Optimization of field scale biopiles for bioremediation of petroleum hydrocarbon contaminated soil at low temperature conditions by response surface methodology (RSM)

Ex-Situ Bioremediation has been increasingly viewed as an appropriate remediation technology for hydrocarbon contaminated soils under cold climates conditions in countries like Canada. A response surface methodology (RSM) based on a factorial design was performed to investigate and optimize the effects of the microbial consortia application rate and amount of mature compost amendment on the TPH removal (964 μg g⁻¹ initial concentration). 18 field-scale biopiles (16 m³ each) were constructed, maintained and subjected to different microbial consortium and mature compost application rates under cold climate conditions over a period of 94 days. TPHs removal rates in the range of 74–82% was observed in the treatments setups where mature compost and microbial consortia were used simultaneously, compared to an average 48% of TPHs removal in control setup.The interaction between these two factors were studied and modelled using a statistical regression model, which showed that the microbial consortia application rate, the mature compost amendment and their interactions had a significant effect on TPHs degradation with a coefficient of determination (R²) of 0.88. Furthermore, using a numerical optimization approach, the optimum rates predicted via RSM were estimated at 4.1 ml m⁻³ and 7% for microbial consortia and compost application rates to obtain a maximum TPH removal of 90.7%.     

Diclofop-methyl increases the proton permeability of isolated oat-root tonoplast

Diclofop-methyl (methyl ester of 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propionate; 100 micromolar) and diclofop (100 micromolar) inhibited both ATP- and PPi-dependent formation of H⁺ gradients by tonoplast vesicles isolated from oat (Avena sativa L., cv Dal) roots. Diclofop-methyl (1 micromolar) significantly reduced the steady-state H⁺ gradient generated in the presence of ATP. The ester (diclofop-methyl) was more inhibitory than the free acid (diclofop) at pH 7.4, but this relative activity was reversed at pH 5.7. Neither compound affected the rate of ATP or PPi hydrolysis by the proton-pumping enzymes. Diclofop-methyl (50, 100 micromolar), but not diclofop (100 micromolar), accelerated the decay of nonmetabolic H⁺ gradients established across vesicle membranes. Diclofop-methyl (100 micromolar) did not collapse K⁺ gradients across vesicle membranes. Both the (+)- and (−)-enantiomers of diclofop-methyl dissipated nonmetabolic H⁺ gradients established across vesicle membranes. Diclofop-methyl, but not diclofop (each 100 micromolar), accelerated the decay of H⁺ gradients imposed across liposomal membranes. These results show that diclofop-methyl causes a specific increase in the H⁺ permeability of tonoplast.     

Interspecific interactions in mixed microbial cultures in a biodegradation perspective

In recent works, microbial consortia consisting of various bacteria and fungi exhibited a biodegradation performance superior to single microbial strains. A highly efficient biodegradation of synthetic dyes, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and other organic pollutants can be achieved by mixed microbial cultures that combine degradative enzyme activities inherent to individual consortium members. This review summarizes biodegradation results obtained with defined microbial cocultures and real microbial consortia. The necessity of using a proper strategy for the microbial consortium development and optimization was clearly demonstrated. Molecular genetic and proteomic techniques have revolutionized the study of microbial communities, and techniques such as the denaturing gradient gel electrophoresis, rRNA sequencing, and metaproteomics have been used to identify consortium members and to study microbial population dynamics. These analyses could help to further enhance and optimize the natural activities of mixed microbial cultures.     

Seasonal Dynamics of Shallow-Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R² = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R² = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the α, β, and γ-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.     

Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis

Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 10³ to 10⁶. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (M_r), and the logarithm of the relative mobility, log (R_l), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, K_R, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (M_r) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (R_l). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (M_r) and log (R_l) or the one between log (M_r) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis.     

