equal volume of 0.2 M HC1 was added, and each suspension was centrifuged and washed twice with 0.2 N HCl. The samples were frozen in liquid nitrogen and lyophilized for 6 h. Samples were titrated with 0.1832 M NaOH (COz-free)standarized against potassium hydrogen phthalate. Data from the pH meter (Orion Model 701A with a combination pH electrode; VWR Scientific, San Francisco, CA) was transferred to a microcomputer system (Tandy Model 111). The computer also controlled a stepper motor to drive the titrant into the test solution (minimum deliverable volume, 0.5 mL). The titration system was thermostated a t 25.0 & 0.1 "C. Solutions being titrated were held under nitrogen gas, which was passed through a scrubber of barium hydroxide to remove any remaining COz and to humidify the gas so as to prevent drying of the sample. Calorimetry. Powdered coal samples, bioextract, benzoic acid, and naphthalene were each pressed into pellets weighing approximately 1 g. Benzoic acid and naphthalene were used to calibrate the colorimeter. Each pellet was burned in a nonadiabatic bomb calorimeter equipped with a computer-interfaced thermistor (accurate to fO.OO1 "C) for temperature measurement. Acid Precipitation of Biodegradation Product. Freezedried biodegraded Leonardite (25 mg) was dissolved in 10 mL of water purified with a Milli-Q water system (Waters Associates, Inc., Milford, MA), and the solution was brought to pH 2 with 5% HC1. The resulting flock was centrifuged, and the supernatant was decanted, washed once with HC1 a t pH 2, and redissolved in 10 mL of a NaOH solution (pH 10). The acid precipitation and washing were repeated, followed by a wash with water. The precipitate was frozen to -80 "C and freeze-dried. The acidprecipitated material constituted 85% of the weight of the biodegraded product. Infrared (IR) Spectroscopy and Elemental Analyses. The IR spectra of KBr pellets of the coal and the product were obtained with a Perkin-Elmer Model 283 grating spectrometer. Analyses of carbon, hydrogen, oxygen, nitrogen, sulfur, and nitrogen (as ammonia) were obtained by Schwartzkopf Microanalytical Laboratories, Woodside, NY. Metal analyses were obtained by X-ray fluorescence, using a Kevex Model O8lOA fluorescence spectrometer, calibrated on single-element, thin-film standards (Micromatter, Inc., East Sound, WA). Sodium and magnesium were determined with a Perkin-Elmer 503 atomic absorption spectrometer. Coal materials were analyzed for iron pyrite with X-ray diffraction. 13C
Nuclear Magnetic Resonance (NMR) Spectroscopy
of Acid-Precipitated Product. The 20-MHz 13CNMR spectrum (Varian FT-80) of a solution of 0.436 g of product in NaOD/D,O (5.2 mL total volume), containing 12.4 mg of dioxane, was obtained. The spectrum was obtained after 70850 scans, with a 50" pulse, 1-s acquisition time, and 2.5-9 pulse delay with proton decoupling off during the pulse delay and the pulse but on during acquisition for suppression of the nuclear Overhauser effect. Use of a 4.5-s pulse delay did not change the integral ratios, a result consistent with the substantial paramagnetic content. Proton NMR Spectroscopy. We obtained 'H NMR spectra of a solution of 14.1 mg of freeze-dried biodegradation product in 0.99 g of 99.995% D-label DzO,containing 7.9 X l0-L g of acetone (HDOacetone; integral ratio, 13.542), at 79.54 MHz with a Varian FT-80 instrument. A solution of 35.8 mg of acid-precipitated product was disaolved in 0.75 mL of NaOD/D,O, prepared by dissolving sodium metal in 99.995% DzO, and the proton NMR spectrum was obtained. After 1 pL (7.9 x lo4 g) of methanol was added, the NMR spectrum was reacquired and integrated, with the methyl integral as an internal standard. Molecular Weight Determinations. The number-average molecular weight of microbial product was determined by va-
Energy & F u e l s , Vol. I , No. I , 1987 81
0'15r-I
0.121
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g
0.09
a
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=
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O o 30I a 0
,
;;
2
4
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,
6
8
1
10
Solvent Polarity
Figure 1. Solubility of the freeze-dried bioextract in various solvents of different polarities (Debye units). The solvents were hexane (a), methylene chloride (b), butanol (c), ethanol (d), methanol (e), and water (f). por-pressure osmometry in aqueous solution by Schwartzkopf Microanalytical Laboratories. Estimates of molecular weight distributions of microbially converted Leonardite were obtained by using ultrafiltration. Aqueous solutions containing about-200 mg/L of organic carbon were slowly filtered, with pressure, through Diaflo ultrafilters (Amicon, Lexington, MA), with use of a stirred cell. Organic carbon was determined in subsamples after filtration through >50000, >10000, >1000, and >500 Da filters with a Coulometric, Inc. (Golden, CO) carbon analyzer. Carbon concentrations that passed individual fiiters were corrected for blanks obtained after filtration of carbon-free water through a filter of the same size.
