Kinetics of chromate reduction during naphthalene degradation in a mixed culture

A mixed culture of Bacillus sp. K1 and Sphingomonas paucimobilis EPA 505 was exposed to chromate and naphthalene. Batch experiments showed that chromate was reduced and naphthalene was degraded by the mixed culture. Chromate reduction occurred initially at a high rate followed by a decrease in rate until chromate reduction ceased. Chromate reduction decreased in the mixed culture when a lower ratio of S. paucimobilis EPA 505 to Bacillus sp. K1 was utilized. A kinetic model incoporating a term for the cell density ratio is proposed to describe chromate reduction in the mixed culture under both chromate limited and electron donor limited conditions. The validity of the model, and its parameter values, was verified by experimental data generated under a variety of initial population compositions and a broad range of chromate concentrations. The consistent result of experimental data with model predictions implies that the model is useful for evaluating the interactions and the use of mixed culture for chromate removal. (c) 1996 John Wiley & Sons, Inc.     

Growth analysis of a mixed culture during the degradation of 4-aminobenzenesulfonic acid

Summary Growth of a mixed culture, Pseudomonas palleronii and Agrobacterium radiobacter using the toxic chemical 4-aminobenzenesulfonic acid was followed by: the measurement of the total cell density by using cell counter and the plate count procedure for enumerating each strain according to the different morphology. The results from these two procedures deviated considerably due to the inability to form colonies on solid agar media.     

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.     

Bacterial degradation of cyclohexane participation of a co-oxidation reaction

Complete biodegradation of cyclohexane was demonstrated to occur in a system containing two strains of Pseudomonad bacteria, viz an n-alkane oxidizer and a microorganism utilizing cyclohexanol. n-Heptane is also present as a substrate for the former. Neither bacterial strain was capable of utilizing cyclohexane individually. This biodegradation of cyclohexane occurs in two steps:

1. the conversion of cyclohexane into cyclohexanol by the n-alkane oxidizer (co-oxidation) and
2. utilization of cyclohexanol by the second strain.

In unsuccessful attempts to accumulate a bacterium which could grow on cyclohexane as its sole source of carbon, we have used some uncommon methods, which are briefly described.     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Anaerobic degradation of cellulose by mixed culture

A mixed culture in which cellulose is capable of being converted to methane and carbon dioxide was obtained from an inoculum procured from a sewage-treatment plant and maintained in a synthetic medium containing tissue paper and an inorganic salt and vitamin mixture. The culture was tested for its ability to degrade 12 different paper and cotton products under batch conditions in 3-l anaerobic fermenters. This culture degraded 6-8 mmol/l per week of cellulose, expressed as glucose equivalents, with total gas yields of 0.3 m3/kg of cellulose degraded. The gas produced contained between 56 and 59% of methane. Maximum cellulose degradation occurred at chemical oxygen demand:nitrogen:phosphorus level of 80:5:1 and was adversely affected by high stirring rate. Also the presence of higher proportions of lignin in cellulose products adversely affected the ability of this culture to degrade cellulose.     

Effect of keratinous waste addition on improvement of crude oil hydrocarbon removal by a hydrocarbon-degrading and keratinolytic mixed culture

The aim of this work was to evaluate the effect of keratinous waste addition on oil-hydrocarbon removal, through a mixed culture of oil-degrading bacteria, with the ability to secrete keratinases. The mixed culture was grown in the media with oil, or oil supplemented with chicken-feathers as the keratinous waste. Residual oil-hydrocarbons were determined as total petroleum hydrocarbons (TPHs) and oil fractions and then quantified by GC–FID and GC–MS.Results showed that in presence of the keratinous waste, the removal of oil-hydrocarbons was 57,400 mg l^?1, meanwhile the treatment without waste presented an oil-hydrocarbons removal of 35,600 mg l^?1. The aliphatic fraction was the most removed in both treatments. In addition, chromatographic profiles indicated that the aliphatic fraction showed different degradation pattern; in the presence of keratinous wastes, the C₁₈ to C₂₈ compounds were preferably removed over the C₁₀ to C₁₇. The addition of keratinous waste not only improved the oil-hydrocarbons removal but, it changed the removal pattern of the target hydrocarbons.     

Kinetic study of the anaerobic degradation of toluene by a mixed culture

Toluene was anaerobically degraded by an enriched mixed culture under methanogenic conditions. The mixed culture was originally developed from cow-dung and sludge from a laboratory reactor, in which benzene was anerobically degraded by sulphate-reducing bacteria. First the mixed culture was enriched on toluene over a year with and without the use of sulphate in the medium. For the evaluation of growth-kinetic and maintenance parameters, namely μ_max, K_s, k_d and Y, the anaerobic degradation of toluene was carried out in batch as well as in continous reactors systems. The gas volume and the methane content in the produced gas was somewhat lover than the theoretical value expected, indicating an incomplete degradation of some of the complex intermediates of the toluene degradation pathway. However, the mixed culture was able to transform 41.3% of the toluene carbon into methane.     

