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.     

Kinetic study and mathematical modelling of killer and sensitive S. Cerevisiae strains growing in mixed culture

This paper presents a kinetic study of two yeasts growing in pure and mixed batch cultures. Two winemaking strains were used: S. cerevisiae K1 possessing the K2 killer character and S. cerevisiae 522D sensitive to the K2 killer toxin. Initially the kinetics of growth of the two strains were analysed in pure culture. In this case, the kinetic profiles of biomass production have shown that the growth rate of the K1 strain is slightly superior to the 522D strain. During the fermentation, the viability for both populations was higher than 90%. Fermentations in mixed culture with an initial percentage in killer strain of 5 and 10% with respect to the total population were carried out. The results showed a more important decrease in the percentage of total viable yeasts when the initial concentration of killer yeast increased. However, the kinetic profiles of total biomass (killer plus sensitive yeasts) were very similar for both fermentations. A mathematical model was proposed to simulate the microbial growth of the killer and sensitive strain developing in pure and mixed cultures. This mathematical model consists in three main reactions: the evolution of the killer toxin in the culture medium, the duplication and the mortality rates for each microbial population. The results of the simulation appeared in agreement with the experimental data.     

Growth Kinetics of Suspended Microbial Cells: From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in K_S for glucose from some 5 mg liter⁻¹ to ca. 30 μg liter⁻¹. The data suggest that a dilemma exists, namely, that either “intrinsic” K_S (under substrate-controlled conditions in chemostat culture) or μ_max (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.     

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

Methylobacterium sp. strain DM4 and Methylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the k_cat values of the enzymes of these strains are 0.6 and 3.3 s⁻¹, respectively, and the K_m values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the K_m of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants.     

Phenol removal from waste gases with a biological filter by Pseudomonas putida

The purpose of this study is to investigate the feasibility of biologically removing phenol from waste gases by means of a biofilter using a Pseudomonas putida strain. Two series of both batch and continuous tests have been performed in order to ascertain the microbial degradation of phenol. For the preliminary batch tests, carried out in order to test the effective feasibility of the process and to investigate their kinetic behavior, two different microbial cultures belonging to the Pseudomonas genus have been employed, a heterogeneous culture and a pure strain of P. putida. The results of these comparative investigation showed that the pure culture is more efficient than the mixed one, even when the latter has undergone three successive acclimatization tests. The continuous experiments have been conducted during a period of about 1 year in a laboratory-scale column, packed with a mixture of peat and glass beads, and utilizing the pure culture of P. putida as microflora and varying the inlet phenol concentration from 50 up to 2000 mg m(-3). The results obtained show that high degrees of conversion can be obtained (0.93/0.996) operating at a residence time of 54 s. (c) 1993 John Wiley & Sons, Inc.     

Dark-Fermentative Biological Hydrogen Production from Mixed Biowastes Using Defined Mixed Cultures

Biological hydrogen (H₂) production from the biowastes is widely recognized as a suitable alternative approach to utilize low cost feed instead of costly individual sugars. In the present investigation, pure and mixed biowastes were fermented by defined sets of mixed cultures for hydrolysis and H₂ production. Under batch conditions, up to 65, 67 and 70 L H₂/kg total solids (2%, TS) were evolved from apple pomace (AP), onion peels (OP) and potato peels (PP) using a combination of hydrolytic mixed culture (MHC5) and mixed microbial cultures (MMC4 or MMC6), respectively. Among the different combinations of mixed biowastes including AP, OP, PP and pea-shells, the combination of OP and PP exhibited maximum H₂ production of 73 and 84 L/kg TS with MMC4 and MMC6, respectively. This study suggested that H₂ production can be effectively regulated by using defined sets of mixed cultures for hydrolysis and H₂ production from pure and mixed biowastes as feed even under unsterile conditions.     

