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Energy & Fuels 1998, 12, 664-671
Articles Biomimetic Solubilization of a Low Rank Coal: Implications for Its Use in Methane Production John A. Bumpus,*,†,‡ John Senko,† Gregory Lynd,† Richard Morgan,‡ Kimberly Sturm,† Jennifer Stimpson,† and Shawn Roe† Department of Chemistry and The Environmental Sciences Program, University of Northern Iowa, Cedar Falls, Iowa 50614 Received September 3, 1997. Revised Manuscript Received April 8, 1998
There is substantial interest in processes that result in solubilization of low rank coal as a potential carbon source for anaerobic production of methane. It is known that certain fungi are able to solubilize selected low rank coals by secreting oxalate ions which chelate metal ions that are present. Apparently, metal ions form ionic bonds which link individual coal macromolecules. Once these metal ions are chelated and the ionic bonds are broken, the relatively polar coal macromolecules are soluble in water. Much research has focused on the coal-solubilizing ability of wood-rotting fungi. The wood-rotting fungi Trametes versicolor and Phanerochaete chrysosporium are known to be able to solubilize extensively an oxidized North Dakota lignite (leonardite). We have used a biomimetic approach to study oxalate-mediated solubilization of this low rank coal. A concentration of approximately 75 mM sodium oxalate was found to be near optimal for leonardite solubilization when the initial concentration of leonardite was 2 mg/ mL. This is of importance because oxalate concentrations in liquid cultures of wood-rotting fungi are typically well below this concentration. Nevertheless, substantial leonardite solubilization was also observed at oxalate concentrations more typical of those found in these fungi. The effect of pH was also studied. It was found that oxalate-mediated leonardite solubilization increased with increasing pH and appeared to be a function of divalent oxalate ion concentration. Similar results were found for dihydrogen phosphate/hydrogen phosphate/phosphate and bicarbonate/ carbonate ions. If the solubilized coal macromolecule from leonardite becomes a viable carbon source for use in anaerobic methane production, these studies suggest that chemical solubilization by common inexpensive Lewis bases would likely be more cost competitive than fungal solubilization processes.
Introduction Low rank coals (lignite including leonardite and other subbituminous coals) account for approximately 58% of the coal reserves in the United States.1 Although the heating capacity and therefore the commercial value of such coal is considerably less than that of bituminous and anthracite coals there is much interest in exploiting low rank coals as fuels. One approach involves chemical or biological solubilization of low rank coal followed by its anaerobic biogasification to methane.2 During the past 17 years there has been considerable interest in processes by which wood-rotting fungi and other fungi mediate solubilization and/or depolymeri* Corresponding author: Department of Chemistry, McCollum Science Hall, University of Northern Iowa, Cedar Falls, IA 50614. † Department of Chemistry. ‡ The Environmental Sciences Program. (1) Faison, B. D. In Microbial Transformations of Low Rank Coals; Crawford, D. L., Ed.; CRC Press: Boca Raton, FL, 1993; pp 1-26. (2) Isbister, J. D.; Barik, S. In Microbial Transformations of Low Rank Coals; Crawford, D. L., Ed.; CRC Press: Boca Raton, FL, 1993; pp 139-156.
zation of low rank coal.3-14 Early investigations demonstrated that certain wood-rotting fungi possess the (3) Fakoussa, R. M. Coal as a Substrate for Microorganisms: Investigation with Microbial Conversion of National Coals. Ph.D. Thesis, Friedrich-Wilhelms University, Bonn, Germany, 1981. (4) Cohen, M. S.; Gabriele, P. D. Appl. Environ. Microbiol. 1982, 44, 23-27. (5) Frederickson, 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. (6) Cohen, M. S.; Gray, E. T., Jr. In Microbial Transformations of Low Rank Coals; Crawford, D. L., Ed.; CRC Press: Boca Raton, FL, 1993; pp 47-64. (7) Cohen, M. S.; Feldman, K. A.; Brown, C. S.; Gray, E. T., Jr. Appl. Environ. Microbiol. 1990, 56, 3285-3291. (8) Wilson, B. W.; Bean, R. M.; Franz, J. A.; Thomas, B. L. Energy Fuels 1987, 1, 80-84. (9) Scott, C. D.; Strandberg, G. W.; Lewis, S. N. Biotechnol. Prog. 1986, 2, 131-139. (10) Torzilli, A. P.; Isbister, J. D. Biodegradation 1994, 5, 55-62. (11) Achi, O. K. Int. Biodeter. Biodeg. 1993, 31, 293-303. (12) Cohen, M. S.; Bowers, W.C.; Aronson, H.; Gray, E. T., Jr. Appl. Environ. Microbiol. 1987, 53, 2840-2843. (13) Stewart, D. L.; Thomas, B. L.; Bean, R. M.; Fredrickson, J. K. J. Ind. Microbiol. 1990, 6, 53-59. (14) Pyne, J. W.; Stewart, D. L.; Fredrickson, J.; Wilson, B. Appl. Environ. Microbiol. 1987, 53, 2844-2848.