Thermodynamic and Kinetic Requirements in Anaerobic Methane Oxidizing Consortia Exclude Hydrogen,Acetate, and Methanol as Possible Electron Shuttles

Anaerobic methane oxidation (AMO) has long remained an enigma in microbial ecology. In the process the net reaction appears to be an oxidation of methane with sulfate as electron acceptor. In order to explain experimental data such as effects of inhibitors and isotopic signals in biomarkers it has been suggested that the process is carried out by a consortium of bacteria using an unknown compound to shuttle electrons between the participants. The overall change in free energy during AMO with sulfate is very small (≈22 kJ mol⁻¹) at in situ concentrations of methane and sulfate. In order to share the available free energy between the members of the consortium, the concentration of the intermediate electron shuttle compound becomes crucial. Diffusive flux of a substrate (i.e, the electron shuttle) between bacteria requires a stable concentration gradient where the concentration is higher in the producing organism than in the consuming organism. Since changes in concentrations cause changes in reaction free energies, the diffusive flux of a catabolic product/substrate between bacteria is associated with a net loss of available energy. This restricts maximal interbacterial distances in consortia composed of stationary bacteria. A simple theoretical model was used to describe the relationship between inter-bacterial distances and the energy lost due to concentration differences in consortia. Key parameters turned out to be the permissible concentration range of the electron shuttle in the consortium (i.e., the concentration range that allows both participants to gain sufficient energy) and the stoichiometry of the partial reactions. The model was applied to two known consortia degrading ethanol and butyrate and to four hypothetical methane-oxidizing consortia (MOC) based on interspecies transfer of hydrogen, methanol, acetate, or formate, respectively. In the first three MOCs the permissible distances between producers and consumers of the transferred compounds were less than two times prokaryotic cell wall diameters. Consequently, it is not possible that a MOC can be based on inter-species transfer of hydrogen, methanol, or acetate. Formate, on the other hand, is a possible shuttle candidate provided the bacteria are attached to one another. In general the model predicts that members of consortia thriving on low energy such as the MOC must adhere to each other and utilize a compound for the exchange of electrons that has a high permissible concentration range and a high diffusion coefficient and transfers as many electrons as possible per molecule.     

Microbial Activity in Aquatic Environments Measured by Dimethyl Sulfoxide Reduction and Intercomparison with Commonly Used Methods

A new method to determine microbial (bacterial and fungal) activity in various freshwater habitats is described. Based on microbial reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), our DMSO reduction method allows measurement of the respiratory activity in interstitial water, as well as in the water column. DMSO is added to water samples at a concentration (0.75% [vol/vol] or 106 mM) high enough to compete with other naturally occurring electron acceptors, as determined with oxygen and nitrate, without stimulating or inhibiting microbial activity. Addition of NaN₃, KCN, and formaldehyde, as well as autoclaving, inhibited the production of DMS, which proves that the reduction of DMSO is a biotic process. DMSO reduction is readily detectable via the formation of DMS even at low microbial activities. All water samples showed significant DMSO reduction over several hours. Microbially reduced DMSO is recovered in the form of DMS from water samples by a purge and trap system and is quantified by gas chromatography and detection with a flame photometric detector. The DMSO reduction method was compared with other methods commonly used for assessment of microbial activity. DMSO reduction activity correlated well with bacterial production in predator-free batch cultures. Cell-production-specific DMSO reduction rates did not differ significantly in batch cultures with different nutrient regimes but were different in different growth phases. Overall, a cell-production-specific DMSO reduction rate of 1.26 × 10⁻¹⁷ ± 0.12 × 10⁻¹⁷ mol of DMS per produced cell (mean ± standard error; R² = 0.78) was calculated. We suggest that the relationship of DMSO reduction rates to thymidine and leucine incorporation is linear (the R² values ranged from 0.783 to 0.944), whereas there is an exponential relationship between DMSO reduction rates and glucose uptake, as well as incorporation (the R² values ranged from 0.821 to 0.931). Based on our results, we conclude that the DMSO reduction method is a nonradioactive alternative to other methods commonly used to assess microbial activity.     

