Active prokaryotic population dynamics exhibit high correlation to reactor performance during methane production from acid hydrolysates of Agave tequilana var. azul bagasse.

AIMS: The purpose of this study was to apply cDNA approach for the characterization of active prokaryotic community to understand microbial scenarios and performance of an AnSBR digester fed with acid hydrolysates of Agave tequilana var. azul bagasse (ATAB). METHODS AND RESULTS: The digester was implemented for methane production under organic loading rate (OLR) disturbances to correlate physicochemical variables with changes in abundance, diversity and population dynamics of active Bacteria and Archaea by principal components analysis (PCA). Results indicated that methane yield increased as well as active syntrophic relationships for interspecies hydrogen/formate (Anaerolinaceae-Methanobacterium beijingense) and acetate (Anaerolinaceae-Methanosaeta concilii) transfers at 8 g-COD l-1  day-1 . However, methane yield was negatively affected at 16 g-COD l-1  day-1 due to the competition for acetate by active Desulfovibrio marrakechensis and volatile fatty acids inhibition. CONCLUSIONS: Microbial scenarios obtained by PCA correlations indicated that methane production from acid hydrolysates of ATAB was feasible at 8 g-COD l-1  day-1 . The digester operation at higher OLR only favoured methanogenesis by the hydrogenotrophic pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: Only cDNA analysis showed Archaea population dynamics, exhibiting high correlation with physicochemical variables towards the understanding of the methanogenic digester performance during OLR disturbances.

Impact of storage conditions on fungal community composition of green coffee beans Coffea arabica L. stored in jute sacks during 1 year.

AIMS: The purpose of this study was to evaluate the effect of warehouse storage conditions on the composition of the fungal community of green coffee beans (GCB) that were stored in jute sacks for 1 year. METHODS AND RESULTS: Molecular characterization of the fungal community composition and population dynamics obtained by Q-PCR, CE-SSCP (Simpson's diversity index D) and Illumina MiSeq sequencing indicated that Saccharomycetales dominated during the first 6 months of storage period with species as Meyerozyma guilliermondii and Pichia kluyveri. However, after 6 months of storage, the filamentous genus Wallemia became dominant. Principal components analysis correlated this fungal dynamic with storage conditions and other variables as chromaticity (colour), water activity, moisture content, reducing sugars concentration, fungal infection and ochratoxin A production. CONCLUSIONS: This study demonstrated that GCB stored in jute sacks after 6 months of storage lead to fungal population dynamics, decreased chromaticity in GCB by bleaching and, then, affected overall quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Storage plays an important role in the quality evolution during the handling of the GCB. In this context, the composition of the microbial community could be considered a marker to assess the trade value of the coffee beans.

Biodegradation of polycyclic aromatic hydrocarbons by an acidophilic Stenotrophomonas maltophilia strain AJH1 isolated from a mineral mining site in Saudi Arabia.

The present study aims at analyzing the degradation of polycyclic aromatic hydrocarbons (PAHs) at acidic conditions (pH = 2) by acidophilic Stenotrophomonas maltophilia strain AJH1 (KU664513). The strain AJH1 was obtained from an enrichment culture obtained from soil samples of mining area in the presence of PAH as sole sources of carbon and energy. Strain AJH1was able to degrade low (anthracene, phenanthrene, naphthalene, fluorene) and high (pyrene, benzo(e)pyrene and benzo(k)fluoranthene) molecular weight PAHs in acidophilic mineral salt medium at pH 2, with removal rates of up to 95% (LMW PAH) and 80% (HMW PAH), respectively. In addition, strain AJH1 treated petroleum wastewater with 89 ± 1.1% COD removal under acidic condition (pH 2) in a continuously stirred reactor. Acidophilic S. maltophilia strain AJH1, hence holds the promise as an effective degrader for biological treatment of PAHs contaminated wastewater at acidic pH.

Dynamics of antibiotic resistance genes and presence of putative pathogens during ambient temperature anaerobic digestion.

AIMS: This study was focused on evaluating the persistency of antimicrobial resistance (AR) genes and putative pathogenic bacteria in an anaerobic digesters operating at mesophilic ambient temperature, in two different year seasons: summer and winter. METHODS AND RESULTS: Abundance and dynamic of AR genes encoding resistance to macrolides (ermB), aminoglycosides (aphA2) and beta-lactams (blaTEM -1 ) and persistency of potentially pathogenic bacteria in pilot-scale anaerobic digesters were investigated. AR genes were determined in the influent and effluent in both conditions. Overall, after 60 days, reduction was observed for all evaluated genes. However, during the summer, anaerobic digestion was more related to the gene reduction as compared to winter. Persistency of potentially pathogenic bacteria was also evaluated by metagenomic analyses compared to an in-house created database. Clostridium, Acinetobacter and Stenotrophomonas were the most identified. CONCLUSIONS: Overall, considering the mesophilic ambient temperature during anaerobic digestion (summer and winter), a decrease in pathogenic bacteria detection through metagenomic analysis and AR genes is reported. Although the mesophilic anaerobic digestion has been efficient, the results may suggest medically important bacteria and AR genes persistency during the process. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to show AR gene dynamics and persistency of potentially pathogenic bacteria through metagenomic approach in cattle manure ambient temperature anaerobic digestion.