S0887-0624(97)00159-X CCC: $15.00 © 1998 American Chemical Society Published on Web 06/11/1998
Biomimetic Solubilization of a Low Rank Coal
ability to mediate solubilization of these coals. Although some studies12,14 suggested that extracellular lignindegrading enzymes may have some role in biosolubilization, more recent investigations demonstrated that oxalate ion has a major role in this process.7 Apparently, macromolecules in low rank coals are held together, at least in part, by ionic linkages in which polyvalent exchangeable cations form bridges between negatively charged groups on constituent coal macromolecules.1 Oxalate ions are able to chelate these cations. Once the ionic linkages are broken, the polar coal macromolecules are relatively water soluble. Although a variety of investigations support the idea that oxalate ion is, indeed, involved in the solubilization process, there is little information available concerning the efficiency, rate, and optimal conditions of oxalatemediated coal solubilization. In the present investigation we have studied the ability of sodium oxalate to solubilize an oxidized North Dakota lignite (leonardite). Because oxalate, secreted by fungi in vivo, is able to mediate solubilization of low rank coal, solubilization of these coals by oxalate salts in vitro may be considered to be a biomimetic process. Methods and Materials Chemicals. Leonardite, an oxidized North Dakota lignite, was supplied as a fine powder by the American Colloid Company, Arlington Heights, IL. This material was dried for 24 h at 105 °C before use. Microorganisms. Phanerochaete chrysosporium (ATCC 24725) and Trametes versicolor (ATCC 12679) were purchased from the American Type Culture Collection, Rockville, MD. Fungi were subcultured and maintained on malt agar and Sabouraud agar, respectively, on Petri plates and on slants in culture tubes. Biological Solubilization of Leonardite by WoodRotting Fungi. Petri plates containing several different solid media (e.g., malt agar, Sabouraud agar, or yeast malt agar) were used. Powdered leonardite (20 mg) was sprinkled as evenly as possible over each Petri plate. Cultures were then inoculated using an agar plug from stock cultures of each fungus. Cultures were visually examined periodically for signs of solubilization. Solubilization was scored as follows: 4+ extensive solubilization in which a black liquid diffused from the solid coal and covered or nearly covered the entire plate; 3+ solubilized coal covered approximately one-half to threequarters of the plate; 2+ solubilization occurred but solubilized coal covered less than one-half of the plate; 1+ a small amount of solubilization occurred but solubilized coal covered less than one-quarter of the plate; - no solubilization was observed. Biomimetic Solubilization of Leonardite. One gram of leonardite was placed in a 125 mL Erlenmeyer flask containing 100 mL of water and 1 g of sodium oxalate. This mixture was stirred for 48 h after which it was centrifuged briefly (1400g, 15 min) to remove particulate material. The dark brown solution then was dialyzed (12-14 kDa molecular weight cutoff (MWCO)) extensively against distilled deionized water, filtered through a 0.22 µm cellulose acetate filter (Corning Costar Corp., Cambridge, MA), lyophilized, and stored at room temperature. This material was characterized by UV-visible, IR, and 1H NMR spectroscopy, and the molecular weight range was determined by GPC-HPLC.15 The relationship between absorbance and mass of soluble leonardite macromolecule at 600 nm was found to be 1.71 au ) 1.0 mg/mL. This relationship was used in some experiments to estimate the concentration of soluble coal macromolecule in aqueous solution. (15) Polman, J. K.; Quigley, D. R. Energy Fuels 1991, 5, 352-353.
