Isolation and characterization of two glycerol-fermenting clostridial strains from a pilot scale anaerobic digester treating high lipid-content slaughterhouse waste.

Two obligately anaerobic bacterial strains were isolated from the contents of a pilot scale, anaerobic digester treating slaughterhouse waste with a high protein and lipid content. The isolates, LIP1 and MW8, were characterized as spore-forming, Gram-positive rods, capable of fermenting glycerol. Isolate LIP1 was also observed to be lipolytic and was able to hydrolyse tallow and olive oil. Both isolates grew optimally at 37 degrees C and formed either acetate and formate (LIP1), or acetate and butyrate (MW8), as major glycerol fermentation products. Both isolates produced ethanol as the major reduced fermentation end-product. Neither MW8 nor LIP1 had growth and metabolism inhibited by the addition of stearic acid at concentrations normally considered bactericidal. Analysis of the 16S rRNA gene sequences, in conjunction with the phenotypic data, confirmed that the isolates are members of the genus Clostridium (sensu lato), clustering with species of clostridial clusters I (MW8) and XIVa (LIP1).

Isolation and characterisation of obligately anaerobic, lipolytic bacteria from the rumen of red deer.

Two Gram-positive, obligately anaerobic, lipolytic bacteria, isolates LIP4 and LIP5, were obtained from the rumen contents of juvenile red deer. These mesophilic bacterial strains were capable of hydrolysing the neutral lipids, tallow, tripalmitin and oliver oil, into their constituent free long-chain fatty acid and glycerol moieties. The latter compound was dissimilated by both isolates, with isolate LIP4 producing propionate as the predominant product, while isolate LIP5 produced acetate, ethanol and succinate. The lactate-utilising isolate LIP4 grew on a limited range of saccharide substrates including glucose, fructose and ribose, and exhibited an unusual cell wall structure and morphology. The isolate LIP5 grew upon a wider range of saccharides, but was unable to use lactate as a substrate. Based upon phenotypic and 16S rRNA gene sequence analyses, isolate LIP4 clusters with species in the genus Propionibacterium, while isolate LIP5 is a member of clostridial cluster XIVa.

Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene to degradation by pure cultures of 1,1-diphenylethylene-degrading aerobic bacteria.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) is the peri-chlorinated derivative of 1,1-diphenylethylene (DPE). Biodegradation of DDE and DPE by bacteria has so far not been shown. Pure cultures of aerobic bacteria involved in biodegradation of styrene and polychlorinated biphenyls (PCB) were therefore screened for their ability to degrade or cometabolize DPE and DDE. Styrene-metabolizing bacteria (Rho-dococcus strains S5 and VLB150) grew with DPE as their sole source of carbon and energy. Polychlorinated-biphenyl-degrading bacteria (Pseudomonas fluorescens and Rhodococcus globerulus) were unable to degrade DPE even in the presence of an easily utilizable cosubstrate, biphenyl. This is the first report of the utilization of DPE as sole carbon and energy source by bacteria. All the tested bacteria failed to degrade DDE when it was provided as the sole carbon source or in the presence of the respective degradable cosubstrates. DPE transformation could also be detected in cell-free extracts of Rhodococcus S5 and VLB150, but DDE was not transformed, indicating that cell wall and membrane diffusion barriers were not limiting biodegradation. The results of the present study show that, at least for some bacteria, the chlorination of DDE is the main reason for its resistance to biodegradation by styrene and DPE-degrading bacteria.

Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) to cometabolic degradation by pure cultures of aerobic and anaerobic bacteria.

Pure cultures of aerobic and anaerobic bacteria capable of oxidation and reductive dehalogenation of chloroethylenes, and aerobic bacteria involved in biodegradation of polychlorinated biphenyls (PCBs) were screened for their ability to cometabolize the persistent pollutant 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE). Bacterial cultures expressing methane monooxygenase (Methylosinus trichosporium), propane monooxygenase (Mycobacterium vaccae) and biphenyl 2,3-dioxygenase enzymes (Pseudomonas fluorescens and Rhodococcus globerulus), as well as bacteria reductively dechlorinating chloroethylenes (Acetobacterium woodii and Clostridium butyricum) could not degrade DDE. Cell-free extracts of M. trichosporium, M. vaccae, P. fluorescens and R. globerulus were also unable to transform DDE, indicating that cell wall and membrane diffusion barriers were not biodegradation limiting. These studies suggest that these bacteria can not degrade DDE, even when provided with cosubstrates that induce chlorophenyl- and dichloroethylene-group transforming enzymes.

Formate and ethanol are the major products of glycerol fermentation produced by a Klebsiella planticola strain isolated from red deer.

The rumen contents of red deer (Cervus elaphus) were used to isolate bacterial capable of fermenting glycerol. The biochemistry, physiology, morphology and phylogeny of one isolate were studied in detail. The isolate (DR3) was tentatively identified as a strain of the species Klebsiella planticola as based on phenotypic characterization. The data obtained from 16S rRNA sequence analysis showed that the deer rumen isolate DR3 was 99.7% similar to the type strain of Kl. planticola (DSM 3069T), thus confirming the results of the phenotypic characterization. During cell growth, it was established that glycerol dissimilation by Kl. planticola DR3 led to the production of formate and ethanol at equimolar levels of 32 mmol 1(-1) and 30 mmol 1(-1), respectively. As a result of the data obtained, a closed carbon balance was constructed for Kl. planticola DR3. This finding represented the first report of the complete end-product profile for glycerol dissimilation by a strain of Kl. planticola isolated from cervine rumen contents.

