Utilization of dimeric lignin model compounds by mixed bacterial cultures

Summary The degradation of dimeric phenylpropanoid lignin model compounds using mixed bacterial cultures was studied. The six model compounds contained the most common linkages of lignin: -O-4, -, -5, and -1. The results indicate that it is possible to enrich bacteria which are able to degrade all these compounds. Bacteria were also able to use these dimers as the sole source of carbon for growth. In view of these results it seems probable that bacterial inability to degrade polymeric lignin is due to the physical properties such as the molecular size of lignin.     

Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.     

Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture

Chlorinated ethenes are toxic substances which are widely distributed groundwater contaminants and are persistent in the subsurface environment. Reports on the biodegradation of these compounds under anaerobic conditions which might occur naturally in groundwater show that these substances degrade very slowly, if at all. Previous attempts to degrade chlorinated ethenes aerobically have produced conflicting results. A mixed culture containing methane-utilizing bacteria was obtained by methane enrichment of a sediment sample. Biodegradation experiments carried out in sealed culture bottles with radioactively labeled trichloroethylene (TCE) showed that approximately half of the radioactive carbon had been converted to 14CO2 and bacterial biomass. In addition to TCE, vinyl chloride and vinylidene chloride could be degraded to products which are not volatile chlorinated substances and are therefore likely to be further degraded to CO2. Two other chlorinated ethenes, cis and trans-1,2-dichloroethylene, were shown to degrade to chlorinated products, which appeared to degrade further. A sixth chlorinated ethene, tetrachloroethylene, was not degraded by the methane-utilizing culture under these conditions. The biodegradation of TCE was inhibited by acetylene, a specific inhibitor of methane oxidation by methanotrophs. This observation supported the hypothesis that a methanotroph is responsible for the observed biodegradations.     

Biodegradation of polyvinyl alcohol by a mixed microbial culture

A mixed culture capable of degrading 1 g l⁻¹ polyvinyl alcohol (PVA) completely was screened from sludge samples at Pacific Textile Factory, Wuxi, China. This mixed culture had stronger capability of degrading PVA with low polymerization and high saponification than degrading PVA with high polymerization and low saponification. Inorganic nitrogen source was more suitable for the mixed culture to grow and degrade PVA than organic nitrogen source. Microorganisms and relative abundance of this mixed culture were explored by terminal restriction fragment length polymorphism (T-RFLP). Small PVA molecules were detected in cell extracts of the mixed culture. This indicated that PVA degradation in the mixed culture was in fact a combined action of extracellular and intracellular enzymes. Two strains producing extracellular PVA-degrading enzyme were isolated from the mixed culture. They could individually degrade PVA1799 with polymerization of 1700 from initial average molecular weight 112,981 to 98,827 Da and 84,803 Da, respectively. However, only small amount of PVA124 in polymerization of 400 could be degraded by these two strains.     

Cellulose degradation by a mixed bacterial culture

Summary An integrated mixed bacterial culture consisting of four strains has been isolated by a batch enrichment technique. The cellulolytic member (strain D) is aCellulomonas sp. and the others are non-cellulolytic. The interaction between strains D and C is pronounced and appears to involve an exchange of reducing sugars and growth factors. The symbiotic relationship of this naturally occurring mixed culture is therefore one of mutualism. The filter paper cellulase and carboxymethyl cellulase activities in extracellular fluid are high, while -glucosidase activity is low. The mixed culture digests a variety of lignocellulosics efficiently and is of fundamental interest in the study of microbial interrelationships.     

Catabolism of 1,2-diarylethane lignin model compounds by two brown-rot fungi

The catabolism of dimethoxybenzil, anisoin and hydroanisoin in nitrogen-limited stationary cultures of the brown-rot fungi Wolfiporia cocos and Gloeophyllum trabeum was analyzed. These three 1,2-diarylethane lignin model compounds, which differ in the degree of oxidation of the alkylic chain, gave rise to p-anisaldehyde in both cultures, suggesting that cleavage between the two aliphatic carbons had occurred. In turn, both strains reduced dimethoxybenzil and anisoin to hydroanisoin, whereas only Wolfiporia cocos was able to oxidize hydro-anisoin to anisoin. On the other hand, chemically derived hydroxyl radical, but not superoxide radical, produced p-anisaldehyde plus other unidentified compounds from anisoin and hydroanisoin. Neither radical modified dimethoxybenzil significantly.Abbreviations HGLN high glucose, low nitrogen - HGHN high glucose, high nitrogen - HPLC high performance liquid chromatography - TLC thin layer chromatography - A anisoin - HA hydroanisoin - DMB dimethoxybenzil - OH Hydroxyl radical - O _inf2 ^sup- superoxide radical     

Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture.