Stability of airborne microbes in the Louvre Museum over time.

The microbial content of air has as yet been little described, despite its public health implications, and there remains a lack of environmental microbial data on airborne microflora in enclosed spaces. In this context, the aim of this study was to characterize the diversity and dynamics of airborne microorganisms in the Louvre Museum using high-throughput molecular tools and to underline the microbial signature of indoor air in this human-occupied environment. This microbial community was monitored for 6 month during occupied time. The quantitative results revealed variations in the concentrations of less than one logarithm, with average values of 10(3) and 10(4) Escherichia coli/Aspergillus fumigatus genome equivalent per m(3) for bacteria and fungi, respectively. Our observations highlight the stability of the indoor airborne bacterial diversity over time, while the corresponding eukaryote community was less stable. Bacterial diversity characterized by pyrosequencing 454 showed high diversity dominated by the Proteobacteria which represented 51.1%, 46.9%, and 38.4% of sequences, for each of the three air samples sequenced. A common bacterial diversity was underlined, corresponding to 58.4% of the sequences. The core species were belonging mostly to the Proteobacteria and Actinobacteria, and to the genus Paracoccus spp., Acinetobacter sp., Pseudomonas sp., Enhydrobacter sp., Sphingomonas sp., Staphylococcus sp., and Streptococcus sp.

Heterogeneity and spatial distribution of bacterial background contamination in pulp and process water of a paper mill.

Identifying the source and the distribution of bacterial contaminant communities in water circuits of industrial applications is critical even when the process may not show signs of acute biofouling. The endemic contamination of facilities can cause adverse effects on process runability but may be masked by the observed daily variability. The distribution of background communities of bacterial contaminants may therefore be critical in the development of new site-specific antifouling strategies. In a paper mill as one example for a full-scale production process, bacterial contaminants in process water and pulp suspensions were mapped using molecular fingerprints at representative locations throughout the plant. These ecological data were analyzed in the process-engineering context of pulp and water flow in the facilities. Dispersal limits within the plant environment led to the presence of distinct groups of contaminant communities in the primary units of the plant, despite high flows of water and paper pulp between units. In the paper machine circuit, community profiles were more homogeneous than in the other primary units. The variability between sampled communities in each primary unit was used to identify a possible point source of microbial contamination, in this case a storage silo for reused pulp. Part of the contamination problem in the paper mill is likely related to indirect effects of microbial activity under the local conditions in the silo rather than to the direct presence of accumulated microbial biomass.

The microbial signature of aerosols produced during the thermophilic phase of composting.

AIMS: The microbial diversity of bioaerosols released during operational activities at composting plants is poorly understood. Identification of bacteria and fungi present in such aerosols is the prerequisite for the definition of microbial indicators that could be used in dispersal and exposure studies. METHODS AND RESULTS: A culture-independent analysis of composting bioaerosols collected at five different industrial open sites during the turning of composting piles in fermentation was performed by building 16S rDNA and 18S rDNA libraries. More than 800 sequences were analysed. Although differences in the phylotypes distribution were observed from one composting site to another, similarities in the structure of microbial diversity were remarkable. The same phyla dominated in the five bioaerosols: Ascomycota among fungi, Firmicutes and Actinobacteria among bacteria. For each phylum, some dominant phylotypes were common to at least four bioaerosols. These common phylotypes belonged to Thermomyces, Aspergillus, Penicillium, Geobacillus, Planifilum, Thermoactinomyces, Saccharopolyspora, Thermobifida and Saccharomonospora. CONCLUSIONS: The microbial signature of aerosols produced during the thermophilic phase of composting was determined. The similarities observed may be explained by the selection of thermophilic and sporulating species. SIGNIFICANCE AND IMPACT OF THE STUDY: Several bacteria and fungi identified in this study may represent potential indicators of composting bioaerosols in air.

Effect of a transient perturbation on marine bacterial communities with contrasting history.