Results Coal Solubilization. Samples of Leonardite coal digested to a black, viscous liquid. Solubilization was visually apparent within 24 h after the Leonardite was added to the cultures. The amount of black liquid product continuously increased until the lignite was completely liquefied or the growth of the fungi ceased, possibly from the effects of toxic products produced during the process of solubilization. The bioextract appeared to contain no particular matter when observed a t a magnification of 400X with a compound microscope. Solubility Tests. When the bioextract was mixed with solvents having polarity indices less than 4, such as hexane (O), methylene chloride (3.4), and 1-butanol (3.9), the solvents appeared to have little, if any, effect on the free dried solid (Figure 1). Mixing the bioextract with solvents that had polarity indices between 4 and 6.2,'such as tetrahydrofuran (4.2), 1-propanol (4.3), and ethanol (5.2), resulted in formation of a light colored, ye?lowish brown solution. This indicated increasing solubility of the bioextract compared with that obtained with the solvents of lower polarity mentioned above. The bioextract was most soluble in water, resulting in an opaque, black solution. Solubility of the starting Leonardite was less than 1 mg/mL for all six solvents tested, even after a 6-day period of solvent exposure. Titration. Two titrations were performed. For one, 0.3254 g of the acid-precipitated material was placed in a beaker with 20.00 mL of water. A total of 1.2500 mL of base was added (5.0 mL increments) a t 5-min intervals, followed by an additional 2.000 mL (10.0-mL increments) a t 6-s intervals. The 6-s period was the minimum needed to insure homogeneity after each addition of titrant. For the second titration, we used 0.012 25 g of the acid-precipitated material in 20.000 mL of water. in this case, 2.1500 mL of titrant was added (10.0-mL increments) at
82 Energy & Fuels, Vol. I, No. 1, 1987 13
Wilson et al. 1
g"
A
20
."'i t
4000
311:'
8
6
3000
2000 1600 1200 800
400
Wavenumber (cm-')
~
4000
3000
2000 1600 1200 800
~~
400
Wavenumber (cm-')
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Figure 3. Infrared spectra of Leonardite and its microbial degradation product, as determined from KBr pellets: (A) Infrared scan of microbial product; (B)Infrared scan of Leonardite.
OH-added . mmoles OH- in water) /g coal
Figure 2. Aqueous titrations of acid-precipitated bidegradation product: curve A, 20-min interval between titration points; curve B, 5 min/point; curve C, extension of curve B at 6 s/point.