Chlorinated ethenes are toxic substances which are widely distributed groundwater contaminants and are persistent in the subsurface environment. Reports on the biodegradation of these compounds under anaerobic conditions which might occur naturally in groundwater show that these substances degrade very slowly, if at all. Previous attempts to degrade chlorinated ethenes aerobically have produced conflicting results. A mixed culture containing methane-utilizing bacteria was obtained by methane enrichment of a sediment sample. Biodegradation experiments carried out in sealed culture bottles with radioactively labeled trichloroethylene (TCE) showed that approximately half of the radioactive carbon had been converted to 14CO2 and bacterial biomass. In addition to TCE, vinyl chloride and vinylidene chloride could be degraded to products which are not volatile chlorinated substances and are therefore likely to be further degraded to CO2. Two other chlorinated ethenes, cis and trans-1,2-dichloroethylene, were shown to degrade to chlorinated products, which appeared to degrade further. A sixth chlorinated ethene, tetrachloroethylene, was not degraded by the methane-utilizing culture under these conditions. The biodegradation of TCE was inhibited by acetylene, a specific inhibitor of methane oxidation by methanotrophs. This observation supported the hypothesis that a methanotroph is responsible for the observed biodegradations.     

Biodegradation of dissolved jet fuel in chemostat by a mixed bacterial culture isolated from a heavily polluted site

A mixed bacterial culture capable of biodegrading of jet fuel was isolated from a heavily polluted site in Tapa, Estonia. Residual concentrations of pollutants in the chemostat culture were determined. The total residual concentrations of dissolved jet fuel in culture medium were 0.42 and 2.1 μg l^-1 at the dilution rates 0.1 and 0.17 h^-1respectively. Benzene, toluene, ethylbenzene, and xylenes were completely degraded and thus not detected in culture broth (detection limit 0.1 μg l^-1)at the dilution rates 0.1 and 0.17 h^-1. The values of apparent substrate saturation constant(K_Sapp) in multisubstrate growth conditions were estimated from the experimental data. The residual concentrations satisfy the regulations in the Republic of Estonia for petroleum hydrocarbons (0.00 mg l^-1 – ‘very good’). Results obtained indicate that use of the biodegradation could be sufficient for the treatment of polluted with kerosene-type jet fuel groundwater up to the acceptable quality. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biodegradation kinetics of stored wastewater substrates by a mixed microbial culture

The anaerobic accumulation of several organic pollutants from industrial wastewaters, as storage substrates, and their subsequent aerobic biodegradation using a wastewater treatment mixed microbial culture for biological nutrient removal has been studied. The amount and the kinetics of substrate accumulation in the anaerobic stage depended on the characteristics of the wastewater fed to the anaerobic stage. Depending on the substrate used, levels of between 27 and 86% of storage polymers were accumulated with respect to the level obtained on feeding with acetate. The biodegradation kinetics were studied by modelling respirometry results. During the aerobic stage, oxygen-consumption data obtained in the respirometric tests were fitted to a model using a non-linear fitting estimation method. The simulation data obtained correlated well with the experimental oxygen-consumption data. The estimated kinetic parameters obtained indicate that each storage polymer was degraded at a different rate. However, the values obtained for the storage polymer half-saturation coefficient, K_S: 16 mg COD l⁻¹, and for the coefficient for endogenous respiration, b: 0.008 h⁻¹, were similar in all the experiments. The results indicate that each substrate produces the synthesis of a specific storage polymer that is degraded at a different rate.     

Biodegradation of methyl tert-butyl ether by a pure bacterial culture.