Influence of the glycolipid-producing bacterium Rhodococcus erythropolis on the degradation of a hydrocarbon mixture by an original soil population

Summary The purpose of this investigation was to show whether or no employing a starter culture of the bacterium Rhodococcus erythropolis that produces 6,6-trehalosedicorynomycolates could replace the addition of purified biosurfactant known to accelerate hydrocarbon degradation by an original soil population in a stirred reactor. The rate of degradation, degree of elimination of hydrocarbons, mineralization and degree of oxidation were determined in order to assess the extent of degradation. In comparison with degradation by soil microorganisms only an acceleration of utilization of the hydrocarbons was observed in cultivations of growing cells of R. erythropolis, although the effect of purified trehalosedicorynomycolates is not reached. Except for a higher degree of oxidation a sufficient amount of trehalosedicorynomycolates bound to autoclaved biomass of R. erythropolis does not have any effect.     

Effect of pH oscillations on a competing mixed culture

Two microorganisms, E. coli and S. cerevisiae, competing for glucose were maintained in a stable cycle of coexistence by alternating the growth advantage between the two organisms by oscillating the pH in a Chemostat. Pure culture experiments found S. cerevisiae to be insensitive to pH between 5 and 4.3 with a maximum specific growth rate (mu(max)) of 0.4/hr; while mu(max) of E. coli decreased from 0.6 h(-1) at pH 5 to 0.1 h(-1) at pH 4.3. Steady-state and cross-inoculation chemostat runs at a dilution rate of 0.17 h(-1) confirmed the expectation that the mixed culture system is unstable at constant pH with E. coli dominating at pH 5 and S. cerevisiae dominating at pH 4.3. Three pH oscillation experiments were performed at D =0.17 h(-1) with 1 g per liter glucose feed. The 16 h/16 h cycle was stable for six periods with a stable alternating cycle of E. coli and S. cerevisiae being quickly established. A 18 h pH 5/14 h pH 4.3 cycle was found to be stable with smaller yeast concentrations. A 6 h/6 h cycle was found unstable with yeast washout. Simulation results were compared with these runs and were used to predict the onset of instability. Oscillations of pH can force stable persistence of a competing mixed culture that is otherwise unstable. Thus, varying conditions are experimentally demonstrated to be one explanation for competitive coexistence.     

Sulfate reduction and anaerobic glycerol degradation by a mixed microbial culture

Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.     

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on crude oil

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on Bow River crude oil was investigated using solid phase microextraction (SPME). Inoculum treatments were examined in relation to C₅–C₁₁ hydrocarbon degradation. Up to 1600 mg/l biomass (dry weight) was tested without achieving significant volatile hydrocarbon partitioning and affecting analysis. Inoculum age rather than concentration had the most profound impact on biodegradation. When late log phase crude oil-grown inocula were used, C₅–C₁₁ biodegradation reached 55–60%; methylcyclohexane and other branched compounds eluting before n-C₈ were recalcitrant. Increasing the late log inoculum concentration from 0.63 to 63 mg/l resulted in a twofold increase in degradation rate without improving the substrate range. Methylcyclohexane recalcitrance was correlated with reduced levels of hydrocarbon-degrading bacteria and volatile hydrocarbon evaporation from the inoculum flasks. A decreased lag phase prior to degradation was observed when using early stationary phase cultures as inocula and most compounds up to C₁₁, including methylcyclohexane, were biodegraded. Journal of Industrial Microbiology & Biotechnology (2001) 26, 356–362. Received 16 November 2000/ Accepted in revised form 17 March 2001     

Anaerobic degradation of phenol using an acclimated mixed culture

Summary Anaerobic methanogenesis of phenol using mixed cultures derived from cow dung and municipal sewage sludge and adapted to phenol was done in batch reactors. The phenol degradation rate depended on the period in which the culture was acclimated to phenol. Interference in phenol uptake by glucose was observed. Consumption of both phenol and acetic acid was observed when an acetate-adapted culure was used. A phenol-acclimated culture was able to degrade dihydroxy phenols thus indicating the feasibility of cross-acclimation. Offprint requests to: P. Ghosh     

Effect of cycling on final mixed culture fate

Cycling in feed substrate concentration and dilution rate is examined as a means of modifying the final fate of a mixed culture. It is shown for the case where the specific growth rate of one species is always greater than that of the second that no cycling strategy will provide the desired extinction of the faster growing species unless time delay is included in the modeling. To account for the time lag in adjusting organism metabolic activities to environmental changes, an adaptability parameter is introduced. Numerical simulations are carried out and an operating diagram indicating the conditions under which the desired extinction occurs is constructed. Cycling in feed substrate concentration and dilution rate are both found to produce the desired result.     