Energy & Fuels, Vol. 12, No. 4, 1998 665 The effect of sodium oxalate concentration on its ability to solubilize leonardite was assessed over a concentration range which varied from 0 to 300 mM sodium oxalate. Mixtures containing sodium oxalate and 20 mg of leonardite in 10 mL of water in 20 mL vials were agitated at 200 rpm in a rotary shaker for 24 h. The amount of coal that was not solubilized then was assayed using a gravimetric procedure in which insoluble particulate material was collected by vacuum filtration and its mass determined on dry tared 47 mm, 0.45 µm, nylon membrane filters (Whatman). The vials were washed with 10 mL of water which was then passed over the nylon membranes. The membranes again were dried, and the mass of the dry membrane and coal that was not solubilized was determined. The amount of coal solubilized was then calculated. Initial experiments revealed that the nylon filters lost a substantial amount of their mass during this procedure and were thus unsuitable for gravimetric determinations unless they were washed prior to use. Thus, in our experiments, nylon filters were pretreated by soaking them in ∼500 mL of water for about 30 min. During this time the filters were gently agitated and the water was changed once. The filters then were dried at 105 °C, 30 min. Filters pretreated in this manner were suitable for use in the gravimetric procedure described above. The effect of leonardite concentration on its own solubilization was assessed in a similar manner. In these experiments, 74.6 mM (1 g/100 mL) sodium oxalate in water containing leonardite concentrations from 50 mg/L to 50 g/L were used. At the highest concentrations studied, the nylon filters became plugged. Thus, these technical difficulties prevented us from using the same volume for each concentration studied. The concentrations studied follow. The volume in parentheses is the volume that was used: 50 g/L (10 mL), 40 g/L (10 mL), 30 g/L (10 mL), 25 g/L (10 mL), 20 g/L (10 mL), 10 g/L (20 mL), 5 g/L (20 mL), 1 g/L (20 mL), 0.5 g/L (20 mL), 0.1 g/L (200 mL), 0.05 g/L (200 mL). In other experiments, the effect of time versus leonardite solubilization was assessed at 0, 1, 10, and 74.6 mM sodium oxalate. The effect of pH on the ability of several Lewis bases to solubilize leonardite was assessed. For sodium oxalate, a concentration of 74.6 mM (1 g/100 mL) was used. For potassium phosphate/hydrogen phosphate/dihydrogen phosphate and sodium bicarbonate/carbonate, a concentration of 75 mM was used. Mixtures containing 10 mL of these solutions at varying pH values and 20 mg of leonardite were shaken for 24 h on a rotary shaker (200 rpm) after which time 1 mL aliquots were centrifuged (4 min, 11000g) in an Eppendorf microcentrifuge, and the absorbance at 600 nm was acquired and plotted against pH. The effect of sodium hydroxide concentration on leonardite solubilization was assessed in a similar manner. Except for sodium bicarbonate/ carbonate, pKa values were corrected for ionic strength to calculate the concentration of acid and conjugate base for each pH value. Solubilization was also assessed gravimetrically at selected pH values.
Results It has been shown that a variety of fungi are able to solubilize selected low rank coals.3-14 We have repeated solubilization studies similar to those reported by others4,9 and have confirmed that P. chrysosporium and T. versicolor are, indeed, able to solubilize leonardite when these fungi are grown on selected agar media. After 50 days of incubation, leonardite solubilization scores of 4+ were observed for P. chrysosporium and T. versicolor when they were grown on Sabouraud agar and yeast malt agar, respectively. In contrast, no solubilization was observed when these fungi were grown on malt agar.
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Figure 1. Effect of pH on the absorbance of soluble coal macromolecule at 600 nm. Mixtures contained 0.53 mg of biomimetically solubilized coal macromolecule in 75 mM potassium phosphate at the indicated pH values.