Biodegradation and detoxification of phenolic compounds by pure and mixed indigenous cultures in aerobic reactors

Degradation and detoxification of a mixture of persistent compounds (2-chlorophenol, phenol and m-cresol) were studied by using pure and mixed indigenous cultures in aerobic reactors. Biodegradation assays were performed in batch and continuous flow reactors. Biodegradation was evaluated by determining total phenols, ultraviolet spectrophotometry and chemical oxygen demand (COD). Microbial growth was measured by the plate count method. Scanning electronic microscopy was employed to observe the microbial community in the reactor. Detoxification was evaluated by using Daphnia magna toxicity tests. Individual compounds were degraded by pure bacteria cultures within 27 h. The mixture of 2-clorophenol (100 mgl⁻¹), phenol (50 mgl⁻¹) and m-cresol (50 mgl⁻¹) was degraded by mixed bacteria cultures under batch conditions within 36 h: 99.8% of total phenols and 92.5% of COD were removed; under continuous flow conditions 99.8% of total phenols and 94.9% of COD were removed. Mineralization of phenolic compounds was assessed by gas chromatography performed at the end of the batch assays and in the effluent of the continuous-flow reactor. Toxicity was not detected in the effluent of the continuous-flow reactor.     

Differences in membrane proteins,cyclic AMP levels,and glucose transport between batch and chemostat cultures ofEscherichia coli

Escherichia coli bacteria were grown in minimal-salt medium with glucose as carbon source under either batch or chemostat culture conditions. The physiological state of chemostatgrown bacteria is significantly different from those grown in batch cultures. Differences were measured in outer membrane protein composition,K _m for glucose uptake, and intracellular cyclic AMP levels. Since chemostat growth conditions more closely resemble conditions bacteria encounter in nature, these physiological differences may be significant for bacterial adaptation and survival.     

Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

Deuterated styrene ([²H₈]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [²H₈]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [²H₈]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

Distinct Microbial Behavior of Re Compared to Tc: Evidence Against Microbial Re Fixation in Aquatic Sediments

Rhenium is enriched in suboxic and anoxic sediments relative to oxic sediments, a characteristic that is being exploited in its use as a paleoredox indicator. Rhenium is fixed at sediment depths where iron reduction and sulfate reduction are the dominant microbial terminal electron-accepting processes. In order to explore mechanisms of its fixation, we investigated perrhenate behavior in pure, batch cultures of two dissimilatory sulfate-reducing strains (Desulfovibrio desulfuricans subsp. desulfuricans and Desulfovibrio desulfuricans ND132) and two iron-reducing strains (Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1). Perrhenate concentrations tested ranged from 0.04 to 12 μM, roughly 4 to 7 orders of magnitude larger than seawater Re concentrations. Within this broad concentration range, none of the organisms tested actively removed Re from solution during one week's growth to stationary phase. Despite these results, the sulfate-reducing cultures appeared to have reached supersaturation relative to ReS_2(s), and the iron-reducing cultures may have reached supersaturation relative to ReO_2(s). We conclude that neither direct nor short-term indirect microbial processes involving these bacteria are likely to explain Re fixation in sediments. Our results cannot exclude the possibility that microbial metabolites, such as Fe(II) or sulfide, do drive abiotic Re fixation over longer periods of time. The lack of perrhenate reduction by dissimilatory sulfate-reducing bacteria and iron-reducing bacteria contrasts with published reports of pertechnetate behavior. Despite many qualitative similarities between Re and Tc, it is clear that these two elements are quantitatively dissimilar, with Re fixation requiring more intensely reducing conditions.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

Biological removal of H2S from the livestock manure using a biofilter

In this paper, the biological removal of H₂S from air had been investigated using a self-made biofilter with efficient bioceramics and a polyhedral hollow ball. The biological removal efficiency of H₂S had been analyzed at different experimental conditions, such as inlet H₂S concentration, residence time, initial pH value, and reaction temperature etc. The results showed that the initial pH value had a slight effect on H₂S removal efficiency from pH 3 to 9. The optimal initial pH value was 5.5, while the H₂S removal efficiency was 100%. The H₂S removal efficiency increased with increases in the nutrient solution spraying rate. The appropriate temperature was 25°C in the temperature range from 15 to 30°C. The H₂S removal efficiency dropped with the increase of air input and inlet H₂S concentration. After being isolated and screened, six strains of heterotrophic sulfide oxidizing bacteria and one strain autotrophic sulfide oxidizing bacteria were determined to be involved in the removal of H₂S within the biofilter. The reaction kinetics of H₂S was in accordance with first order reaction kinetics.     

