Biphenomycin A production by a mixed culture.

Production of biphenomycin A by Streptomyces griseorubiginosus 43708 was stimulated by a mixed culture with a partner strain, Pseudomonas maltophilia 1928. This stimulatory effect on biphenomycin A accumulation by the mixed culture was caused by the enzyme activity which strain 1928 possessed. It is suggested that in a mixed culture strain 43708 produces a precursor of biphenomycin A in culture broth and that strain 1928 converts the precursor to biphenomycin A.     

Influence of organic nutrients and cocultures on the competitive behavior of 1,2-dichloroethane-degrading bacteria.

The effects of organic nutrients and cocultures on substrate removal by and competitive behavior of 1,2-dichloroethane-degrading bacteria were investigated. Xanthobacter autotrophicus GJ10 needed biotin for optimal growth on 1,2-dichloroethane. In continuous culture, dilution of biotin to a concentration below 0.2 nM resulted in washout. Growth could be restored by inoculation with the 2-chloroethanol utilizer Pseudomonas sp. strain GJ1, leading to a new steady state in which about 1% of the mixed culture consisted of cells of strain GJ1. This indicates that strain GJ1 excreted biotin or a precursor for its synthesis. Inoculation of the mixed culture with Ancylobacter aquaticus AD25 did not result in washout of strain GJ10, although strain AD25 has a 10-fold-lower Ks for growth on 1,2-dichloroethane. Strain AD25 did not become dominant because of the lack of vitamins, which are necessary for its optimal growth. The results indicate that medium composition and the presence of other species strongly influence the effect of substrate limitation on the composition of a bacterial population that degrades a xenobiotic compound in a continuous culture.     

Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.     

Weak mixed lymphocyte culture response in the Mongolian gerbil (Meriones unguiculatus).

A study was made to determine whether the mixed lymphocyte culture test could detect histocompatibility differences between two strains of the Mongolian gerbil, Meriones unguiculatus. A semi-micro mixed lymphocyte culture was developed using 6 X 10(5) stimulating and responding cells per 0.12 ml culture volume at a ratio of 1:1. A culture period of 120 hours was found to be optimal. Although a weak allogeneic response was demonstrated with the Uclp:(MON) strain responding to stimulating cells from the MON/Tum strain, the reverse was not seen. A mixed lymphocyte reaction to xenogeneic (mouse) cells was demonstrated, and response to the mitogen, phytohemagglutinin, was strong. These data and the low stimulation index obtained in allogeneic culture supported the view that histocompatibility differences among different strains of the Mongolian gerbil are weak.     

Growth competition of macrolide-resistant and -susceptible Helicobacter pylori strains

We examined population dynamics in a mixed culture of clonally related macrolide-resistant and -susceptible Helicobacter pylori strains isolated from a single patient. The resistant strain had a macrolide resistance-conferring A2143G mutation in the 23S rRNA gene. The growth rate of these two strains did not apparently differ when cultured separately. On the other hand, by conducting sequential passage of a mixed culture of the resistant and the susceptible strains, the ratio of the resistant strain to the susceptible strain in the culture typically decreased per passage, indicating that the resistance imposed a significant disadvantage on bacterial fitness in the population.     

南方红豆杉细胞双液相培养中强化紫杉醇生产的研究

研究了南主红豆杉(Taxuschinensisvar.maireiChengetFu)细胞双液相培养中前体饲喂和混合糖源作为生产培养基糖源对细胞生长和紫杉醇合成的影响。结果表明,前体饲喂(苯丙氨酸、苯甲酰胺和醋酸钠)或混合糖(麦芽糖和蔗糖)作为生产培养基糖源对南方红豆杉细胞单液相培养、更重要的是对其双液相培养的紫杉醇产量提高有显著促进作用,而且前体饲喂和混合糖源的协同作用对其双液相培养的紫杉醇产量提高有进一步的促进作用。与对照组(蔗糖作为糖源和无前体的单液相培养)相比,在南方红豆杉细胞双液相培养中,第10天加入前体,第11天加入混合糖源,紫杉醇产量提高4倍多     

Study on Enhanced Production of Taxol from Taxus chinensis var. mairei in Biphasic-liquid Culture

The effects of precursor feeding and mixed sugar supplements on the growth and taxol production of Taxus chinensis var. mairei Cheng et Fu in the biphasic-liquid culture were studied. Precursor feeding (phenylanine, benzamide and sodium acetate) and mixed sugars (maltose and sucrose) resulted in significant increase of taxol production not only in the monophasic-liquid culture but also in the biphasic-liquid culture of T. chinensis var. mairei. The synergistic function of precursor feeding on day 10 and mixed sugars on day 11 in the biphasic-liquid culture revealed more significant increase of taxol production, which was four times that of the control (the monophasic-liquid culture without precursors or mixed sugars).     

Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms

An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C.     

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.     

Unaltered Nodulation Competitiveness of a Strain of Bradyrhizobium sp. (Lotus) after a Decade in Soil

A Bradyrhizobium sp. (Lotus) strain that formed a soil population that was highly competitive for nodulation of Lotus pedunculatus 11 years after its introduction into a field soil and a culture of the same strain stored lyophilized were compared with an antibiotic-resistant mutant in respect of their nodulation competitiveness. The mutant was less competitive than the wild-type strain it was isolated from and had to be present at a cell ratio of 5.76:1 in mixed inoculum in sand culture to form 50% of the nodules on L. pedunculatus (50% nodulation value, 5.76). The 50% nodulation values for a soil population of the mutant mixed with soil populations of the lyophilized and field soil strain were, respectively, 6.83 and 5.77, indicating that the field soil strain was not significantly different from the lyophilized strain in nodulation competitiveness. A 50% nodulation value of 11.18 obtained when soil containing a recently established mutant population was mixed with the field soil containing the population established 11 years before, indicating that the plant infection technique underestimated cell numbers of the field soil population by 100%. Nodulation competitiveness was unaffected by the size of the strain populations in the range of 100 to 1,000 cells per g of soil; at 10 cells per g a significant correlation between strain ratios in nodules and in soil was still evident. The results indicated that apparently superior nodulation competitiveness of a well-established soil population relative to that of a subsequently introduced strain may not necessarily reflect the intrinsic competitive abilites of the strain(s) involved. The soil strain did not differ from laboratory-maintained cultures in antigenic properties, effectiveness, or whole cell protein electrophoresis profiles.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures.

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.     

A novel microbial interaction: obligate commensalism between a new gram-negative thermophile and a thermophilic Bacillus strain

Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated. From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60°C. The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1. In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase. Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium. For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium. The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1. During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase. Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60°C. Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp. strain SK-1. Received: November 18, 1999 / Accepted: December 2, 1999     

Production of metabolites in the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of Mycobacterium sp. and Sphingomonas sp

The effects of the mixed culture of Mycobacterium sp. strain A1-PYR and Sphingomonas sp. strain PheB4 on the degradation characteristics of single polycyclic aromatic hydrocarbon were investigated. In the mixed bacterial culture, phenanthrene, fluoranthene and pyrene were degraded by 100% at Day 3, 71.2% and 50% at Day 7, respectively. Compared to their respective pure cultures, the degradation of phenanthrene and fluoranthene decreased, but that of pyrene increased significantly. Based on GC-MS analysis, eight and six new metabolites were produced from the biodegradation of phenanthrene and fluoranthene, respectively, while only two new metabolites were formed from pyrene. To our knowledge, this is the first report that the mixed bacterial culture could increase the diversity of metabolites from PAH, but the diverse metabolite pattern was not necessarily beneficial to the degradation of the recalcitrant PAH. The enhancement on pyrene degradation was possibly attributed to the rapid growth of strain PheB4.     

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil-charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Continuous production of mixed lactic starters containing probiotics using immobilized cell technology

The production of a mixed lactic culture containing Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707 was studied during a 17-day continuous immobilized-cell culture at different temperatures between 32 and 37 degrees C. The two-stage fermentation system was composed of a first reactor (R1) containing cells of the two strains separately immobilized in kappa-carrageenan/locust bean gum gel beads and a second reactor (R2) operated with free cells released from the first reactor. The system allowed continuous production of a concentrated mixed culture with a strain ratio whose composition depended on temperature and fermentation time. A stable mixed culture (with a 22:1 ratio of L. diacetylactis and B. longum) was produced at 35 degrees C in the effluent of R2, whereas the mixed culture was rapidly unbalanced in favor of B. longum at a higher temperature (37 degrees C) or L. diacetylactis at a lower temperature (32 degrees C). Strain redistribution in beads originally immobilizing pure cultures of L. diacetylactis or B. longum was observed. At the end of culture, the strain ratio (7:1 L. diacetylactis/B. longum) in bulk bead samples was similar to that of individual beads. The determination of the spatial distribution of the two strains in gel beads by immunofluorescence and confocal laser-scanning microscopy showed that bead cross-contamination was limited to a 100 microm peripheral layer. Data from this study validate a previous model for population dynamics and cell release in gel beads during mixed immobilized-cell cultures.     

Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

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.