Anaerobic Degradation of Soluble Fractions of [14C-Lignin]Lignocellulose

[¹⁴C-lignin]lignocellulose 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.     

Thermophilic anaerobic biodegradation of [C]lignin, [C]cellulose, and [C]lignocellulose preparations

Thermophilic (55 degrees C) anaerobic enrichment cultures were incubated with [C-lignin]lignocellulose, [C-polysaccharide]lignocellulose, and kraft [C]lignin prepared from slash pine, Pinus elliottii, and C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Thermophilic Anaerobic Biodegradation of [14C]Lignin, [14C]Cellulose, and [14C]Lignocellulose Preparations

Thermophilic (55°C) anaerobic enrichment cultures were incubated with [¹⁴C-lignin]lignocellulose, [¹⁴C-polysaccharide]lignocellulose, and kraft [¹⁴C]lignin prepared from slash pine, Pinus elliottii, and ¹⁴C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Methanogenic Decomposition of Ferulic Acid, a Model Lignin Derivative

Ferulic acid, a model lignin derivative, was observed to be biodegradable to methane and carbon dioxide under strict anaerobic conditions. This conversion appears to be carried out by a consortium of bacteria similar to that previously described for the methanogenic degradation of benzoic acid. A temporary buildup of acetate in these cultures indicates that it is a likely intermediate and precursor for methane formation. An analog of coenzyme M, 2-bromoethanesulfonic acid (BESA), inhibited gas production and enhanced the buildup of propionate, butyrate, isobutyrate, and isovalerate. Phenylacetate, cinnamate, 3-phenylpropionate, benzoate, cyclohexane carboxylate, adipate, and pimelate were also detected in BESA-inhibited cultures. A pathway is proposed which includes these various acids as possible intermediates in the methanogenic degradation of ferulic acid. This model overlaps previously described benzoic acid degradation pathways, suggesting that this type of anaerobic degradation may be common for aromatic compounds.     

Microbial acetate conversion to methane: kinetics,yields and pathways in a two-step digestion process

Summary Organic waste is converted in a two-stage process to methane and carbon dioxide by mixed cultures of microorganisms. Acetate, a product of acidogenic and acetogenic bacteria and the main substrate for methanogenic bacteria, is an important intermediate of the anaerobic degradation process, which results in the generation of methane. It was shown by labelling experiments using (U-¹⁴C) acetate that as much as 65%–96% of the total methane produced came from the acetate. The first order utilization rate for acetate in the methanogenic stages of a two-stage digestion process was between 0.17 h^-1 and 0.5 h^-1. The kinetics as well as the mass flow and yields of acetate and the methyl group of acetate were determined by pulse-labelling experiments with (U-¹⁴C) acetate and (2-¹⁴C) acetate without a significant rise of the total concentrations. Up to 58% of the acetate carbon was transformed to methane, and about 30% to carbon dioxide; only 4%–15% was incorporated into the biomass. There are at least two parallel degradation mechanisms in the metabolic transformation of acetate to methane: acetate is cleaved either to form methane and carbon dioxide or to form hydrogen and carbon dioxide, which can be transformed by an additional reaction to methane. Labelling experiments with (2-¹⁴C) acetate show that both mechanisms took place at similar order.     

Methanogenic Degradation of Poly(3-Hydroxyalkanoates)

Poly(3-hydroxybutyrate) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) were fermented to methane and carbon dioxide within 16 days by an anaerobic sewage sludge consortium. The cultures adapted quickly to metabolize these polymeric compounds, and between 83 and 96% of the substrate carbon was transformed to methane and carbon dioxide.     

Anaerobic microbial associations degrading aromatic amino acids

Anaerobic microbial associations have been isolated that degrade aromatic amino acids to methane and carbon dioxide at high rates. Significant differences between the morphological, cytological, and physiological traits of cultures isolated from samples of adapted and unadapted sludge are shown. The effects of cultivation temperature, illumination, and presence of mineral nitrogen and bicarbonate in the medium upon adaptation of enrichment cultures to substrates and subsequent behavior of the anaerobic associations have been studied. Intermediate and final products of degradation of aminoaromatic compounds and the sequence of their formation in the cultures have been determined. We have also studied the effects of exogenous electron acceptors and additional carbon sources on the degradation of aminoaromatic compounds.     

Oxidation of methane in the absence of oxygen in lake water samples.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.     

Oxidation of methane in the absence of oxygen in lake water samples.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.     

Microbial transformation of styrene by anaerobic consortia

Methanogenic microbial consortia, originally enriched from anaerobic sewage sludge with ferulic acid or styrene (vinylbenzene) as sole organic carbon and energy sources, were used to study transformation of styrene under strictly anaerobic conditions. Styrene, which was added as the substrate in a range of concentrations from 0.1 to 10 mmol/l, was extensively degraded but no methane production was observed during incubation for eight months. The addition of yeast extract during the enrichment stage completely inhibited degradation of styrene. Gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), high performance liquid chromatography (HPLC) analyses of the culture fluid, and GC analyses of the anaerobic headspace, indicated that the transformation of this arylalkene was initiated through an oxidation-reduction reaction and that the favoured mechanism was most likely the addition of water across the double bond in the alkenyl side-chain. The degradation proceeded through to carbon dioxide, the final product. Benzoic acid and phenol were transient compounds found in highest concentrations in the spent culture fluid and are suggested as the key intermediates of the transformation process. The tentative routes of anaerobic transformation partially overlap with those previously proposed for aromatic hydrocarbons such as toluene. Several pure cultures, which were tentatively identified as Clostridium spp. and Enterobacter spp., were isolated from the styrene-degrading consortia. Two of these cultures were demonstrated to grow on styrene as sole carbon and energy source. Additionally, a pure culture of Enterobacter cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.     