The spectral characteristics (data not shown) of biomimetically solubilized coal macromolecule were very similar to those reported for coal macromolecule solubilized biologically by T. versicolor.8 The molecular weight of biomimetically solubilized coal macromolecule ranged from 14 000 to ∼66 000. It should be noted that some low molecular weight material was also generated by our solubilization protocol using sodium oxalate. However, this material was removed during the dialysis (12-14 kDa MWCO) step that was used to remove sodium oxalate. The characteristics of biomimetically solubilized coal macromolecule are consistent with those of humic acid, an acidic aromatic polymer normally found in peat, brown coal, and lignite. In some of our investigations, solubilization of leonardite was estimated by monitoring the increase in absorbance that occurs in the visible range of this material. The UV-visible spectrum of this material is nondescript, gradually increasing in intensity through the visible spectrum into the UV. There are no distinct peaks. The absorbance at 600 nm was selected and used for these estimations. The use of this wavelength allowed many of our spectral determinations to be made without having to dilute the sample. Because several experiments assessed leonardite solubilization as a function of pH, it was necessary to determine the effect of pH on the absorbance of soluble coal macromolecule. Figure 1 shows that absorbance at 600 nm of soluble coal macromolecule increased between pH 4.7 and 11.9. Because of this pH-dependent increase, absorbance was converted to concentration only in a few experiments performed in aqueous solution at pH ∼5.6 for which a conversion factor had been determined. In those experiments in which leonardite solubilization was studied as a function of pH, relative solubilization was monitored by determining the absorbance at 600 nm as well as gravimetrically. It is now apparent that the oxalate ion that is secreted by fungi5-7 has an important role in solubilization of low rank coal. However, little information exists concerning the identification of conditions that optimize or enhance oxalate solubilization of such coal. The effect of sodium oxalate concentration, incubation time, re-
Figure 2. Effect of sodium oxalate concentration on leonardite solubilization. In 20 mL reaction vials each reaction mixture contained 20 mg of leonardite and 10 mL of sodium oxalate at the indicated concentrations. Reaction vials were placed on a rotary shaker for 24 h. Particulate material was then collected on 0.45 µm filters and washed with 10 mL of water. The absorbance of the filtrate at 600 nm was then acquired, multiplied by 2 to account for the dilution which occurred during washing, and plotted against sodium oxalate concentration (Figure 2a). The mass of the particulate material was determined gravimetrically as described in methods and materials. The percent of leonardite solubilized was then calculated. These values were plotted versus sodium oxalate concentration (Figure 2b). Experiments were performed in triplicate.
peated extraction with sodium oxalate, and initial concentration of leonardite on leonardite solubilization are presented in Figures 2, 3, 4, and 5, respectively. Leonardite solubilization (initial concentration ) 2 mg/ mL) was shown to be dependent on sodium oxalate concentration from 0 to ∼75 mM sodium oxalate (Figure 2). The use of greater concentrations of sodium oxalate did not result in a leonardite solubilization that was statistically different from that which occurred at 75 mM. Although concentrations of 75 mM and above of sodium oxalate resulted in the greatest amount of leonardite solubilization, such concentrations of oxalate ion are well above those which occur in liquid cultures
Biomimetic Solubilization of a Low Rank Coal
of wood-rotting fungi.16 It should be noted, however, that substantial amounts of leonardite were solubilized at oxalate concentrations that are more representative of those that are produced by these fungi. The time course (Figure 3a) for oxalate-mediated leonardite (initial concentration ) 10 mg/mL) solubilization at two such concentrations (1 and 10 mM) and at a concentration of 74.6 mM indicated that following an initial rapid rate solubilization of leonardite continued at a slower rate for the duration of the incubation. Results obtained using 1 and 10 mM sodium oxalate were relatively straightforward. However, at 74.6 mM sodium oxalate, a second rapid increase of absorbance at 600 nm occurred after several days of incubation. Demonstration of this second burst of apparent solubilization was repeatable. However, the precise time at which it occurred was less predictable. To further examine this phenomenon, control experiments were performed in which solubilization was measured gravimetrically. In these experiments (solid bars in Figure 3b), the percent of leonardite solubilized after 10, 20, and 26 days was shown to be 45.3 ( 0.05, 48.0 ( 0.03, 48.05 ( 0.04, respectively. Of interest, however, was the observation that after 26 days the A600 per mg/mL of coal macromolecule in water was found to be 2.75, which is substantially greater than the relationship that we determined and used to calculate the concentration of coal macromolecule. Subsequent investigations (data not shown) indicate that the increase in this ratio was due, at least in part, to an increase in pH from 5.2 to ∼8.9 which occurred during the incubation. These studies show that calculations based solely on absorbance during prolonged incubation can led to overestimation of the solubilization of leonardite that occurs. Although the time course data tend to overestimate the degree of coal solubilization that occurred, these results are useful in that, taken together, they demonstrate that substantial (i.e., ∼50%) leonardite solubilization occurs in the presence of 74.6 mM sodium oxalate. They also suggest that after the first few days, oxalate-mediated solubilization of leonardite is a relatively slow process. In another set of experiments, a 1 g sample of leonardite was subjected to 10 repeated daily extractions with 100 mL of 74.6 mM sodium oxalate (Figure 4). Of interest is the fact that a measurable, albeit small, amount of leonardite was solubilized even on the 10th day of this treatment. This again confirms that, following an initial rapid rate, oxalate-mediated solubilization of leonardite is a relatively slow process. The effect of leonardite concentration on its own solubilization was also studied (Figure 5). At a sodium oxalate concentration of 74.6 mM, the amount of leonardite solubilized increased linearly over a range in which the initial concentration of leonardite increased from 50 mg/L to 50 g/L of water. However, the relative percent of leonardite solubilized initially increased and then decreased over this concentration range. It has been known for many years that certain low rank coals can be solubilized in the presence of 1 M sodium hydroxide.17 It also has been suggested that (16) Dutton, M. V.; Evans, C. S.; Atkey, P. T.; Wood, D. A. Appl. Microbiol. Biotechnol. 1993, 39, 5-10. (17) Fowkes, W. W.; Frost, C. M. Leonardite: a lignite byproduct. Report of investigations 5611; Bureau of Mines, U.S. Department of the Interior: Washington, D. C., 1960; pp 1-12.