Total biodegradation of the oestrogenic mycotoxin zearalenone by a bacterial culture.

A mixed culture of bacteria, enriched from soil collected at a coal gasification site, proved capable of removing the potent oestrogenic mycotoxin zearalenone from culture media. The bacteria grew rapidly when zearalenone was provided as the sole source of carbon and energy. HPLC and ELISA analysis of culture extracts revealed no zearalenone or zearalenone-like products. Fourteen bacterial isolates from the mixed culture were identified and purified. The ability to degrade zearalenone was lost upon purification and recombination of the bacterial members of the mixed culture. A strain of Pseudomonas fluorescens capable of degrading polychlorinated biphenyls was unable to degrade zearalenone. This is the first report of the complete degradation of zearalenone by bacteria. The present study suggests the potential of mixed cultures in the biodegradation of zearalenone.

Comparative fate of 1,1-diphenylethylene (DPE), 1,1-dichloro-2,2-bis(4-Chlorophenyl)-ethylene (DDE), and pentachlorophenol (PCP) under alternating aerobic and anaerobic conditions.

Bacterial degradation of 1,1-dichloro-2,2-bis-(4-chlorophenyl)-ethylene (DDE) and its dehalogenated derivative 1,1-diphenylethylene (DPE) has not yet been shown and may require culture adaptation and special culture conditions. We compared the degradability of DPE, DDE, and pentachlorophenol (PCP) in aerobic/anaerobic sequenced batch reactor systems. Reactors operated under aerobic/methanogenic and aerobic/denitrifying conditions were inoculated with bacterial consortia from anaerobic granular sludge, long-term PCP- and DDE-contaminated soil, and pulp and paper waste pond sediment. The culture was gradually acclimatized to low concentrations of DPE, DDE, and PCP in defined minimal growth media with benzoate, phenol, ethanol, and formate as primary carbon sources. DDE remained refractory for 105 days, whereas DPE and PCP were degraded. This suggests that DDE is extremely recalcitrant to degradation by aromatic organochlorine-degrading bacteria from long-term polluted soils and sediments. The results confirm that the chlorination of DDE is a major biodegradation barrier for adapted bacteria under aerobic and anaerobic conditions.

Ecoengineering high rate anaerobic digestion systems: analysis of improved syntrophic biomethanation catalysts.

High performance biomethanation granules with operational specific COD removal rates of 7 kg COD removed/kg SS/d were obtained by ecoengineering conventional, granular, UASB digester sludge using a designed protocol of starvation and selection on a defined volatile fatty acid (VFA) based mineral medium. Addition of low (0.15 mM) sulfate levels to this VFA medium increased the maximum shock-load COD removal rate of the ecoengineered biomethanation granules to 9 kg COD/kg SS/d with specific acetate, propionate, and butyrate removal rates of 111, 28, and 64 mol/g SS/d. Addition of moderate (26 mM) calcium levels inhibited growth and altered the structure of granules. The general cellular, growth, stability, and performance features of these ecoengineered granules are described and discussed in relation to their use as improved biomethanation starter cultures.

Control of interspecies electron transfer flow during anaerobic digestion: dynamic diffusion reaction models for hydrogen gas transfer in microbial flocs.

Dynamic reaction diffusion models were used to analyze the consequences of aggregation for syntrophic reactions in methanogenic ecosystems. Flocs from a whey digestor were used to measure all model parameters under the in situ conditions of a particular defined biological system. Fermentation simulations without adjustable parameters could precisely predict the kinetics of H(2) gas production of digestor flocs during syntrophic methanogenesis from ethanol. The results demonstrated a kinetic compartmentalization of H(2) metabolism inside the flocs. The interspecies electron transfer reaction was mildly diffusion controlled. The H(2) gas profiles across the flocs showed high H (2) concentrations inside the flocs at any time. Simulations of the syntrophic metabolism at low substrate concentrations such as in digestors or sediments showed that it is impossible to achieve high H(2) gas turnovers at simultaneously low steady-state H(2) concentrations. This showed a mechanistic contradiction in the concept of postulated low H(2) microenvironments for the anaerobic digestion process. The results of the computer experiments support the conclusion that syntrophic H(2) production may only be a side reaction of H(2) independent interspecies electron transfer in methanogenic ecosystems.

The anion-exchange substrate shuttle process: A new approach to two-stage biomethanation of organic and toxic wastes.

A novel biomethanation process configuration is described which uses improved biocatalysts to enhance the productivity and stability of waste biomethanation systems. The design facilitates the maintenance of acetogenic and methanogenic bacteria under optimum substrate concentrations far above their K(s) values in a two-stage biomethanation system with closed loop reactors. Volatile fatty acid anions (VFA anions) are provided as substrates for the methanogenic stage by using an anion exchange unit coupled to the acidogenic stage. The slow growing and sensitive acetogenic and methanogenic bacteria are protected from oxygen, cationic pollutants, toxins, and microbial contamination by use of the substrate shuttle process. Due to the closed loop process configuration, washout of the ecoengineered biocatalysts is excluded. The modular system configuration allows industrial mass production of the system components. The new biomethanation process enhances both the BTU content of the gas and the methane production over state-of-the-art anaerobic digestor bioreactor concepts.