Conversion of glucose to fatty acids and methane: roles of two mycoplasmal agents.

Two species of obligately anaerobic mycoplasmas were the major components of a methanogenic glucose-limited enrichment culture. In pure culture, one of these organisms, tentatively named Anaeroplasma sp. strain London, was shown to be responsible for the fermentation of glucose to fatty acids, hydrogen, and carbon dioxide; the other mycoplasma was shown to produce methane from hydrogen and carbon dioxide and was named Methanoplasma elizabethii. This same methanogenic mycoplasma contained a low-molecular-weight fluorescent cofactor which had a maximum light absorbance at 430 nm. When both species of mycoplasmas were grown together on glucose, fermentation products included fatty acids and methane. For the first time, mycoplasmas are implicated as agents of anaerobic degradation and methanogenesis in a sewage sludge digester.     

Anaerobic Biodegradation of Indole to Methane

Methane gas was produced from a laboratory, granular activated carbon, anaerobic filter treating a synthetically prepared mixture of polycyclic N-aromatic compounds. The biodegradability of the individual polycyclic N-aromatic compound present in the mixture was investigated. Experimental results obtained from test bottles containing methanogenic enrichment cultures suggested that indole was degraded to methane and carbon dioxide under strict anaerobic conditions.     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia.

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.     

Microbial transformation of styrene by anaerobic consortia

Methanogenic microbial consortia, originally enriched from anaerobic sewage sludge with ferulic acid or styrene (vinylbenzene) as sole organic carbon and energy sources, were used to study transformation of styrene under strictly anaerobic conditions. Styrene, which was added as the substrate in a range of concentrations from 0.1 to 10 mmol/l, was extensively degraded but no methane production was observed during incubation for eight months. The addition of yeast extract during the enrichment stage completely inhibited degradation of styrene. Gas chromatog-raphy (GC), gas chromatography/mass spectrometry (GC/MS), high performance liquid chromatography (HPLC) analyses of the culture fluid, and GC analyses of the anaerobic headspace, indicated that the transformation of this arylalkene was initiated through an oxidation-reduction reaction and that the favoured mechanism was most likely the addition of water across the double bond in the alkenyl side-chain. The degradation proceeded through to carbon dioxide, the final product. Benzoic acid and phenol were transient compounds found in highest concentrations in the spent culture fluid and are suggested as the key intermediates of the transformation process. The tentative routes of anaerobic transformation partially overlap with those previously proposed for aromatic hydrocarbons such as toluene. Several pure cultures, which were tentatively identified as Clostridium spp. and Enterobacter spp., were isolated from the styrene-degrading consortia. Two of these cultures were demonstrated to grow on styrene as sole carbon and energy source. Additionally, a pure culture of Enterobacter cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.     

硝酸盐和硫酸盐厌氧氧化甲烷途径及氧化菌群

甲烷属于温室气体,厌氧氧化甲烷有效地减少了大气环境中甲烷的含量。依据吉布斯自由能变,以SO42、Mn4+、Fe3+、NO3等作为电子受体,厌氧条件下甲烷可以转化为CO2。重点阐述以SO42和NO3为电子受体时甲烷厌氧氧化的机理、反应发生的环境条件以及甲烷厌氧氧化菌的特点。针对目前研究存在的主要问题,提出了今后的发展方向。SO42为电子受体时,甲烷厌氧氧化的可能途径包括:逆甲烷生成途径、乙酰生成途径以及甲基生成途径。甲烷的好氧或厌氧氧化协同反硝化是以NO3为电子受体的甲烷氧化的可能途径。环境中的甲烷、硫酸盐或硝酸盐的浓度,有机质的数量,以及环境条件对甲烷的厌氧氧化有显著影响。     

Anaerobic degradation of toluene and o-xylene by a methanogenic consortium.

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.     

Anaerobic Microbial Associations Degrading Aminoaromatic Acids

Anaerobic microbial associations have been isolated that degrade aminoaromatic acids to methane and carbon dioxide at high rates. Significant differences between the morphological, cytological, and physiological traits of cultures isolated from samples of adapted and unadapted sludge are shown. The effects of cultivation temperature, illumination, and presence of mineral nitrogen and bicarbonate in the medium upon adaptation of enrichment cultures to substrates and subsequent behavior of the anaerobic associations have been studied. Intermediate and final products of degradation of aminoaromatic compounds and the sequence of their formation in the cultures have been determined. We have also studied the effects of exogenous electron acceptors and additional carbon sources on the degradation of aminoaromatic compounds.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 422–428.Original Russian Text Copyright © 2005 by Kotova, Savel’eva, D’yakonova, Sklyar, Kalyuzhnyi, Stams, Netrusov.     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.