Interactions in a mixed bacterial population growing on methane in continuous culture

The growth of a mixed methane-utilizing culture in a continuous flow fermenter has been studied under both methane and oxygen limitation. Small additions of methanol have been shown to inhibit the methane-utilizing moiety in the culture and it has been shown that the Hyphomicrobium sp. in the mixed culture removes any inhibitory methanol. The interaction between the methane-utilizing Pseudomonas sp., and the Hyphomicrobium sp. has been explained and a model of the continuous mixed culture under oxygen limitation has been formulated. Qualitative predictions of transient phenomena by the model have been verified experimentally.     

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.     

Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures.

The microbial degradation of phenol by pure and mixed cultures of Pseudomonas putida was studied in batch, phenol-stat, and continuous culture systems. In the continuous culture runs, both steady state and transient experiments were performed. From these experiments, a model for the kinetic behavior of the organisms was evolved and an analysis performed on the stability and dynamic behavior of pure and mixed cultures. The results indicate that it should be possible to achieve phenol removal from wastewaters down to levels of 1-2 ppm in a single state system. However, because of the effect of substrate inhibition on kinetic behavior of the microorganisms, long lasting transients can occur. The transient behavior of such systems cannot be solely determined from mumax or Ks parameters, but must include a consideration of the transient size and response characteristic of the organism.     

Batch- and continuous-culture studies of a methane-utilizing mixed culture

A methane-utilizing mixed culture isolated from activated sludge by selective enrichment at 45°C was found to consist of three interacting species: a methaneutilizing bacterium, a citrate-utilizing bacterium, and a methanol-utilizing bacterium. All three species grew well at 45°C. Three different stable mixed cultures were reconstituted by various combinations of these pure cultures. The nutritional requirements and substrate ranges for each pure culture were determined. The nutritional requirements and substrate ranges for each pure culture were determined. The saturation constant for the methane-utilizing bacterium on methane (K) and for the methanol-utilizing bacterium on methanol (K) were 1.73 × 10^?6M and 4.51 × 10^?7M, respectively. The volumetric mass transfer coefficient for methane (K_L a) was determined to be 65.6 hr^?1.     

Effects of PCB on interspecific competition in natural and gnotobiotic phytoplankton communities in continuous and batch cultures

The toxicity of polychlorinated biphenyls (PCB) to the diatomThalassiosira pseudonana (formerlyCyclotella nana), grown in pure and mixed cultures, was greatest when in competition with other species. Continuous cultures were superior to batch cultures for studying competitive interactions, and PCB caused greater alteration of species composition in continuous cultures than it did in batch cultures. Natural phytoplankton communities from Vineyard Sound, maintained in continuous culture, responded to PCB stress the same as did gnotobiotic communities, withT. pseudonana showing similar responses in both communities. A PCB concentration of 0.1 μg/liter (0.1 part per billion), a level not uncommon in natural waters, did not affect algal growth in pure cultures but caused substantial disruption of continuous culture communities. The possible impact of PCB pollution on natural phytoplankton communities is discussed. Contribution No. 3181 from the Woods Hole Oceanographic Institution.     

Stability analysis of commensal and mutual relations with competitive assimilation in continuous mixed culture

The population dynamics of continuous mixed cultures with pure commensalism, commensalism plus competitive assimilation, pure mutualism, and mutualism plus competitive assimilation was disused. The population does not display oscillatory phenomena where there is a single interaction of commensalism or mutualism. Damped oscillations take place when two interactions (such commensalism and competitive assimilation or mutualism and competitive assimilation) coexist in the continuous mixed culture. The stability of these systems was discussed in detail.     

Wall growth in mixed bacterial cultures growing on methane

The effects of wall growth are described for a mixed methane-utilizing bacterial population growing in both batch and continuous culture. These effects are similar to those predicted previously by a theoretical analysis (Topiwala and Hamer, 1971).     

Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system

AIMS: To characterize a methane-utilizing poly-beta-hydroxybutyrate (PHB)-producing microbial community. METHODS AND RESULTS: Three different approaches based on microbiology, analytical chemistry and molecular biology were used to determine the composition of the mixed culture. The dominant species, Methylocystis sp. GB25, represents more than 86% of the total biomass. Seven accompanying bacterial species are present in the mixed culture of which two are methylotrophic bacteria and five are utilizers of complex carbon sources. Both these groups were found to be present at the same ratio with respect to each other. Results of fatty acid analysis and PCR-DGGE fingerprints reflect the stability of the mixed-culture composition in the open system during multiple continuous growth and polymer formation processes throughout a period of 29 months. The consistently high quality of the accumulated polymer further corroborates this finding. CONCLUSION: The methane-utilizing mixed culture has the potential of self-regulation resulting in a stable composition even under non-aseptic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Avoiding the necessity of sterile conditions, as demonstrated in this paper, is an important step towards the development of a viable large-scale process for the production of PHB using cheap substrates like methane from natural or renewable sources. This is the first report characterizing a bacterial mixed culture being used for the biotechnological production of a high-value product in an open system.     