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Figure 3. Effect of incubation time on oxalate-mediated solubilization of leonardite. Reaction mixtures contained 1 g of leonardite in 100 mL of water or sodium oxalate in water at the indicated concentrations. Reaction mixtures were stirred on a magnetic stirrer and A600 was determined at the times indicated (Figure 3a). In three experiments (closed squares, open squares, closed circles), the initial concentration of sodium oxalate was 74.6 mM. In other experiments, incubation mixtures contained 10 mM sodium oxalate (closed triangles), 1 mM sodium oxalate (open circles), and water (open trianlges). In one experiment (Figure 3b) solubilization was determined spectrophotometrically (using the relationship A600/mg ) 1.71) and gravimetrically. Spectrophotometrically acquired data is represented by the open columns in Figure 3b while gravimetrically acquired data is represented as the solid columns in Figure 3b. Error bars represent (1 standard deviation. In this experiment, triplicate samples containing 1 g of leonardite in 100 mL of 74.6 mM sodium oxalate were stirred for 10, 20, and 26 days. Samples were centrifuged for 30 min at 11000g. The A600 of the supernatant of each sample was then determined. The pellets were then dried. The mass of each pellet was determined, and the amount of leonardite solubilized was calculated. See text for further explanation.
biological solubilization of low rank coal may be due, at least in part, to the ability of certain microorganisms to increase the pH of their incubation medium thereby causing base-mediated solubilization.18 Cohen and his associates6,19 showed that the use of phosphate buffers can complicate the interpretation of biosolubilization (18) Quigley, D. R. In Microbial Transformations of Low Rank Coal; Crawford, D. L., Ed.; CRC Press: Boca Raton, FL, 1993; pp 27-46.
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Figure 4. Effect of repeated oxalate treatment on leonardite solubilization. Reaction mixtures contained 1 g of leonardite and 100 mL of 74.6 mM sodium oxalate in water. Reaction mixtures were stirred for 24 h and centrifuged (1400g, 15 min). A 1 mL aliquot was then removed and centrifuged at 11000g for 4 min, and the A600 of the supernatant was determined. The pellet was then resuspended in 100 mL of 74.6 mM sodium oxalate, and this process was repeated nine more times.
Figure 5. Effect of leonardite on its own solubilization. Reaction vessels containing the requisite amounts of leonardite and volumes of 74.6 mM sodium oxalate were placed on a rotary shaker for 24 h after which time the amount of leonardite solubilized was determined gravimetrically. Experiments were performed in triplicate.
experimental data and were the first to note the fact that in such studies the divalent hydrogen phosphate ion (HPO42-) functions as a Lewis base (an electron pair donor) and, like oxalate, is a good metal chelator and is able to solubilize leonardite. It is, therefore, necessary to better understand the role of pH in solubilization of low rank coal and to be able to distinguish between base (hydroxide ion)-mediated solubilization and solubilization that is due to ions functioning as Lewis bases. The effect of pH on leonardite solubilization in the presence of sodium oxalate, potassium phosphate, and (19) Wilson, B. W.; Pyne, J. W.; Bean, R. M.; Stewart, D. L.; Lucke, R. B.; Thomas, B. L.; Thomas, M. T.; Campbell, J. A.; Cohen, M. S. In Proc. 12th Annu. EPRI Contractors Conference on Fuel Science and Conversion; Electric Power Research Institute: Palo Alto, CA, 1988; Vol. 3, p 1.