AIMS: To evaluate the importance of the bacterial composition on the resilience of the organic matter assimilation in the sea. METHODS AND RESULTS: Chemostats were inoculated with coastal and offshore bacterial communities. Bacterial density and protein synthesis increased before stabilizing, and this response to confinement was more marked in the offshore chemostats. Before the toluene perturbation the community structure in the coastal chemostats remained complex whereas the offshore chemostats became dominated by Alteromonas sp. After the perturbation, bacterial protein synthesis was inhibited before peaking briefly at a level fivefold to that observed before the perturbation and then stabilizing at a level comparable to that before the perturbation. Alteromonas dominated both the coastal and the offshore communities immediately after the perturbation and the coastal communities did not recover their initial complexity. CONCLUSIONS: Cell lysis induced by the toluene perturbation favoured the growth of Alteromonas which could initiate growth rapidly in response to the nutrient pulse. Despite their different community structure in situ, the resilience of protein synthesis of coastal and offshore bacterial communities was dependent on Alteromonas, which dominated in the chemostats. SIGNIFICANCE AND IMPACT OF THE STUDY: Here we show that although Alteromonas sp. dominated in artificial offshore and coastal communities in chemostats, their response time to the shock was different. This suggests that future perturbation studies on resilience in the marine environment should take account of ecosystem history.

Synergetic effect of pH and biochemical components on bacterial diversity during mesophilic anaerobic fermentation of biomass-origin waste.

AIMS: To investigate the synergetic effect of pH and biochemical components on bacterial community structure during mesophilic anaerobic degradation of solid wastes with different origins, and under acidic or neutral conditions. METHODS AND RESULTS: The bacterial community in 16 samples of solid wastes with different biochemical compositions and origins was evaluated during mesophilic anaerobic degradation at acidic and neutral pH. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) were used to compare the communities. Multivariate analysis of the DGGE and SSCP results revealed that most of the dominant microbes were dependent on the content of easily degradable carbohydrates in the samples. Furthermore, the dominant microbes were divided into two types, those that preferred an acid environment and those that preferred a neutral environment. A shift in pH was found to change their preference for medium substrates. Although most of the substrates with similar origin and biochemical composition had similar microbial diversity during fermentation, some microbes were found only in substrates with specific origins. For example, two microbes were only found in substrate that contained lignocellulose and animal protein without starch. These microbes were related to micro-organisms that are found in swine manure, as well as in other intestinal or oral niches. In addition, the distribution of fermentation products was less sensitive to the changes in pH and biochemical components than the microbial community. CONCLUSIONS: Bacterial diversity during anaerobic degradation of organic wastes was affected by both pH and biochemical components; however, pH exerted a greater effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study reveal that control of pH may be an effective method to produce a stable bacterial community and relatively similar product distribution during anaerobic digestion of waste, regardless of variation in the waste feedstocks.

Structural and functional responses of sewage microbial communities used for the treatment of a complex mixture of volatile organic compounds (VOCs).

AIMS: The aim of this work was to assess the impact of the applied mass loading on the selection of an efficient microbial community able to degrade a complex mixture of volatile organic compounds (VOCs). METHODS AND RESULTS: Two reactors were used and were supplied with a gaseous effluent containing 11 VOCs with different concentrations. The response of the microflora was monitored as a function of time: biodegradation activity, bacterial density and diversity. The results showed that the applied mass loading seems to have an impact on the functioning and the genetic structure of the bacterial community. CONCLUSIONS: A high mass loading seems to induce a low efficient functioning in terms of elimination efficiency and a simplification of the genetic structure of the total bacterial community with the apparition of a dominant microflora. A low mass loading seems to favour a better functioning and allows to keep a healthier bacterial diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: In the treatment processes of gaseous effluents, it would be judicious to define the functioning parameters of the process to keep the diversity of important functional bacterial groups. These results provide also useful information about changes in microbial communities following natural or anthropogenic alterations in different ecosystems.

Structural divergence of bacterial communities from functionally similar laboratory-scale vermicomposts assessed by PCR-CE-SSCP.

AIMS: To evaluate bacterial community structure and dynamics in triplicate vermicomposts made from the same start-up material, along with certain physico-chemical changes. METHODS AND RESULTS: The physico-chemical parameters (pH, temperature, carbon, nitrogen, soluble substances and cellulose) evolved similarly in the triplicate vermicomposts, indicating a steady function. The 16S bacterial gene abundance remained constant over time. To monitor changes in the bacterial community structure, fingerprinting based on capillary electrophoresis single-strand conformation polymorphism was employed. A rise in bacterial diversity occurred after precomposting and it remained stable during the maturation phase. However, a rapid shift in the structure of the bacterial community in the vermicompost replicates was noted at the beginning that stabilized with the process maturation. Multivariate analyses showed different patterns of bacterial community evolution in each vermicompost that did not correlate with the physico-chemical changes. CONCLUSIONS: The broad-scale functions remained similar in the triplicates, with stable bacterial abundance and diversity despite fluctuation in the community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated that microbial fingerprinting with multivariate analysis can provide significant understanding of community structure and also clearly suggests that an ecosystem's efficacy could be the outcome of functional redundancy whereby a number of species carry out the same function.

