Biodegradation of lignin-derived molecules under ...

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LH-20 gel (Sigma Chemical Co., St. Louis, MO). Elution was by .... MCCARTY, P. L., L. Y. YOUNG, J. M. GOSSETT, D. C.. STUCKEY, and J. B. HEALY, JR. 1976.
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BARRETTO DE MENEZES, T. J., D. F. SPLITTSTOESSER, and J. zation of wines fermented with various malo-lactic bacteria. R. STAMER. 1972. Induced malo-lactic fermentation of New Appl. Microbiol. 14: 608-618. York State wines. N.Y. Food Life Sci. 5: 24-26. 1976. Stimulatory effect of malo-lactic fermentation CASTINO,M., L. USSEGLIO-TOMASSET, and A. GANDINI. on the growth rate of Leuconostoc oenos. Appl. Environ. 1975. Factors which affect the spontaneous initiation of Microbiol. 32: 405-408. malo-lactic fermentation in wines. The possibility of RADLER, F. 1967. Etude microbiologique des bacteries de la fermentation malo-lactique. Connaissance Vigne Vin, 3: transmission by inoculation and its effect on organoleptic properties. In Lactic acid bacteria in beverages and foods. 73-91. Academic Press, New York. pp. 139-148. ROGOSA,M., R. F. WISEMAN,J. A. MITCHELL,M. N. DEWEY,M., and J. CONKLIN.1960. Starch gel electro1953. Species differentiaDISRAELY, and A. J. BEAMAN. phoresis of lactic dehydrogenase from rat kidneys. Proc. tion of oral lactobacilli from man including descriptions of Soc. Exp. Biol. Med. 105: 492. Lactobacillus salivarius nov. spec. and Lactobacillus IMAMOTO,S., T . AMACHI,and H. YOSHIZUMI.1973. cellobiosus nov. spec. J . Bacterial. 65: 681-699. Syntheses of some derivatives of glycosyl panthothenic STRUHL,K., D. T . STINCHCOMB, S. SCHERER,and R. W. acids, analogues of growth factor for MLF bacteria. Agric. DAVIS. 1979. High-frequency transformation of yeast: Biol. Chem. 37: 544-55 1. autonomous replication of hybrid DNA molecules. Proc. KUNKEE,R. E. 1967. Malo-lactic fermentation. Adv. Appl. Natl. Acad. Sci. U.S.A. 73: 1035- 1039. Microbiol. 9: 235-279. VAN DER WESTHUIZEN, L. M., W. A. AGENBACH, M. A. 1968. A simplified chromatographic procedure for the Loos, and N. F. SCHOOMBEE.1981. Comparison of detection of malo-lactic fermentation. Wines Vines, 49(3): procedures for isolation of malo-lactic bacteria from wine. 23-24. Am. J. Enol. Vitic. 32:.168-170. PILONE,G. J., and R. E. KUNKEE.1966. Chemical characteri-

Biodegradation of lignin-derived molecules under anaerobic conditions P. J. COLBERG AND L. Y. YOUNG' Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford, CA, U.S.A. 94305 Accepted March 16, 1982 COLBERG, P. J., and L. Y. YOUNG.1982. Biodegradation of lignin-derived molecules under anaerobic conditions. Can. J. Microbiol. 28: 886-889. Soluble fragments of lignin origin obtained by thermochemical treatment of [14C]lignin-labeled lignocellulose served as substrate for anaerobic enrichment cultures. This study demonstrates that oligolignols, thought to be refractory under anaerobic conditions, can be degraded to lower molecular weight compounds and to methane and carbon dioxide. COLBERG, P. J., et L. Y. YOUNG.1982. Biodegradation of lignin-derived molecules under anaerobic conditions. Can. J Microbiol. 28: 886-889. Des fractions solubles de lignine obtenues par traitement thermochimique de la lignocellulose marquk de [14C]lignineont Cti utiliskes cornme substrat pour l'enrichissement anakrobie de cultures. Cette Ctude demontre que les oligolignols, que l'on croyail rkfractaires aux conditions d'anakrobiose peuvent &tred6gradCs en composCs de poids molCculaire plus faible, en mCthane et er bioxyde de carbone. [Traduit par le journal: Almost half the global carbon which is annually fixed b y photosynthesis is incorporated into the lignocellulosic cell walls of arborescent ~ l a n t s(Bassham 1975). ' 'Author to whom coflespondence should addressed. Present address: Department of Environmental Medicine, New York University Medical Center, 550 First Avenue, New York, NY, U.S.A. 10016.

