phenol esters during degradation of a lignosulfonate prepa- ... Preparation of lignin-derived substrate. ... fraction by Crawford (9) for a Douglas fir preparation. To.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1985, p. 345-349 0099-2240/85/020345-05$02.00/0 Copyright C 1985, American Society for Microbiology
Vol. 49, No. 2
Anaerobic Degradation of Soluble Fractions of [14C-Lignin]Lignocellulose P. J. COLBERG1t AND L. Y. YOUNG2*
Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford, California 94305,1 and Department of Environmental Medicine and Department of Microbiology, New York University Medical Center, New York, New York 100162 Received 13 August 1984/Accepted 26 November 1984
[14C-ligninJlignocellulose was solubilized by alkaline heat treatment and separated into different molecular size fractions for use as the sole source of carbon in anaerobic enrichment cultures. This study is aimed at determining the fate of low-molecular-weight, polyaromatic lignin derivatives during anaerobic degradation. Gel permeation chromatography was used to preparatively separate the original 14C-lignin substrate into three component molecular size fractions, each of which was then fed to separate enrichment cultures. Biodegradability was assessed by monitoring total carbon dioxide and methane production, evolution of labeled gases, loss of 14C-activity from solution, and changes in gel permeation chromatographic elution patterns. Results indicated that the smaller the size of the molecular weight fraction, the more extensive the degradation to gaseous end products. In addition, up to 30% of the entire soluble lignin-derived carbon was anaerobically mineralized to carbon dioxide and methane. Lignin is a major source of reduced carbon in the biosphere, and its recalcitrance to biodegradation is considered to restrict the cycling of other photosynthate carbons, both in the natural environment and in biomass conversion systems. Although significant quantities of lignin-containing plant materials eventually enter anaerobic zones and are believed to be the rate-limiting components in methane production processes, most studies to date have considered lignocellulose degradation under aerobic conditions only. Recently, Benner et al. (1) reported that the lignin component of intact softwoods and hardwoods is partially degraded to gaseous end products under anaerobic conditions. Work by Healy and Young (11, 12) and Healy et al. (13) has shown that a consortium of anaerobic bacteria enriched from digester sludge is capable of cleaving the aromatic rings of 11 simple lignin derivatives. In addition, the aromatic substrates are stoichiometrically fermentable to carbon dioxide and methane. Recent studies by Schink and Pfennig (17) and Kaiser and Hanselmann (13a) have examined the biodegradation of several substituted aromatic compounds by enrichments and isolates from lake and marine muds. Aerobic studies indicate that lignin-degrading fungi and actinomycetes are capable of releasing aromatic and oligolignol compounds of reduced molecular size during catabolism of lignin-containing substrates. Haars and Huttermann (10) detected small-molecular-weight (MW) phenols and phenol esters during degradation of a lignosulfonate preparation by a member of the class Basidiomycete. Clayton and Srinivasan (2) detected intermediate MW aromatic compounds (two to three rings) by gel permeation chromatography (GPC) during batch culture growth of a Candida sp. on a Kraft lignin waste. Reid and Pettersson (Seminar in Biotechnology in the Pulp and Paper Research Industry, Pulp and Paper Research Institute of Canada, Quebec, 14 to 16 September, 1981) report significant release of water-sol-
uble products from ground wood by the white-rot fungus Phanerochaete chrysosporium, whereas Phelan et al. (16) and Crawford et al. (6) detected solubilization of lignocellulose by Streptomyces spp. Crawford et al. have recently characterized some of these lignin fragments (5, 8). Soluble lignin fragments, therefore, can be released into the environment by natural and industrial processes. We recently have demonstrated in our laboratory that a mixture of oligolignols is degraded by a mixed bacterial population under strictly anaerobic culture conditions to yield end products of methanogenesis (3). The oligolignol mixture was prepared from natural lignocellulose and spanned the MW range of 200 to 1,400. We now report the results of a study in which the lignin-derived substrate is separated into three component molecular size fractions. Each fraction is then used as the sole source of carbon for the degradation studies by strictly anaerobic enrichment cultures. MATERIALS AND METHODS Preparation of lignin-derived substrate. [14C-lignin]lignocellulose was prepared from Douglas fir by the method of Crawford et al. (6). Phenylalanine was fed as aqueous L-[U-14C]phenylalanine to freshly cut twigs of Douglas fir, with alternating light and dark uptake periods for 1 week. The bark and needles were removed and discarded. The inner wood was dried at 100°C and ground in a Wiley Mill to pass 20 mesh, followed by a series of soxhlet extractions, including alcohol-benzene to remove toxic extractables (e.g., resins, fats, waxes), ethanol to remove tannins, and water to remove unincorporated label and other soluble organics. Klason analysis indicated that 67% of the recovered 14C-label is in the acid-insoluble lignin fraction. This is in excellent agreement with the 66% reported as recovered in the lignin fraction by Crawford (9) for a Douglas fir preparation. To produce a size range of soluble lignin-derived compounds, fragments of the natural, 14C-labeled softwood were prepared for degradation experiments in the following manner. A slurry of '4C-labeled wood was treated with NaOH (400 meq/liter) and heated at 200°C for 1 h in a bomb-type auto-
* Corresponding author. t Present address: Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH-8600 Dubendorf, Switzerland.