Figure 6. Effect of pH on leonardite solubilization in the presence of several Lewis bases. In 20 mL reaction vials each reaction mixture contained 20 mg of leonardite and, at the pH values indicated, 10 mL of sodium oxalate (74.6 mM), potassium phosphate/hydrogen phosphate/dihydrogen phosphate (75 mM), sodium bicarbonate/carbonate (75 mM), or the indicated concentration of sodium hydroxide. Reaction vials were placed on a rotary shaker for 24 h after which time aliquots were centrifuged and the absorbance at 600 nm was determined (Figure 6a). In separate experiments the amount of leonardite solubilized was determined gravimetrically at the pH values indicated (Figure 6b). Gravimetric experiments were performed in triplicate.
sodium bicarbonate/carbonate is presented in Figure 6. Solubilization as a function of sodium hydroxide concentration is also presented in Figure 6. The oxalate ion system (monovalent and divalent oxalate ion) was selected, of course, because of its importance in the biological solubilization of coal by wood-rotting fungi. The phosphate/hydrogen phosphate/dihydrogen phosphate and bicarbonate/carbonate systems were selected because of their importance and abundance as buffers in the environment. Sodium hydroxide was studied because, as noted above, it is important to be able to distinguish between base-mediated solubilization that is caused by high concentrations of hydroxide ion and solubilization that is mediated by oxalate, phosphate, and bicarbonate/carbonate ions functioning as Lewis bases (i.e., as metal chelators). The effect of pH on leonardite solubilization in the presence of each of the three Lewis bases studied is presented individually in
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Figure 9. Effect of pH on leonardite solubilization in the presence of potassium phosphate. The data describing phosphate/hydrogen phosphate/dihydrogen phosphate-mediated solubilization of leonardite from Figure 6 was replotted to illustrate the effect of ionization on solubilization. Figure 7. Effect of pH on leonardite solubilization in the presence of sodium oxalate. The data describing oxalatemediated solubilization of leonardite from Figure 6 was replotted to illustrate the effect of ionization on solubilization.
Figure 8. Effect of pH on leonardite solubilization in the presence of sodium bicarbonate/carbonate. The data describing bicarbonate/carbonate-mediated solubilization of leonardite from Figure 6 was replotted to illustrate the effect of ionization on solubilization.
Figures 7, 8, and 9, respectively. In all cases, results showed that for each Lewis base studied, solubilization of leonardite increased as pH increased. Furthermore, it was shown that optimal solubilization occurred at pH values at which the species in question existed as the divalent (or higher valent) anion. Of importance is the fact that substantial solubilization occurred at pH values well below those at which hydroxide-mediated solubilization takes place.
Discussion Early coal solubilization investigations by Cohen and Gabriele4 focused on the ability of lignin-degrading fungi to solubilize a North Dakota lignite. The selection of lignin-degrading fungi appeared to be a logical choice based on the perceived structural resemblance of lignite and lignin; the idea being that lignin-degrading enzymes might also be responsible for solubilization of lignite. Although lignite and lignin do bear a number of overall similarities, it is important to stress that there are also a number of important differences. For example, most
proposed structures1,20,21 of low rank coal suggest that aromatic substructures of lignite and other low rank coals are somewhat more condensed than those found in lignin. Also, phenyl ether linkages do not appear to be as important in the structure of lignite1,20,21 as they are in lignin.22 A profound difference between lignin and lignite is the fact that in low rank coal polyvalent cations form ionic bonds between coal substructures. Comparable substructures are not found in lignin. As noted previously, it has been shown that fungi secrete oxalate ions which, in turn, chelate polyvalent cations in low rank coal, and it is this process that renders the macromolecules water soluble.7 Although it has been reported that laccase, or possibly other fungal enzymes, might have a role in solubilization of low rank coal, it is now generally accepted that chelation by oxalate (or possibly other chelators) is the predominant mechanism responsible for solubilization of low rank coal by T. versicolor and other fungi, including P. chrysosporium. Similarly there is no