Isolation of UVC-Tolerant Bacteria from the Hyperarid Atacama Desert, Chile

Martian surface microbial inhabitants would be challenged by a constant and unimpeded flux of UV radiation, and the study of analog model terrestrial environments may be of help to understand how such life forms could survive under this stressful condition. One of these environments is the Atacama Desert (Chile), a well-known Mars analog due to its extreme dryness and intense solar UV radiation. Here, we report the microbial diversity at five locations across this desert and the isolation of UVC-tolerant microbial strains found in these sites. Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA sequences obtained from these sites showed banding patterns that suggest distinct and complex microbial communities. Analysis of 16S rDNA sequences obtained from UV-tolerant strains isolated from these sites revealed species related to the Bacillus and Pseudomonas genera. Vegetative cells of one of these isolates, Bacillus S3.300-2, showed the highest UV tolerance profile (LD₁₀?=?318 J?m²), tenfold higher than a wild-type strain of Escherichia coli. Thus, our results show that the Atacama Desert harbors a noteworthy microbial community that may be considered for future astrobiological-related research in terms of UV tolerance.     

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Polycyclic Aromatic Hydrocarbon-Induced Structural Shift of Bacterial Communities in Mangrove Sediment

Mangrove sediment is well known for its susceptibility to anthropogenic pollution, including polycyclic aromatic hydrocarbons (PAHs), but knowledge of the sediment microbial community structure with regards to exposure to PAHs is limited. The study aims to assess the effects of PAHs on the bacterial community of mangrove sediment using both 16s rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and traditional enrichment methods. Both the exposure time and the PAH concentration reduced the microbial diversity, as determined by the DGGE bands. Although PAHs could act as carbon sources for microorganisms, PAHs, at a concentration as low as 20 mg l⁻¹, posed a toxic effect to the microbial community. Sequencing of DGGE bands showed that marine bacteria from the genera of Vibrio, Roseobacter, and Ferrimonas were most abundant after PAH exposure, which suggests that both marine and terrestrial bacteria coexisted in the mangrove sediment, but that the marine microbes were more difficult to isolate using the traditional culture method. DGGE determination further demonstrated that the consistency among triplicates of the enriched consortia was significantly less than that of the sediment slurries. The present study reveals that the mangrove sediment microbial structure is susceptible to PAH contamination, and complex microbial community interactions occur in mangrove sediment.     

Effect of pH on the metabolic flux of Klebsiella oxytoca producing 2,3-butanediol in continuous cultures at different dilution rates

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h^?1) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.     

Cation Transport in Escherichia coli : III. Potassium fluxes in the steady-state

The present study is concerned with the measurement of the unidirectional K flux in E. coli. Methods are described by means of which a fairly dense suspension of cells may be maintained in a well defined steady-state with respect to the intracellular K concentration and the pH of the medium. The kinetics of K⁴² exchange under these conditions are consistent with the presence of a single intracellular K compartment with a unidirectional K flux of 1 pmol/(cm² sec.). This rate is independent of the extracellular K concentration over the range studied. The simultaneous rate of H secretion averages 16 pmols/(cm² sec.) indicating that in the steady-state the efflux of metabolically produced H is not linked mole for mole to K movement.     

Perturbation of Chara Plasmalemma Transport Function by 2[4(2',4'-Dichlorophenoxy)phenoxy]propionic Acid

Electrophysiological measurements on internodal cells of Chara corallina Klein ex Willd., em. R.D.W. revealed that in the presence of (2-[4-(2′,4′-dichlorophenoxy)phenoxy]propionic acid) (diclofop) the membrane potential was very sensitive to the pH of the bathing medium. At pH 5.7, 100 micromolar diclofop caused a slow reduction in the electrogenic component of the membrane potential to the value of −123 ± 5 millivolts. Membrane resistance initially decreased, recovered transiently, then stabilized at approximately 65% of the control value. At pH 7.0, the potential appeared to plateau around −200 millivolts before rapidly declining to −140 ± 4 millivolts; removal of diclofop resulted in recovery of the electrogenic component. Diclofop reduced cytoplasmic ATP levels by 96.4% and 36.6% at pH 5.7 and 7.0, respectively. At pH 8.2, diclofop did not change the ATP concentration significantly, but induced a hyperpolarization of the membrane potential to near −250 millivolts, and also reduced or inhibited the dark-induced hyperpolarization; the light-induced depolarization was reduced to a lesser extent. DCMU applied in the light elicited the same response at the plasmalemma as placing cells in the dark. When K⁺ channels were opened and cells depolarized with 10 millimolar K⁺, diclofop induced a further depolarization of approximately 30 millivolts. Cells decoupled with HPO₄⁻² were still sensitive to diclofop. Currents associated with OH⁻ efflux and HCO₃⁻ influx, as measured with a vibrating probe technique, became spatially destabilized and reduced in magnitude in the presence of diclofop. After 60 minutes, most of the cell surface was engaged in a low level of OH⁻ efflux activity. The results indicate that diclofop may be a proton ionophore at pH 7.0 and 5.7. At pH 8.2, diclofop may inhibit the operation of the H⁺-ATPase and OH⁻ efflux systems associated with HCO₃⁻ transport by perturbing the control processes that integrate the two, without a reduction in ATP concentration.     