Table I. Elemental Analyses of Starting Coal and Coal Products
20-min intervals. The data are shown in Figure 2. Values for the abscissa were calculated from
acid-precipitated productC
I
(mL of OH-)
[OH-] -
(
element microbial producta Leonarditeb C 46.09 f 0.11" 54.95 f 0.08 H 3.80 0.03 3.60 f 0.08 N 4.34 f 0.15 0.80 f 0.10 0 30.87 f 0.11 30.24 f 0.50 S 1.08 0.01 1.06 f 0.02 ash 7.27 f 0.28 8.32 f 0.05
*
X
K,(total
anal.. '70
;::
solution) ))/g
of sample
where K, was determined experimentally to be 1.035 X M2 from titrating water under the same conditions as coal. This quantity is equivalent to the number of millimoles of OH- reacting per gram of coal sample. There were no distinct endpoints in the titrations, indicating the presence of weak acids having a wide range of acid constants. In the early stages of the titration, some of the buffering capacity was due to the base-assisted dissolution of the solid. Once the solution became basic, there was evidence for a slow reaction of hydroxide ion with the coal ions in solution. The plot of the slower titration (Figure 2) indicates that the hydroxide ion continued to react slowly with the sample. After slow addition of more than 20 mmol of OH-, sample pH remained at 11. The kinetic effect can be seen in the comparison of the two titrations (Figure 2). The data a t 5 min/point have more free hydroxide (a higher pH) earlier in the titration, with an even more pronounced effect when the base was introduced as fast as possible. The titration curves indicate the approximately 4-5 mol of base were needed to neurtralize lo00 g of the coal sample, an amount corresponding to an equivalent weight of 200-250 g/equiv. Calorimetry. The average energy content of the freeze-dried Leonardite coal sample was 4.394 kcal/g (7378 Btu/lb). Average energy content for the bioextract was 4.208 kcal/g, equivalent to 95.7% of the original energy content of the lignite on a gram-for-gram basis (1g of feed coal yielded less than 1 g of solid bioextract in solution). The maximum error of these measurements was 1.2%, determined by measuring the energy content of naphthalene. IR Spectroscopy and Elemental Analyses. IR spectra of the Leonardite feed coal and bioextracted product are shown in parts A and B of Figure 3. The CH2 stretch that is evident at 3000 cm-' in the feed is not
*
53.53 3.83 2.07 30.70 1.06 3.48
'Microbial product (Cl~B.sNo.s06.0So.l). * Leonardite (C10H7.8dValues indiN0.104.1S0.1). Acid precipitate (C10H8.~N~.~0~,1So,1). cate range for duplicate determinations. Table 11. X-ray Fluorescence Analyses of Starting Coal and Coal Products anal., 70 Na K Mg Ca Si A1 C1 Fe S lignite a 0.02 a 0.41 0.47 0.15 b 0.38 0.69 biodegraded 2.77c 0.55 0.35c 0.47 b 0.43 0.34 1.45 1.04 acid a 0.03 a 0.1 b 0.30 2.3 1.41 1.0 precipitated
'Not determined.
Not detected.
By atomic absorption.
distinguishable in the bioextracted spectrum. Furthermore, a new absorbance peak, consistent with COOand/or NH4+is apparent a t 1400 cm-' in the spectrum of the product (Figure 4B). Elemental compositions, determined by combustion analysis, for the feed coal, bioextract, and acid-precipitated bioextract are shown in Table I. Analysis of ammonium ions on two different samples of product indicated that ammonium represented almost half (40.0 and 49.8%) of the product nitrogen. Results from X-ray fluorescence determinations for trace metals in the bioextract and the feed Leonardite are shown in Table 11. Nuclear Magnetic Resonance (NMR) Spectroscopy. The I3C NMR spectrum of the acid-precipitated product dissolved in NaOD/D20, is shown in Figure 4A. Integral ratios were 14:36:4:20 for the 200-160, 160-100, 67.4 (dioxane), and 60-10 ppm regions. With the dioxane integral as an internal standard, the integrals indicate detection of 0.12 g of carbon (28% by weight, compared to 46% (0.20 g) by elemental analysis) and 0.06 g of carboxylate oxygen (14% by weight, not including phenolic or other oxygen types, compared to 31% (0.14 g) total oxygen) by elemental analysis. The carboxylate carbon detected (0.024 g) cor-
Energy & Fuels, Vol. 1, No. 1, 1987 83
Microbial Conversion of Low-Rank Coal
a 3.0 pH 6.4
0 pH
pH 10.4
>50
50-10
10.1
1-5