Kinetic behavior of mixed populations of activated sludge

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage isothermal continuous flow completely mixed aeration tank. A series of experiments were carried out at various dilution rates using glucose as the growth limiting substrate. The steady-state behavior of the system was observed at each dilution rate and the results were found to fit fairly well with the steady-state equation bayed on the Monod model with an endogenous respiration term included, i.e., μ = μ_mS/(K_s + S) ? K_d. The growth kinetics of cells harvested at steady state for each dilution rate were studied using batch experiments. The multiple response data of the system as functions of time were used to estimate the parameter values in the above kinetic model. It was found that values of the growth parameters changed significantly and systematically with cell population. For example, values of μ_m were high at high dilution rates and low at low dilution rates. It was also found that only those batch growth parameters from cells obtained at fairly high dilution rates are comparable with those estimated by the results of steady-state operations. The results of this investigation suggest that (1) different cell populations pre dominated at different steady-state dilution rates, with high dilution rates resulting in predominantly fast-growing organisms and low dilution rates resulting in predominantly slow-growing cells, and (2) risk exists in any randomly picked batch experiment to predict the steady-state behavior of the system when heterogeneous microbial populations must be used.     

Impact of initial conditions on extant microbial kinetic parameter estimates: application to chlorinated ethene dehalorespiration

Monod kinetics are the foundation of mathematical models of many environmentally important biological processes, including the dehalorespiration of chlorinated ethene groundwater contaminants. The Monod parameters—q _max, the maximum specific substrate utilization rate, and K _S, the half-saturation constant—are typically estimated in batch assays, which are superficially simple to prepare and maintain. However, if initial conditions in batch assays are not chosen carefully, it is unlikely that the estimated parameter values will be meaningful because they do not reflect microbial activity in the environmental system of interest, and/or they are not mathematically identifiable. The estimation of q _max and K _S values that are highly correlated undoubtedly contributes significantly to the wide range in reported parameter values and may undermine efforts to use mathematical models to demonstrate the occurrence of natural attenuation or predict the performance of engineered bioremediation approaches. In this study, a series of experimental and theoretical batch kinetic assays were conducted using the tetrachloroethene-respirer Desulfuromonas michiganensis to systematically evaluate the effects of initial batch assay conditions, expressed as the initial substrate (S ₀)-to-initial biomass concentration (X ₀) ratio (S ₀/X ₀) and the S ₀/K _S ratio on parameter correlation. An iterative approach to obtain meaningful Monod parameter estimates was developed and validated using three different strains and can be broadly applied to a range of other substrates and populations. While the S ₀/X ₀ ratio is critical to obtaining kinetic parameter estimates that reflect in situ microbial activity, this study shows that optimization of the S ₀/K _S ratio is key to minimizing Monod parameter correlation.     

Development of a miniaturized selective counting strategy of lactic acid bacteria for evaluation of mixed starter in a model cassava fermentation

A miniaturized most probable number (MPN) method for the selective enumeration of three bacteria species ( Lactobacillus plantarum A6, Leuconostoc mesenteroides and Lactococcus lactis ) is described. This selective count method, based on specific consumption of carbon substrate and resistance to antibiotics, was used for the quantitative assessment of the three bacteria during mixed cultures in a model cassava fermentation. A typical microbial succession pattern was observed: (i) Lactococcus lactis and Leuc. mesenteroides dominated during the first hours of fermentation as their growth was very rapid ; (ii) from hour 12, Lactobacillus plantarum replaced the two latter strains and Lactococcus lactis disappeared gradually, followed by Leuc. mesenteroides . The growth rates of each strain appeared to be independent of the others, while acidification rates increased strongly in mixed cultures compared with pure cultures. No positive interactions resulting from the amylolytic character of Lactobacillus plantarum A6, and no negative interactions resulting from the Nis⁺ property of Lactococcus lactis , were revealed between the three strains under the model conditions used.     

Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

Background

Pure bacterial strains give better yields when producing H₂ than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H₂ production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.

Results

Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20?L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H₂ production (sequence 1); and the H₂-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H₂ yields were obtained for starch fermentations, and the highest yield of 2.91?mol?H₂/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5?L biogas/ h was observed for the co-culture (1). In general co-cultures produced H₂ at higher rates than the pure Clostridium cultures, without negatively affecting the H₂ yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H₂. In the second part of the study the co-culture (3) was used to produce H₂ during 13?days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.

Conclusions

The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H₂ using glucose and starch as carbon sources. The artificial co-cultures produced H₂ at higher rates than the pure strains, while the H₂ yields were only slightly affected.     

Stable-isotope-based labeling of styrene-degrading microorganisms in biofilters

Deuterated styrene ([(2)H(8)]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [(2)H(8)]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [(2)H(8)]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

Rates of Transformation of Methyl Parathion and Diethyl Phthalate by Aufwuchs Microorganisms

Using batch cultures, we determined transformation rates for low concentrations of two toxicants—an insecticide, methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), and a plasticizer, diethyl phthalate—by aufwuchs, aquatic microbial growth attached to submerged surfaces or suspended in streamers or mats. Aufwuchs samples were collected from field sites, an indoor channel, and a continuous-flow fermentor. Aufwuchs fungi, protozoa, and algae did not transform methyl parathion or diethyl phthalate, but bacteria rapidly transformed both chemicals. Second-order transformation rate coefficients, K_b, based on total plate counts of bacteria in aufwuchs, were determined for potential use in a mathematical model capable of predicting the transport and fate of chemicals in aquatic systems. K_b for both methyl parathion and diethyl phthalate decreased as the concentration of total bacteria, [B], increased in aufwuchs. This effect resulted from the proportion of nontransformer to transformer bacteria increasing as [B] increased and from the rate of transformation per transformer cell decreasing as [B] increased. First-order transformation rate coefficients, K₁, were relatively stable per unit of surface area colonized by aufwuchs, because K_b decreased as [B] increased (K₁ = K_b × [B]).     

Biodegradation of benzene by pure and mixed cultures of <Emphasis Type="Italic">Bacillus</Emphasis> spp.

Benzene has a wide range of industrial applications, but it is also a major source of environmental pollution. The most eco-friendly/cost-effective method of remediation is biodegradation. In the present study, we used a variety of microbial strains in different combinations on a selection of substrate concentrations to determine the most effective degradation processes. Bacterial strains of pure culture (L₄, N₃, and N₆) were isolated from oil sludge in both Luria–Bertani buffer (LB) and nutrient broth media, and identified by 16S-rRNA analysis (≥98% similarity). The degradation experiments were performed using different combinations of bacterial strains (L₄, N₃, N₆, L₄ + N₃, L₄ + N₆, N₃ + N₆, and L₄ + N₃ + N₆) in modified carbon-free media with different concentrations of benzene as a carbon source (60, 100, and 160 mg l⁻¹) at 30 °C. The isolates of L₄ (Acc no: FJ686821), N₃ (FJ686825) and N₆ (FJ868628) were identified as Bacillus spp. using 16S-rRNA gene sequence analysis. All combinations of isolates have the capacity to degrade benzene. However, the L₄ + N₃ combination was more efficient than the other mixed or single cultures. In the presence of N₆ isolate, the degradation rate of benzene decreased, possibly due to inter- and/or intra species interaction amongst the bacteria. The kinetic parameters ‘K _m’ of the Lineweaver–Burk regressions conducted as part of this experiment showed that the lower the level of K _m was, the better the biodegradation achieved. The results of this study showed that the use of Bacillus strains in benzene decomposition is feasible. In addition, different strain combinations exhibited different degradation patterns, which are attributed to the most efficient mixed cultures of Bacillus spp.