Continuous anaerobic phenol degradation using an adapted mixed culture

A mixed culture derived from cow dung and sewage sludge and adapted to phenol was used for anaerobic phenol degradation. The phenol degradation rate depended on the period of adaptation of the mixed culture to phenol. In the continuous process, a higher degradation rate (2500 mg.1^-1 d^-1) and better reactor stability was achieved with a granular activated-carbon-packed bed reactor than with a stirred tank reactor.The authors are with the Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, Delhi Hauz Khas, New Delhi, India.     

Effect of the addition of microbial surfactants on hydrocarbon degradation in a soil population in a stirred reactor

Summary The hydrocarbon degradation rate could be doubled by the addition of sophorose lipids as biosurfactants in a model system containing 10% soil and a 1.35% hydrocarbon mixture of tetradecane, pentadecane, hexadecene, 1,2,4-trimethylcyclohexane, pristane (2,6,10,14-tetramethylpentadecane) phenyldecane and naphthalene suspended in mineral salts medium. The adaptation phases for two degradation phases were shortened, and the extent of degradation and final biomass were increased. The added biosurfactants were degraded after they had facilitated degradation of all hydrocarbon components.     

Enhanced anaerobic degradation of benzene by enrichment of mixed microbial culture and optimization of the culture medium

A heterogeneous mixed culture, originally collected from two different sources, namely cow-drug and sludge from the mineral medium containing 1% glucose and then adapted on benzene as the carbon and energy source. Under anaerobic conditions benzene was degraded via benzoic acid as a major intermediate in the benzene degradation pathway. The degradation rate of benzene was improved stepwise by the number of enrichments and optimization of the culture medium. The effects of microaerobic conditions and/or physicochemical treatment with H₂O₂ prior to anaerobic degradation were studied with respect to variations in benzene degradation rate, growth of biomass and gas produced is less than the theoretical value expected and the percentage of methane in the product gas was very small (3%–3.5%). The reason for this is not well understood but it is presumed that the major group of benzene-degrading bacteria present in the culture medium are sulphate reducers and the mixed consortium is unable to degrade certain complex aromatic intermediates in the benzene degradation pathway under the experimental conditions. For an actual explanation of the situation arising in this study, further investigations must be carrie out. However, the mixed culture is capable of oxidizing benzene more rapidly to intermediate compounds and also partly into gas under the culture conditions, compared to the published data for the anaerobic degradation of benzene.     

Partial degradation of p-aminoazobenzene by a defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia

A defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia was used to accomplish the partial biodegradation of the azo-dye p-aminoazobenzene (pAAB). Kinetic experiments were conducted, under aerobic conditions, to study the mineralization of p-aminoazobenzene by the above-defined mixed culture, under aerobic conditions. The combination of two previously developed models, (Zissi et al., 1997), which describes pAAB biodegradation by Bacillus subtilis into aniline and p-phenylenediamine, and (Zissi and Lyberatos, 1999), which describes aniline biodegradation by Stenotrophomonas maltophilia, is shown to predict well the anticipated mixed culture growth and partial biodegradation of pAAB. In previous work (Zissi et al., 1997) it was observed that pphenylenediamine was unstable during the experiments therefore the fate of p-phenylenediamine was not studied. The overall kinetic model of the defined mixed culture was then used to study the behavior of the mixed culture system in a range of operating conditions in the chemostat. The partial degradation of pAAB (regarding one of the two products, aniline) was described by an interaction between the two bacteria with competitive and commensalistic elements. The two bacteria are shown to coexist in a CSTR for some ranges of the operating variables.     

Biodegradation of the mixture of 2,4,6-trichlorophenol, 4-chlorophenol,and phenol by a defined mixed culture

Two new strains, Pseudomonas sp. TCP114 degrading 2,4,6-trichlorophenol (TCP) and Arthrobacter sp. CPR706 degrading 4-chlorophenol (4-CP), were isolated through a selective enrichment procedure. Both strains could also degrade phenol. The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 could not degrade any of the components present. This restriction on degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through cooperative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three-component mixture. Immobilization not only accelerates the degradation rate, but also enables reuse of the cell mass several times without losing the cells' degrading capabilities.     

The strategy of strain selection for a mixed culture performing rapid conversion of a mixture of polyaromatic compounds

The strain Sphingomonas sp. VKM V-2434 converts the mixture of seven polyaromatic compounds (PACs): fluorene, dibenzothiophene, carbazole, phenanthrene, anthracene, fluoranthene, and pyrene. The effect of each of the above PACs on the rate of mixture conversion was determined. The following two strains, which utilize the substances inhibiting the studied process, were added to the culture: strain FON-11 utilizing 9-fluorenone (fluorene metabolite) and strain CBZ-21 utilizing carbazole. In the case of the mixed culture of three strains, conversion rates were 1.5 and 1.2–3.8 times higher for the PAC mixture and its individual components, respectively, than the rates for Sphingomonas sp. VKM V-2434 monoculture. The degree of degradation of PAC conversion products increased from 32 to 44%. The rate of PAC conversion by the mixed culture exceeded the sum of conversion rates for the individual component strains; this cooperative effect was particularly marked for anthracene and pyrene.