Bioorganic Fertilizer Enhances Soil Suppressive Capacity against Bacterial Wilt of Tomato

Tomato bacterial wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases. Many strategies have been taken to improve soil suppressiveness against this destructive disease, but limited success has been achieved. In this study, a novel bioorganic fertilizer revealed a higher suppressive ability against bacterial wilt compared with several soil management methods in the field over four growing seasons from March 2011 to July 2013. The application of the bioorganic fertilizer significantly (P<0.05) reduced disease incidence of tomato and increased fruit yields in four independent trials. The association among the level of disease incidence, soil physicochemical and biological properties was investigated. The soil treated with the bioorganic fertilizer increased soil pH value, electric conductivity, organic carbon, NH₄ ⁺-N, NO₃ ^--N and available K content, microbial activities and microbial biomass carbon content, which were positively related with soil suppressiveness. Bacterial and actinomycete populations assessed using classical plate counts were highest, whereas R. solanacearum and fungal populations were lowest in soil applied with the bioorganic fertilizer. Microbial community diversity and richness were assessed using denaturing gel gradient electrophoresis profile analysis. The soil treated with the bioorganic fertilizer exhibited higher bacterial community diversity but lower fungal community diversity. Redundancy analysis showed that bacterial community diversity and richness negatively related with bacterial wilt suppressiveness, while fungal community richness positively correlated with R. solanacearum population. We concluded that the alteration of soil physicochemical and biological properties in soil treated with the bioorganic fertilizer induced the soil suppressiveness against tomato bacterial wilt.     

Ectyoplasia ferox, an Experimentally Tractable Model for Vertical Microbial Transmission in Marine Sponges

The oviparous sponge Ectyoplasia ferox is commonly found in Florida and the Bahamas. Every year in August and/or September about 6 days after a full moon, E. ferox will shed embryo-containing spawning material into the seawater from which hundreds to thousands of larvae will hatch per host individual. In order to investigate vertical microbial transmission in E. ferox, 16S rRNA gene library construction and denaturing gradient gel electrophoresis was employed. Microbial symbionts from six phyla and the unknown lineage SAUL were shown to be vertically transmitted. The identification of 21 VT clusters, of which 19 were situated within sponge-specific or sponge-coral-specific clusters, indicated that a large fraction of the symbiotic microbial consortium was present in the sexual reproductive stages. Spawning led to a 50 % reduction of microbial numbers in the adult sponge mesohyl. We furthermore provide the first evidence that the symbiotic microbial consortia of E. ferox were generally metabolically active within the reproductive stages. Finally, we propose E. ferox as a model system for vertical transmission owing to the ease of experimental access to all sexual reproductive stages, and to experimental tractability in the laboratory including the possibility of rearing symbiont-free juvenile sponges.     

Enrichment and characterization of an anaerobic cellulolytic microbial consortium SQD-1.1 from mangrove soil

