Mixed Culture Of Anaerobic Bacteria Research Articles

Identification and Characterization of Natural Habitats of Electrochemically Active Bacteria

Aims: This study aims to define criteria for the main physical and chemical characteristics of the environmental niches populated with electrochemically active microorganisms, capable to perform anaerobic respiration and potentially used in Bio-electrochemical systems such as Microbial Fuel Cells. 
 Study Design: In this study, specific parameters of the environment in water bodies (such as lakes, streams etc.) and their bottom layers are analyzed. The main parameters of interest include the concentration of dissolved oxygen in the water column, the organic matter content in the sediments and the presence of alternative electron acceptors (such as iron and manganese ions) to support anaerobic respiration. Sediment microorganisms are characterized for their electrochemical and biodegradation activity.
 Place and Duration of Study: The tested sediment and water samples were collected from "Poda" Protected Site located on the outfall of Lake “Uzungeren”, south of City of Burgas, Bulgaria.
 Methodology: The samples were analyzed employing TGA, ICP and microbiological methods focusing on chemical, physical and biological conditions available for anaerobic respiration in this ecological niche.
 Results: The results show very low concentrations of dissolved oxygen (from 1.4 to 2.2 mg/dm3 in the various locations). The conductivity and the pH values ​​measured were relatively high and the mean values obtained are 5230 μS/cm and 8.2 respectively. The sediment samples demonstrated very high organic matter content (22.5% of the dry mass) and relatively high levels of iron and manganese.
 Microbial fuel cell powered by mixed bacterial culture isolated from the tested sediment samples demonstrated stable performance reaching power density of 3.5 W/m2 and the COD removal rate of 42 mgO2/dm3 per day.
 Conclusion: The result confirms the initial hypothesis that electrochemically active microorganisms are available in environments with high concentration of organic matter, iron and manganese in combination with low availability of dissolved oxygen. Mixed culture of anaerobic bacteria isolated from the tested sediment sample was successfully implemented to power Microbial Fuel Cell.

Electron transfer interpretation of the biofilm-coated anode of a microbial fuel cell and the cathode modification effects on its power

Biofilm-coated electrodes and outer cell membrane-mimicked electrodes were examined to verify an extracellular electron transfer mechanism using Marcus theory for a donor–acceptor electron transfer. Redox couple-bound membrane electrodes were prepared by impregnating redox coenzymes into Nafion films on carbon cloth electrodes. The electron transfer was believed to occur sequentially from acetate to nicotinamide adenine dinucleotide (NAD), c-type cytochrome, flavin mononucleotide (FMN) (or riboflavin (RBF)) and the anode substrate. Excellent polarisation and power density characteristics were contributed by the modification of the cathode with a high-surface-area ordered mesoporous carbon or a hollow core–mesoporous shell carbon. The maximum power density of the microbial fuel cell (MFC) could be improved by a factor of two mainly due to the accelerated electron consumption by modifying the cathode surfaces within three-dimensionally interconnected mesoporous carbon particles, and the anode was coated with a mixed culture of anaerobic bacteria.

English

Several studies have been carried out to produce biofuels by anaerobic digestion of stillage from ethanol fermentation. This type of process, known as ABE Fermentation, generates by-products such as acetone, butanol and ethanol. This fermentation can use pure or mixed cultures of anaerobic bacteria, mainly of the genus Clostridium. In this context, there is the need for deeper studies for proper disposal of the residue resulting from Anaerobic Fermentation of ethanolic stillage, hereinafter referred to as AF stillage (stillage from Anaerobic Fermentation), obtained after this fermentation. Thus, the aim of this study was to evaluate the biodegradation and toxicity of stillage from Anaerobic Fermentation. The biodegradation of AF stillage in soil samples in the presence and absence of commercial inoculum was evaluated using a respirometric method described by Bartha & Pramer, according to the standard NBR 14283 (ABNT, 1999). The production of CO2 was evaluated for 57 days. Additionally, quantification of microorganisms was carried out at the beginning and end of the experiment. Toxicity tests were performed with the microcrustacean Daphnia similis using raw AF stillage and leachate from the respirometry test, according to the standard NBR 12713 (ABNT, 2009). The results of the respirometry test showed that, after 13 days of incubation, the biodegradation efficiency of all samples was above 30%. Using the Friedman statistical test, results showed that adding the inoculum caused no statistically significant difference in the biodegradation of AF stillage. The acute toxicity tests were performed on raw AF stillage and on the leachates showed that toxicity was removed after biodegradation in soil at all concentrations used. Thus, discharge of AF stillage into soil is an alternative viable disposal. Key words: Biodegradation, stillage, anaerobic fermentation, toxicity.