evidence that the other major oxidative enzymes important in lignin degradation (lignin peroxidases, Mn peroxidases) have a substantial role in solubilization of low rank coal. It should be noted, however, that several research groups have reported that lignin peroxidases and Mn peroxidases from white rot fungi have a role in the subsequent depolymerization of soluble coal macromolecule.23-26 In the present investigation, we have confirmed that T. versicolor and P. chrysosporium are able to solubilize an oxidized North Dakota lignite (leonardite). A biomimetic approach was used in an effort to better understand the role of oxalate ion in coal solubilization. Solubilization was shown to be a function of oxalate concentration between 0 and ∼75 mM oxalate, and 75 mM appeared to be near optimal under the conditions used in this study. These results are important from a (20) Huttinger, K. J.; Michenfelder, A. W. Fuel 1987, 66, 1164-1165. (21) Gorbaty, M. L. Fuel 1994, 73, 1819-1828. (22) Freudenberg, K. Science 1965, 148, 595-600. (23) Wondrak, L.; Szanto, M.; Wood, W. A. Appl. Biochem. Biotechnol. 1989, 20/21, 765-780. (24) Ralph, J. P.; Graham, L. A.; Catcheside, D. E. A. Appl. Microbiol. Biotechnol. 1996, 46, 226-232. (25) Hofrichter, M.; Fritsche, W. Appl. Microbiol. Biotechnol. 1997, 47, 419-424. (26) Hofrichter, M.; Fritsche, W. Appl. Microbiol. Biotechnol. 1997, 47, 566-571.
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biological perspective because the wood-rotting fungi that have been studied do not, in liquid culture, produce concentrations of oxalate near, or even approaching, those optimal for low rank coal solubilization. For example, Dutton et al.16 studied the comparative ability of stationary liquid cultures of white rot and brown rot fungi to produce oxalate when grown on FahreusReinhammer medium using 1% glucose as the carbon source. They showed that oxalate concentration varied from 0.04 to 10.0 mM for the white rot fungi and from 2.0 to 20.0 mM for the brown rot fungi. These concentrations are less than optimal, but some are still high enough to solubilize substantial amounts of low rank coal. It should be noted, however, that oxalate concentrations produced by wood-rotting fungi in cultures grown on solid or agar media are likely to be much greater than those found in liquid media. This is not necessarily because more oxalate is secreted but because of the low amounts of water present and the fact that diffusion of secreted oxalate would occur relatively slowly (unlike the situation in liquid cultures). These two factors would be expected to combine to produce high local concentrations of oxalate. Indeed, several successful coal solubilization experiments have been performed in agar media.4,9 In addition to wood-rotting fungi, many other fungi are able to solubilize low rank coals. Penicillium spp. and Aspergillus spp. appear to be particularly effective.9,13 These studies9,13 were performed before the discovery of oxalate involvement in low rank coal solubilization. In retrospect it is not surprising that Penicillium spp. and Aspergillus spp. are good coal solubilizers as these species have long been known to produce prodigious amounts of oxalate (ref 27 and references therein). This, of course, raises the possibility that fungi other than wood-rotting fungi may be more effective coal-solubilizing agents. This also suggests that, if biosolubilization procedures are to be pursued, it may be prudent to screen for coal solubilization those fungi that are known to produce high concentrations of oxalate. In particular, Sclerotium spp. may be good candidates. For example, the plant pathogen Sclerotium glucanicum has been reported to be able to produce oxalate in concentrations up to 142 mM when cultured on 87.6 mM sucrose.28 This represents conversion of 27% of the total available carbon source to oxalate. Lichens, a symbiotic relationship between algae and fungi, also produce large amounts of oxalate. However, they grow slowly (ref 27 and references therein). The effect of pH on low rank coal solubilization is important. Fowkes and Frost17 used 1 M sodium hydroxide (pH 14) to solubilize several different lignites. This is an effective method. However, it seems that it would be preferable to develop conditions by which low rank coals are solubilized under less harsh conditions. Indeed, this is one reason there is considerable interest in the use of microorganisms for such purposes. (27) Hodgkinson, A. Oxalic Acid in Biology and Medicine; Academic Press: New York, 1977; p 325. (28) Pierson, P. E.; Rhodes, L. H. Mycologia 1992, 84 (3), 467469.
Bumpus et al.