Biodegradation of methyl tert-butyl ether (MTBE) by the hydrogen-oxidizing bacterium Hydrogenophaga flava ENV735 was evaluated. ENV735 grew slowly on MTBE or tert-butyl alcohol (TBA) as sole sources of carbon and energy, but growth on these substrates was greatly enhanced by the addition of a small amount of yeast extract. The addition of H(2) did not enhance or diminish MTBE degradation by the strain, and MTBE was only poorly degraded or not degraded by type strains of Hydrogenophaga or hydrogen-oxidizing enrichment cultures, respectively. MTBE degradation activity was constitutively expressed in ENV735 and was not greatly affected by formaldehyde, carbon monoxide, allyl thiourea, or acetylene. MTBE degradation was inhibited by 1-amino benzotriazole and butadiene monoepoxide. TBA degradation was inducible by TBA and was inhibited by formaldehyde at concentrations of >0.24 mM and by acetylene but not by the other inhibitors tested. These results demonstrate that separate, independently regulated genes encode MTBE and TBA metabolism in ENV735.     

Biodegradation of methyl tert-butyl ether by a bacterial pure culture.

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 x 10(6) cells ml(-1) were 0.07, 1.17, and 3.56 microg ml(-1) h(-1) for initial concentrations of 5, 50, and 500 microg MTBE ml(-1), respectively. When incubated with 20 microg of uniformly labeled [(14)C]MTBE ml(-1), strain PM1 converted 46% to (14)CO(2) and 19% to (14)C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE(-1). Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 microg of MTBE ml(-1) added to the core material. The rate of MTBE removal increased with additional inputs of 20 microg of MTBE ml(-1). These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.     

Biodegradation of mixed phenolic compounds by Aspergillus awamori NRRL 3112

The ability of the fungus Aspergillus awamori NRRL 3112 to degrade mixtures of some common phenolic compounds, namely phenol, catechol, 2,4-dichlorphenol and 2,6-dimethoxyphenol was investigated in the present study. For all combinations in which dichlorophenol was incorporated, it took equal time for the nearly complete degradation of the compound—4 days. Phenol was decomposed almost completely (99.5%) in a combination with dimethoxyphenol, to a lesser extent (88%) in a combination with catechol and to the least degree (25%) in the presence of 2,4-dichlorophenol. Catechol experienced a more substantial biotransformation (64%) when mixed with phenol and weaker (45%)—in a combination with dichlorophenol. 2,6-Dimethoxyphenol was better decomposed (69%) in mixtures containing phenol, while its biodegradation in a combination with 2,4-dichlorophenol was considerably poor (only 5%).     

Biodegradation kinetics of methyl iso-butyl ketone by acclimated mixed culture

Methyl iso-butyl ketone (MIBK) is a widely used volatile organic compound (VOC) which is highly toxic in nature and has significant adverse effects on human beings. The present study deals with the removal of MIBK using biodegradation by an acclimated mixed culture developed from activated sludge. The biodegradation of MIBK is studied for an initial MIBK concentration ranging from 200–700 mg l⁻¹ in a batch mode of operation. The maximum specific growth rate achieved is 0.128 h⁻¹ at 600 mg l⁻¹of initial MIBK concentration. The kinetic parameters are estimated using five growth kinetic models for biodegradation of organic compounds available in the literature. The experimental data found to fit well with the Luong model (R ² = 0.904) as compared to Haldane model (R ² = 0.702) and Edward model (R ² = 0.786). The coefficient of determination (R ²) obtained for the other two models, Monod and Powell models are 0.497 and 0.533, respectively. The biodegradation rate found to follow the three-half-order kinetics and the resulting kinetic parameters are reported.     

Thermophilic methanogenesis from pectin by a mixed defined bacterial culture

Thermophilic degradation of pectin was studied in batch cultures at 55°C by different associations of anaerobic bacteria, includingClostridium thermocellum, Methanobacterium sp., andMethanosarcina sp.Clostridium thermocellum alone produced large amounts of methanol along with some isopropanol and H₂. The inoculation ofMethanobacterium sp. in the culture did not affect the metabolism ofC. thermocellum; this demonstrates the absence of interspecies hydrogen transfer. In the presence of the methylotrophicMethanosarcina sp., methanol was reduced to methane without effect on pectin hydrolysis; a small amount of the H₂ produced was also used to reduce methanol.     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

Thermophilic methanogenesis from gelatin by a mixed defined bacterial culture

Summary The fermentation of gelatin by different associations of bacteria, including Thermobacteroides proteolyticus, Methanobacterium sp. and Methanosarcina MP was studied. Experimental vessels were incubated at 55°C. T. proteolyticus growing axenically produced acetate, isovalerate, H₂ and CO₂. Traces of propionate and isobutyrate were detected. Cocultures of T. proteolyticus and Methanobacterium sp. showed an increase in propionate and isobutyrate production. The Thermobacteroides-Methanosarcina association had no effect on metabolism of T. proteolyticus, and acetate was not used.In triculture, growth of Methanosarcina MP occurred on acetate in coculture with T. proteolyticus and Methanobacterium sp. Utilization of H₂ by Methanobacterium sp. in the triculture lowered the H₂ concentration sufficiently to permit acetate utilization by Methanosarcina. Maximum methane production was obtained with the triculture system.     