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clave (minireactor no. 4562; Parr Instrument Co., Moline, Ill.). The reactor was flushed with nitrogen and stirred continuously, and temperature was monitored by a thermocouple. The reaction mixture was settled, and the supernatant liquid was decanted, filtered, and neutralized to pH 7. About 66% of the lignin (Klason analysis) is solubilized in this manner. This alkaline treatment readily solubilizes lignin, whereas little solubilization of cellulose occurs. This was confirmed by testing for free glucose in solution by the procedure of Moore and Johnson (15). Furthermore, when samples of the 14C-labeled substrate were incubated with cellulase, no glucose release was detected. This lignin solubilization procedure resulted in oligolignols in the same MW range as the lignin fragments observed in the degradation of lignin by P. chrysosporium (B. Pettersson, personal communication). The lignin-derived substrate was preparatively separated into its component molecular size fractions by GPC. The column (1.5 by 50 cm) was packed with Sephadex LH-20 (Sigma Chemical Co., St. Louis, Mo.). Elution was by descending chromatography in 1:1 dioxane-water at a flow rate of 1 ml/min. Elution patterns of lignin-derived compounds were obtained by liquid scintillation counting of collected fractions in Aquagel (Packard Instrument Co., Downers Grove, Ill.). Numerous replicate runs were made on the column to obtain enough of each fraction to be used as a carbon source. Collected aliquots of similar molecular size were pooled into three separate molecular size fractions, dried under vacuum by rotary evaporation at 50 to 55°C, and reconstituted in a defined mineral salts medium (11) for use as substrates in the biodegradation studies. The total organic carbon (TOC) of the dried, reconstituted fractions was determined with a TOC analyzer (Dohrman Div., Envirotech Corp., Santa Clara, Calif.). Approximately 300 to 350 mg of C/liter was fed as the substrate. Biodegradation cultures. A serum bottle modification of the Hungate technique was adapted from Miller and Wolin (4). Prereduced, defined medium was inoculated with 10% (vol/vol) seed from a laboratory anaerobic digester which was fed waste activated sludge. The defined medium and its preparation have been previously described (11). Serum bottles (capacity, 120 ml) were flushed with oxygen-free
nitrogen for 5 to 10 min before the addition of media, substrate, and inocula. The bottles were closed with butyl rubber stoppers and crimped aluminum seals. The headspace was composed of 70% nitrogen and 30% carbon dioxide. The cultures were incubated in the dark at 35°C. Each of the molecular size fractions was used as the sole source of carbon in duplicate cultures. Background gas controls were not fed any carbon source, and autoclaved control cultures were run for each fraction. Gas analyses. A wetted glass syringe was inserted directly into the culture bottle for gas volume measurements. Produced gas was measured as the displaced volume of the syringe plunger with a reproducibility of ±1%. Gas-tight syringes were used for all other analyses. Gas composition was determined by GPC (model 25 V; Fisher Scientific Co., Pittsburgh, Pa.). Certified gas standards were used for standardization of methane, carbon dioxide, and nitrogen. One milliliter of gas from the headspace was removed with a Pressure Lok Syringe (Precision Sampling Co., Baton Rouge, La.) and analyzed for ['4C]methane by a direct liquid scintillation counting method (18). Another milliliter of headspace gas was removed for determination of ['4C]carbon dioxide (19). Radioactivity was counted in a Tri-Carb 3330 Liquid Scintillation Spectrophotometer (Packard Instrument Co.). Quench corrections were made by the channels-ratio method. RESULTS
Figure 1 illustrates the GPC profile of the original 14C-preparation before degradation. A series of repeated GPC separations on Sephadex LH-20 were necessary to collect sufficient quantities of the individual fractions to pool for use as substrates for anaerobic enrichment cultures. Fraction 1 is composed of oligolignols which elute in the approximate MW range of 1,000 to 1,400; oligolignols in fraction 2 are in the MW range of 400 to 1,000; and molecules in fraction 3 are in the MW range of less than 400. Fractions 1, 2, and 3 comprise 27, 60, and 13%, respectively, of the TOC of the total soluble lignin substrate. Figure 2 depicts the resultant GPC patterns when measured aliquots were taken from the cultures fed fractions 1, 2, and 3 and again chromatographed on Sephadex LH-20 gel
ANAEROBIC DEGRADATION OF [14C-LIGNIN]LIGNOCELLULOSE
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after 30 days of anaerobic incubation. The individual profiles have been plotted together for evaluation. After degradation, fraction 1 exhibited a change in its elution pattern, suggesting that the average MW of the original substrate had been reduced, thus yielding compounds of smaller MW. Fractions 2 and 3 do not display such a shift but do show some reductions in total area. These data are in good agreement with our previous results (3), in which the fractions were not separated, and the entire soluble lignin, comprised of all three fractions, was fed as the sole source of carbon to anaerobic enrichment cultures. The cumulative moles of carbon dioxide and methane produced by the enrichment cultures from each of the fractions are illustrated in Fig. 3. Total gas produced appears
to be inversely related to molecular size; that is, the smallest MW fraction (fraction 3) yielded the most gas, whereas the largest MW fraction (fraction 1) yielded the least. Approximately 21, 32, and 40% of the total carbon in fractions 1, 2, and 3, respectively, underwent complete conversion to gaseous end products. Gas evolution from the largest MW fraction appears to level off first on day 11, whereas fraction 2 produces gas until day 17. The smallest MW fraction did not reach a maximum until day 39. The relative rates of cumulative gas production for fractions 1 and 2 are statistically greater than that for fraction 3. Figure 4 depicts methane production per unit of substrate carbon added for each of the three molecular size fractions. Of the total gas produced (Fig. 3), methane accounts for ca.
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30% of fractions 1 and 2 and 63% of fraction 3. This suggests that soluble lignin fragments or oligolignols of MW 1,400 and 1,400 and smaller can serve as a methane source in biomass conversion processes and in the anaerobic regions of the natural environment. It also appears that during the mineralization of the smallest MW fraction (MW,