Method for evaluating anaerobic digester performance

The degradation rate and efficiency of digestion processes is typically measured by introducing a substrate or pollutant into a digester and then monitoring the effluents for the pollutant or substrate, a costly and slow process. A new method for rapid measurement of the rates and efficiencies of anaerobic degradation of pollutants and lignocellulose substrates from various pretreatments is described. The method uses micro-reactors (10-30mL) containing a mixed culture of anaerobic bacteria obtained from a working anaerobic digester. The rate of degradation of pollutants and metabolic heat rate are measured in parallel sets of micro-reactors. Measurement of metabolic rate and pollutant degradation simultaneously is an effective means of rapidly examining pollutant degradation on a micro-scale. Calorimetric measurements alone allow rapid, relative evaluation of various substrate pretreatment methods.

Phylogenetic characterization of bacterial consortia obtained of corroding gas pipelines in Mexico

Characterization of the microbial populations formed in gas pipelines is essential to understand the metallic surface-microbe interaction, their role in metal corrosion, and to implement efficient monitoring and control strategies. Microbial community analysis in a corroded gas pipeline in a petroleum-producing facility in the Southeast region in Mexico was performed by traditional cultivation techniques and identification based on 16S rRNA gene sequence. In all samples, thin bacterial biofilms were observed and pitting corrosion was reveled after removing the biofilms. Six pure or mixed cultures of anaerobic bacteria were obtained and their 16S rRNA libraries were constructed, respectively. At least two members of each RFLP profile were sequenced and the phylogenetic affiliations of cloned bacterial 16S rRNA genes indicated that native biofilms were mainly colonized by Desulfovibrio vulgaris and Desulfovibrio desulfuricans, sulfate-reducing bacteria members; Citrobacter freundii, an Enterobacteriaceae member; Clostridium celerecrescens and Clostridium sporogenes, spore-forming anaerobic species and Cetobacterium somerae, a microaerotolerant, non-spore-forming fusobacteria. Some of these species have been observed consistently in other steel pipelines previously, but Cetobacterium members and C. celerecrescens are described for the fist time in this corroded gas pipeline. The potential role of each species in biofilm formation and steel corrosion is discussed.

Anaerobic decolorization of an azo dye by a mixed culture

Wool dyeing wastewater contains xenobiotic compounds that can be removed by biotechnological processes. Studies on various dyes showed that anaerobic processes are suitable to alter azo dyes as a first step of the biodegradation process. These compounds are reduced by anaerobic consortia to aromatic amines and its ultimate degradation can be achieved by a further aerobic treatment. Studies on degradation rate of an wool acid dye were performed in batch systems inoculated with anaerobic biomass. A commercial diazo dye, Acid Red 73, was added to the synthetic medium in which glucose was used as sole carbon source. Results indicated that the Acid Red 73 was partially degraded by a mixed culture of anaerobic bacteria and a decolorization of 90% was obtained. Kinetics studies on removal of the colour showed that the decolorization rate was several times faster than the degradation rate of glucose for a range of dye concentrations between 60 mg/L and 400 mg/L. A first order kinetic model was used for dye concentrations up to 200 mg/L. For higher concentrations a model similar to the Michaelis‐Menten equation was better fitted to the experimental data.

Electricity generation in microbial fuel cells using neutral red as an electronophore.

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.

Inhibition of methanogenesis by methyl fluoride: studies of pure and defined mixed cultures of anaerobic bacteria and archaea

Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.

Rapid bacterial degradation of polysaccharides in anoxic marine systems

Extracellular hydrolysis of organic macromolecules is often assumed to be the slow step in remineralization of organic matter. We tested this assumption by comparing the degradation of four polysaccharides (pullulan, laminarin, and two polysaccharides isolated from the marine cyanobacterium Synechococcus WH7335) to determine whether size, linkage position, or anomeric linkage affected rates or mechanisms of carbohydrate degradation by mixed cultures of anaerobic bacteria enriched from marine sediments. Gel permeation chromatography and nuclear magnetic resonance spectroscopy (NMR) were used to follow the extracellular conversion of high molecular weight polysaccharides to lower molecular weight polysaccharides and oligosaccharides which were subsequently remineralized. In all cases, substrate degradation was rapid. NMR spectra showed that preferential hydrolysis occurred at specific chemical linkages, and extracellular enzymatic hydrolysis of polysaccharides occurred far more rapidly than bacterial uptake and remineralization of the lower molecular weight oligosaccharides produced through enzymatic hydrolysis. Substrate size was not a significant determinant of remineralization rate: high molecular weight does not always correlate with slow degradation rate. The hypothesis that extracellular enzymatic hydrolysis is a slow step in the degradation of macromolecular organic matter in marine systems needs to be critically re-examined.