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.     

Biodegradation of ortho-cresol by a mixed culture of nitrate-reducing bacteria growing on toluene.

A mixed culture of nitrate-reducing bacteria degraded o-cresol in the presence of toulene as a primary growth substrate. No degradation of o-cresol was observed in the absence of toluene or when the culture grew on p-cresol and 2,4-dimethylphenol. In batch cultures, the degradation of o-cresol started after toluene was degraded to below 0.5 to 1.0 mg/liter but continued only for about 3 to 5 days after the depletion of toluene since the culture had a limited capacity for o-cresol degradation once toluene was depleted. The total amount of o-cresol degraded was proportional to the amount of toluene metabolized, with an average yield of 0.47 mg of o-cresol degraded per mg of toluene metabolized. Experiments with [ring-U-14C]o-cresol indicated that about 73% of the carbon from degraded o-cresol was mineralized to CO2 and about 23% was assimilated into biomass after the transient accumulation of unidentified water-soluble intermediates. A mathematical model based on a simplified Monod equation is used to describe the kinetics of o-cresol degradation. In this model, the biomass activity toward o-cresol is assumed to decay according to first-order kinetics once toluene is depleted. On the basis of nonlinear regression of the data, the maximum specific rate of o-cresol degradation was estimated to be 0.4 mg of o-cresol per mg of biomass protein per h, and the first-order decay constant for o-cresol-degrading biomass activity was estimated to be 0.15 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single cell protein from bagasse pith by a mixed bacterial culture

A spontaneous association of Cellulomomas sp. with another bacterial strain was studied for its capabilities for single cell protein (SCP) production from bagasse pith. The associated strain was identified as Pseudomonas sp. and further characterized for its physiological properties. The effect of the initial proportions of both strains, the way of propagation, and the effect of pH on the growth of the mixed culture on bagasse pith was studied. Separate propagation of both strains before the fermentation step (“controlled mixed culture”), a range of proportions Cellulomonas-Pseudomonas from 4:1 to 100: 1, and pH 7.0, were found to be the most appropriate conditions of growth. A mutualistic symbiotic relationship was demonstrated to take place between both strains during the mixed growth on bagasse pith, the Cellulomonas supplying the carbon source (glucose produced from bagasse degradation) to the Pseudomonas, and the latter producing the vitamin supplements necessary for the Cellulomonas growth, allowing the growth of the mixed culture in a minimal medium, without any growth factor supplement. Fed-batch cultivation of the mixed culture on this substrate was successful, giving rise to high biomass production (19.4 g/l), thus increasing the productivity of the system. Due to its improved productivity, high biomass production, inexpensiveness of the culture medium, (without any vitamin supplement), and good stability, this culture presents economical advantages and constitutes an attractive choice for lignocellulosic substrate utilization.     

Biodegradation of fuel oil hydrocarbons by a mixed bacterial consortium in sandy and loamy soils

The aerobic degradation of light fuel oil in sandy and loamy soils by an environmental bacterial consortium was investigated. Soils were spiked with 1 or 0.1% of oil per dry weight of soil. Acetone extracts of dried soils were analyzed by GC and the overall degradation was calculated by comparison with hydrocarbon recovery from uninoculated soils. In sandy soils, the sum of alkanes n-C(12) to n-C(23) was degraded to about 45% within 6 days at 20 degrees C and to 27-31% within 28 days, provided that moisture and nutrients were replenished. Degradation in loamy soil was about 12% lower. The distribution of recovered alkanes suggested a preferential degradation of shorter chain molecules (n-C(12) to n-C(16)) by the bacterial consortium. Partial 16S rDNA sequences indicated the presence of strains of Pseudomonas aeruginosa, Pseudomonas citronellolis, and Stenotrophomonas maltophilia. Toxicity tests using commercial standard procedures showed a moderate inhibition of bacterial activity. The study showed the applicability of a natural microbial community for the degradation of oil spills into soils at ambient temperatures.