Lignin constitutes 20-30% of the dry weight of a1 vascular plant tissues (Reddy 1978) a n d ranks second tc cellulose as the most abundant. naturally occuninr polymer in the biosphere (see c r a w f o r d 1981). ~ e c a u ; of its unique smcrural properties (Reddy and Fomel 1978) lignin is believed to function a s a b a m e r to thc decomposition of plant carbohydrates. Consequently

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lignin is an integral part of the carbon cycle because it is a major repository of reduced carbon and impedes the turnover of other photosynthate carbon. Lignin is considered highly resistant to biological degradation. The white-rot fungi (Kirk 1971, 1975;Kirk et al. 1977) and several genera of aerobic bacteria (Crawford 1978; Crawford and Crawford 1976; Haider et al. 1978; Reddy 1978; Robinson and Crawford 1978; Sorenson 1962; Trojanowski et al. 1977) have been characterized as active lignin degraders. Consequently, most studies have examined the degradation of lignin under aerobic conditions only. However, significant quantities of lignin-containing plant detritus, agricultural residues, and forest litter eventually enter anaerobic environments. In addition, several recent studies report considerable solubilization of [14C]lignin-labeledlignocellulose preparations by the natural flora of a leaf-pack 1 community in a freshwater stream (K. H. Baker, personal communication) by lignin-degrading Streptomyces spp. isolated from soils and other lignocellulose-containing habitats (Phelan et al. 1979) and by the white-rot fungus Phanerochaete chrysosporium (B. Pettersson and I. D. Reid, personal communictions). This solubilization results in the release of ligninderived compounds of reduced molecular size. We now I report laboratory evidence for the anaerobic degradation of oligolignols derived from natural lignin. Phenylalanine, major precursor to lignin, was fed as aqueous L-[U-14C]phenylalanineto freshly cut twigs of Douglas fir (Pseudotsuga menziesii) (Crawford and Crawford 1976), 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 Wiley-milled to pass a 20-mesh grid, followed by a series of soxhlet extractions, including alcohol-benzene to remove toxic extractibles (e.g., resins, fats, waxes) and ethanol to remove tannins. An independent Klason -analysis indicated that 67% of the recovered label was in the lignin. This is in excellent agreement with the 66% reported by Crawford (1981). In order to produce a size range of lignin-derived compounds, soluble fragments of the natural 14C-labeled softwood were prepared for degradation experiments. Sodium hydroxide was added to a slurry of 14C-labeled wood to a final concentration 400 mequiv. /L and heat treated at 2 0 r C for-.lh in a mb-type autoclave (Parr Instrument Co., minireactor . 4562) (McCarty et al. 1976). The reactor was shed with nitrogen and stirred continuously the The ture was monitored by a mixture was and the "pernatant liquid anted* and pH 7. This atment is known to readily solubilize lignin, while no lulose solubilization occurred. This was confirmed testing for free glucose in solution according to the

procedure of Moore and Johnson (1967). In addition, when aliquots of the 14c-labeled substrate were incubated in the presence of cellulase, no glucose release was detected. This solubilization resulted in molecularsize fractions in the same molecular weight range as those reported as soluble intermediates in the degradation of lignin by Phanerochaete chrysosporium (B. Pettersson. 1981. Paper presented at Semin. Biotechnol. Pulp Pap. Ind., Pulp and Paper Research Institute of Canada, Pointe Claire, P.Q., 14- 16 September; I. D. Reid, G . D. Abrams, and J. M. Pepper. 1981. Paper presented at the TAPPI annual meeting, Chicago, IL, 2-5 March). Using a serum bottle variation of the Hungate technique (Miller and Wolin 1974), this preparation served as the sole source of carbon in anaerobic enrichment cultures (Healy and Young 1978, 1979; Healy et al. 1980). Cultures were seeded with inocula from an anaerobic, mesophilic digestor fed wasteactivated sludge. Unseeded controls and controls with no added 14c-labeled substrate were set up for all experiments. A comparison of gel filtration chromatography elution patterns of the 14C-labeled substrate both before and after anaerobic degradation is shown in Fig. 1. The column (1.5 cm X 50 cm) was packed with Sephadex LH-20 gel (Sigma Chemical Co., St. Louis, MO). Elution was by descending chromatography in a dioxme-water solvent system (1: 1, v/v) and void volume was calculated by passage of blue dextran. Retention times of reference standards of approximate molecular weights 200 and 600 were also determined. Elution patterns of lignin-derived molecules were determined by

elution volume (m L ) 1. Gel permeation chromatography profiles an e"chrnent culture fed solubilized ["c]ligni-labeled lignocellulose. Elution was both before (0)and after 30 days (0) of anaerobic incubation. The void volume (vv) was determined by passage of blue dextran. Ferulic acid (molecular weight 194) and syringic acid (molecular weight 198) were used as reference standards of molecular weight range 200; p d i phenylaminesulfonic acid (molecular weight 634) was used as the 600 molecular weight standard. FIG.

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counting 1-rnL aliquots of the collected fractions by liquid scintillation counting in Aquagel (Packard Instrument Co., Downers Grove, IL). Background counts were subtracted from all measurements. The original profile consists of three peaks with molecular weights in the ranges of 1400,700, and 300, respectively. After 30 days, the profile results in a total of eight peaks, two of which, though reduced in area, correspond to retention times of the original peaks of molecular weights 700 and 300. These data indicate that these soluble lignin components were degraded anaerobically, resulting in the production of smaller molecular fractions, as evidenced by the appearance of peaks which eluted later than the original peaks. These new peaks are in the molecular weight ranges of 900, 400, 200, and