Although coal solubilization is mediated under strongly basic conditions, it has been noted that certain Lewis bases are also able to mediate solubilization of low rank coal at pH values considerably lower than those used to effect solubilization by strong base.6,29 We studied the effect of pH on the ability of oxalate, phosphate, and bicarbonate/carbonate ions to mediate solubilization of leonardite. Of interest is the observation that, in all cases, optimal solubilization occurs at pH values at which the species in question exists as the divalent (or higher valent) anion. At higher pH (>11), the increase appears to be be due to solubilization due to the presence of strong base (i.e., hydroxide ion). These results are important from a biological perspective. For example, many fungi grow at acidic pH; the optimum physiological pH of P. chrysosporium for growth and for lignin degradation has been reported to be between pH 4 and 5.5.30 Clearly this pH range is below that which is optimal for oxalate-mediated solubilization of leonardite. In contrast, Dutton et al.16 reported that when grown on Fahraeus-Reinhammer medium P. chrysosporium achieved and maintained a pH of ∼7.7, a pH value that would be more amenable to oxalate-mediated solubilization of leonardite. Elucidation of the details surrounding solubilization and biodegradation of low rank coal and other hard-todegrade organic materials is of interest and importance in its own right. However, because soluble coal macromolecule has been proposed for use as a substrate for methane production, the economics of coal solubilization for this purpose must be considered. This issue has been addressed by Isbister and Barik2 who note that two general strategies have been pursued in an attempt to convert low rank coal to methane. During indirect biogasification, coal is first solubilized chemically or biologically followed by anaerobic fermentation to form methane and carbon dioxide. The solubilization step is logical because solubilization of solid substrates is usually the rate-limiting step in their degradation.2,31 It has been demonstrated that conversion of soluble carbon in coal to methane can be reasonably high (ref 2 and references therein). However, when based on total carbon (soluble + insoluble carbon), conversion to methane has been reported to be “unacceptably low”.2,32 For this reason, a second strategy, direct biogasification of coal, has been pursued in which methanogenic bacteria are used to treat solid coal without an initial solubilization step. Direct systems for coal conversion are prone to problems with management of bacterial consortia and problems inherent in mixed phase bioreactors. They are also prone to have long retention times.33 Despite such problems, direct biogasification of coal by microorganisms, at present, appears to be the most promising approach.2 Our results might be important to both strategies. For indirect biogasification, solubilization by a very (29) Fakoussa, R. M. Fuel Process. Technol. 1994, 40, 183-192. (30) Kirk, T. K.; Schultz, E.; Conners, W. J.; Lorenz, L. F.; Zeikus, J. G. Arch. Microbiol. 1978, 117, 277-285. (31) Grethlein, H. E. In Bioprocessing and Biotreatment of Coal; Wise, D. L., Ed.; Marcel Dekker: New York, 1990; pp 73-81. (32) Department of Energy, Pittsburgh Energy Technology Center Final Report: Development of a novel approach for coal bioconversion to alcohol fuels. Contract No. DE-AC22-88PC88815, May 1991. (33) Klasson, K. T.; Ackerson, M. D.; Clausen, E. C.; Gaddy , J. L. In Microbial Transformations of Low Rank Coal; Crawford, D. L., Ed.; CRC Press: Boca Raton, FL, 1993; pp 93-110.
Biomimetic Solubilization of a Low Rank Coal
inexpensive Lewis base would be economically advantageous. For direct biogasification processes, inclusion of a Lewis base at moderate pH values could reasonably be expected to decrease bioreactor retention times (i.e., by increasing the rate of methanogenesis) because the rapid solubilization would result in increased and rapid availability of a more easily metabolized substrate. Perhaps our most important observation is that the sodium bicarbonate/carbonate system effectively solubilizes leonardite. Sodium bicarbonate/carbonate is abundant and very inexpensive, and the bicarbonate/ carbonate ion could also function as a substrate during methanogenesis. On the other hand, the large amounts of solubilizing agents required might be prohibitive from a materials-handling perspective on an industrial scale. If, however, the solubilized coal macromolecule from
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leonardite does become a viable carbon source for use in anaerobic methane production, these studies suggest that chemical solubilization by common inexpensive Lewis bases would likely be more cost competitive than fungal solubilization processes. Acknowledgment. This research was sponsored by the United States Department of Energy (Grant DEFG22-94PC94209). Gregory Lynd, Kimberly Sturm, and John Senko were supported by the United States Department of Energy University Coal Research Internship Program. Partial support from NSF Grant CHE 9531791 is also gratefully acknowledged. EF9701596