开放条件下高聚β-羟基丁酸酯含量的甲烷氧化混合菌群的富集

目的:在开放条件下以甲烷为底物富集高聚β-羟基丁酸酯(PHB)含量的甲烷氧化菌群,获得能够利用低成本碳源高产PHB的菌种。方法:采用丰盛-饥饿模式间歇供料,以甲烷为底物,好氧开放式培养甲烷氧化混合菌群,利用苏丹黑染色法动态检测丰盛和饥饿阶段胞内PHB含量的变化,以此为基础考察丰盛-饥饿期比例对富集高PHB含量的甲烷氧化菌群的影响。结果:丰盛-饥饿期比例为1∶3时,微生物PHB含量从17.7%增加到35.5%,且开放培养过程中菌群结构稳定。结论:通过丰盛-饥饿模式间歇供料开放式培养所得的高PHB含量的稳定的甲烷氧化菌群,具有工业生产PHB的应用价值。     

Studies on an acetate-fermenting strain of Methanosarcina.

An acetate-fermenting strain of Methanosarcina was isolated from an acetate enrichment culture inoculated with anaerobic sludge from a waste treatment digestor. In pure culture, this organism fermented acetate in the absence of added hydrogen at rates comparable in magnitude to those found in digestor systems. This rate was significantly higher than previously obtained for pure cultures of this genus. Mineral components of yeast extract were highly stimulatory for cultures growing on methanol. Comparable stimulation was not observed for cultures growing on acetate. Labeling studies indicated that acetate was converted to methane and CO2 as predicted by previous studies on mixed cultures. Total oxidation or reduction of acetate was not the mechanism of conversion of acetate to methane by the pure culture. The ability of this strain to form colonies or to produce methane from acetate was apparently influenced by the choice of substrate and conditions used for growing the inoculum.     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture.

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Auxotrophy and intrapopulation complementary in the ‘interactome’ of a cultivated freshwater model community

Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet‐uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high‐quality metagenome‐assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin‐ and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage‐based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to ‘social’ interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring ‘interactomes’ and to overcome our inability to isolate and grow the microbes dominating in nature.     

Role of heterotrophic bacteria in complete mineralization of trichloroethylene by Methylocystis sp. strain M.

Biodegradation experiments with radioactively labeled trichloroethylene showed that 32% of the radioactive carbon was converted to glyoxylic acid, dichloroacetic acid and trichloroacetic acid and that the same percentage was converted to CO2 and CO after 140 h of incubation by a pure culture of a type II methane-utilizing bacterium, Methylocystis sp. strain M, isolated from a mixed culture, MU-81, in our laboratory. In contrast, these water-soluble (14C)trichloroethylene degradation products were completely or partially degraded further and converted to CO2 by the MU-81 mixed culture. This phenomenon was attributed to the presence of a heterotrophic bacterium (strain DA4), which was identified as Xanthobacter autotrophicus, in the MU-81 culture. The results indicate that the heterotrophic bacteria play an important role in complete trichloroethylene degradation by methanotrophs.     

Role of heterotrophic bacteria in complete mineralization of trichloroethylene by Methylocystis sp. strain M.

Biodegradation experiments with radioactively labeled trichloroethylene showed that 32% of the radioactive carbon was converted to glyoxylic acid, dichloroacetic acid and trichloroacetic acid and that the same percentage was converted to CO2 and CO after 140 h of incubation by a pure culture of a type II methane-utilizing bacterium, Methylocystis sp. strain M, isolated from a mixed culture, MU-81, in our laboratory. In contrast, these water-soluble (14C)trichloroethylene degradation products were completely or partially degraded further and converted to CO2 by the MU-81 mixed culture. This phenomenon was attributed to the presence of a heterotrophic bacterium (strain DA4), which was identified as Xanthobacter autotrophicus, in the MU-81 culture. The results indicate that the heterotrophic bacteria play an important role in complete trichloroethylene degradation by methanotrophs.     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Kinetics of pure cultures of hydrogen-oxidizing denitrifying bacteria and modeling of the interactions among them in mixed cultures

In this study we report the isolation of four denitrifying bacteria from a batch reactor, where the progress of hydrogenotrophic denitrification was examined. Only three of the strains had the ability to use hydrogen as electron donor. In the present work, kinetic batch experiments were carried out in order to study the dynamic characteristics of pure and defined mixed cultures of hydrogen-oxidizing denitrifying bacteria, under anoxic conditions, in a defined synthetic medium, in the presence of nitrates. Kinetic models were developed and the kinetic parameters were determined from the batch experiments for each bacterium separately. The behavior of mixed cultures and the interactions between the bacteria were described using kinetic models based on the kinetic models developed for each bacterium separately and their predictions were compared with the results from mixed culture experiments. The mathematical models that were developed and validated in the present work are capable of describing the behavior of the bacteria in pure and mixed cultures, and in particular, the kinetics of nitrate and nitrite reduction and cell growth.