Methanogenesis in granular sludge exposed to oxygen

Substrate competition between methanogenic and facultative bacteria under highly aerobic conditions was investigated in batch experiments. Natural mixed cultures of anaerobic bacteria immobilized in granular sludge were able to concurrently utilize oxygen and produce methane when supplied with ethanol as substrate. The most oxygen tolerant sludge converted 3 to 25% of substrate chemical oxygen demand to methane after 3 days while 23 to 2 mg 1−1 of dissolved oxygen were present in the media. The tolerance of methanogens to oxygen and their coexistence with facultative bacteria were evident even after long periods of oxygen exposure. Eventually, methane oxidizing bacteria developed in the co-culture. The consumption of oxygen by facultative bacteria, creating anaerobic microniches inside the granules, is hypothesized to protect the methanogens.

Methanogenesis in granular sludge exposed to oxygen

Substrate competition between methanogenic and facultative bacteria under highly aerobic conditions was investigated in batch experiments. Natural mixed cultures of anaerobic bacteria immobilized in granular sludge were able to concurrently utilize oxygen and produce methane when supplied with ethanol as substrate. The most oxygen tolerant sludge converted 3 to 25% of substrate chemical oxygen demand to methane after 3 days while 23 to 2 mg 1−1 of dissolved oxygen were present in the media. The tolerance of methanogens to oxygen and their coexistence with facultative bacteria were evident even after long periods of oxygen exposure. Eventually, methane oxidizing bacteria developed in the co-culture. The consumption of oxygen by facultative bacteria, creating anaerobic microniches inside the granules, is hypothesized to protect the methanogens.

Biotransformation of chloromethane to methanethiol

Monochloromethane (MeCl) was biotransformed by a mixed culture of anaerobic bacteria which had been enriched to utilize dichloromethane as the sole source of carbon and energy. Cells grown in dichloromethane-salts medium catalyzed the transformation, whereas sucrose-grown cells were inactive. MeCl disappeared almost totally, and the methyl group was recovered in a yield of 80-90% as methanethiol and traces of dimethyl sulfide, whose identities were confirmed by GC-MS. The methyl transfer was heat-labile, and free aquocobalamin did not promote the reaction, which was apparently enzymically catalyzed. 37 refs., 1 fig., 2 tabs.

Transformation of indole by methanogenic and sulfate-reducing microorganisms isolated from digested sludge

In the present study, mineralization of an aromaticN-heterocyclic molecule, indole, by microorganisms present in anaerobically digested sewage sludge was examined. The first step in indole mineralization was the formation of a hydroxylated intermediate, oxindole. The rate of transformation of indole to oxindole and its subsequent disappearance was dependent on the concentration of inoculum and indole and the incubation temperature. Methanogenesis appeared to be the dominant process in the mineralization of indole in 10% digested sludge even in the presence of high concentrations of sulfate. Enrichment of the digested sludge with sulfate as an electron acceptor allowed the isolation of a metabolically stable mixed culture of anaerobic bacteria which transformed indole to oxindole and acetate, and ultimately to methane and carbon dioxide. This mixed culture exhibited a predominance of sulfate-reducers over methanogens with more than 75% of the substrate mineralized to carbon dioxide. The investigation demonstrates that indole can be transformed by both methanogenic and sulfate-reducing microbial populations.

Fermentation of Glycollate by a Mixed Culture of Anaerobic Bacteria

Summary A mixed culture of anaerobic bacteria was able to ferment glycollate in the presence of yeast extract. Under these conditions three cell forms were present in approximately equal numbers. Substrates other than glycollate supported growth of two of the observed organisms (a vibrio and a long thin rod), but the third member of the mixed culture (an oval-shaped organism in chains) was seen only in the presence of glycollate. Glycollate was fermented to acetate, propionate and CO 2 concomitant with growth (μ = 0.13 h -1 at 34°C). The growth yield was estimated at 4.7 g dry weight per mol of glycollate. Glycollate fermentation was not inhibited under a hydrogen headspace, indicating that hydrogen transfer between organisms was not occurring.

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

AbstractStudies to examine the microbial fermentation of coal gasification products (CO2, H2 and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H2/mol CO2. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO2 and hydrogen.