Enrichment of microbial consortia provides an approach to simulate and investigate microbial communities in natural environments. In this study, a cellulolytic microbial consortium SQD-1.1 was enriched from mangrove soil of Qinglan port (Hainan, China) by 27 times continuous subcultivation under anaerobic static conditions. The consortium could completely degrade 0.2 % (w/v) filter paper within 3 days and utilized it as the sole carbon source. PCR-denaturing gradient gel electrophoresis analysis revealed a stable microbial community structure in the incubation process of 10 days and in the procedure of subcultivation. Twenty-four operational taxonomic units belonging to seven phyla were obtained from the full-length 16S rRNA gene library. Five clones, closest related to the genera Alkaliflexus, Clostridium, Alistipes, Spirochaeta, and Trichococcus, were the predominant ones. Among them, M117, phylogeneticly showing high similarity (16S rRNA gene identity, 95.3 %) with the cellulolytic anaerobic bacterium Clostridium straminisolvens CSK1^T, was the potential key cellulolytic bacterium. Using the plate cultivation method, 12 strains, including one potential new species and four potential new species of new genera, were isolated. The strain P2, corresponding to the most frequently detected clone (M05) in the 16S rRNA gene library, showed both CMCase and xylanase activity and may be another important cellulolytic bacterium. The findings of cellulase activity in cell pellet and cohesion and dockerin domains in metagenome data further suggested the potential of utilization of cellulosomes by the consortium to degrade cellulose. Consortium SQD-1.1 provides a candidate for investigating the mechanism of cellulose degradation under anoxic conditions in natural environments.     

Core Fluxome and Metafluxome of Lactic Acid Bacteria under Simulated Cocoa Pulp Fermentation Conditions

In the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive ¹³C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel ¹³C studies with [¹³C₆]glucose, [1,2-¹³C₂]glucose, and [¹³C₆]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains of L. fermentum and L. plantarum revealed major differences in their fluxes. The L. fermentum strains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas only L. fermentum NCC 575 used fructose to form mannitol. In contrast, L. plantarum strains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of different L. fermentum and L. plantarum strains indicated a dominant (96%) contribution of L. fermentum NCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures. L. fermentum NCC 575 might be one candidate due to its superior performance in flux activity.     

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

The microbial communities established in mesophilic and thermophilic expanded granular sludge bed reactors operated with sulfate as the electron acceptor were analyzed using 16S rRNA targeted molecular methods, including denaturing gradient gel electrophoresis, cloning, and phylogenetic analysis. Bacterial and archaeal communities were examined over 450 days of operation treating ethanol (thermophilic reactor) or ethanol and later a simulated semiconductor manufacturing wastewater containing citrate, isopropanol, and polyethylene glycol 300 (mesophilic reactor), with and without the addition of copper(II). Analysis, of PCR-amplified 16S rRNA gene fragments using denaturing gradient gel electrophoresis revealed a defined shift in microbial diversity in both reactors following a change in substrate composition (mesophilic reactor) and in temperature of operation from 30°C to 55°C (thermophilic reactor). The addition of copper(II) to the influent of both reactors did not noticeably affect the composition of the bacterial or archaeal communities, which is in agreement with the very low soluble copper concentrations (3–310 μg l⁻¹) present in the reactor contents as a consequence of extensive precipitation of copper with biogenic sulfides. Furthermore, clone library analysis confirmed the phylogenetic diversity of sulfate-reducing consortia in mesophilic and thermophilic sulfidogenic reactors operated with simple substrates.     

Temporal Stability and Biodiversity of Two Complex Antilisterial Cheese-Ripening Microbial Consortia

The temporal stability and diversity of bacterial species composition as well as the antilisterial potential of two different, complex, and undefined microbial consortia from red-smear soft cheeses were investigated. Samples were collected twice, at 6-month intervals, from each of two food producers, and a total of 400 bacterial isolates were identified by Fourier-transform infrared spectroscopy and 16S ribosomal DNA sequence analysis. Coryneform bacteria represented the majority of the isolates, with certain species being predominant. In addition, Marinolactobacillus psychrotolerans, Halomonas venusta, Halomonas variabilis, Halomonas sp. (10⁶ to 10⁷ CFU per g of smear), and an unknown, gram-positive bacterium (10⁷ to 10⁸ CFU per g of smear) are described for the first time in such a consortium. The species composition of one consortium was quite stable over 6 months, but the other consortium revealed less diversity of coryneform species as well as less stability. While the first consortium had a stable, extraordinarily high antilisterial potential in situ, the antilisterial activity of the second consortium was lower and decreased with time. The cause for the antilisterial activity of the two consortia remained unknown but is not due to the secretion of soluble, inhibitory substances by the individual components of the consortium. Our data indicate that the stability over time and a potential antilisterial activity are individual characteristics of the ripening consortia which can be monitored and used for safe food production without artificial preservatives.