Involvement of microbial populations during the composting of olive mill wastewater sludge.

Olive mill waste water sludge obtained by the electro-Fenton oxidation of olive mill waste water was composted in a bench scale reactor. The evolution of microbial species within the composter was investigated using a respirometric test and by means of both cultivation-dependent and independent approaches (Polymerase Chain Reaction-Single Strand Conformation Polymorphism, PCR SSCP). During the period of high respiration rate (7-24 days), cultivation method showed that thermophilic bacteria as well as actinomycetes dominated over eumycetes. During the composting process, the PCR-SSCP method showed a higher diversity of the bacterial community than the eukaryotic one. After 60 days of composting, the compost exhibited a microbial stability and a clear absence of phytotoxicity.

The microbial signature of drinking waters: myth or reality?

This paper presents a new software developed for analyzing single strand conformation polymorphism (SSCP) electrophoresis patterns delivered by the genetic analyzer ABI310 (Applied Biosystems). SSCP is a molecular typing technique based on the PCR amplification of microbial 16S rDNA and used for the monitoring of complex microbial ecosystems dynamics. The software--a home-made MATLAB toolbox called MODIMECO--developed for the analysis of SSCP patterns is presented. MODIMECO includes a number of basic signal processing abilities as well as largely used statistical tools such as the well known principal component analysis. The use of the SSCP for assessing the hypothesis of the existence of a microbial signature of drinking waters illustrates the typical advantages of using such software tools. Results are discussed and conclusions drawn.

Purification and properties of the alpha-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118.

alpha-Acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118 was expressed at low levels in cell extracts and was also unstable. The purification was carried out from E. coli in which the enzyme was expressed 36-fold higher. The specific activity was 24-fold enhanced after purification. The main characteristics of alpha-acetolactate decarboxylase were: (i) activation by the three branched chain amino acids leucine, valine and isoleucine; (ii) allosteric properties displayed in absence and Michaelis kinetics in the presence of leucine. The enzyme is composed of six identical subunits of 26,500 Da.

Identification of Staphylococcus carnosus and Staphylococcus warneri isolated from meat by fluorescent in situ hybridization with 16S rRNA-targeted oligonucleotide probes.

16S rRNA targeted oligonucleotide probes were designed by sequence analysis of an rRNA database to discriminate S. carnosus, S. warneri, and S. saprophyticus species. After establishing hybridization conditions by RNA dot blot hybridization with reference species, our probes were shown to be specific. By in situ hybridization only S-S-S.carno-0440-a-A-23 and S-S-S.war-0180-a-A-23 can specifically detect S. carnosus and S. warneri, respectively. The detection of old cells of S. carnosus 833 was more limited by the permeabilisation than by the low rRNA content. One day old cells could be permeabilized with lysostaphin, whereas young cells were permeabilized with lysozyme.

16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure.

A laboratory-scale continuously stirred anaerobic thermophilic batch digester was inoculated with cattle manure. Bacterial and archaeal communities, as well as digester performances, were analysed during reactor start-up for about 20 days. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used for overall detection and for study of the dynamics of microbial populations. Dominant bacteria and archaea 16S rDNAs were sequenced from the sample on day 12. Ten bacteria and 3 archaea OTUs (operational taxonomic units) were identified from the 52 clones sequenced. Sequences corresponding to the dominant bacterial SSCP peak were phylogenetically close to the 16S rDNA sequence of Bacillus thermoterrestris, whereas sequences corresponding to the two dominant archaeal SSCP peaks were phylogenetically close to the 16S rDNA sequence of Methanoculleus thermophilicus and Methanosarcina thermophila.

A molecular method to study population and activity dynamics in anaerobic digestors.

The applicability of a new molecular fingerprinting method (Single Strand Conformation Polymorphism) to study the microbial populations of anaerobic digestors was investigated. After extraction of total nucleic acids, the 16S rDNA and 16S rRNA molecules were amplified and the amplicons were separated by SSCP electrophoresis. Characteristic and complex peak patterns were obtained, where each peak could be correlated with the 16S rDNA sequence of one micro-organism. The rDNA peak patterns should consist of the most abundant sequences and thus would reflect the diversity of prominent species of different digestors. Ribosomal DNA patterns were compared to rRNA patterns and revealed the bacteria that were the most active metabolically. The SSCP method also revealed dynamic changes in the presence and activity of populations, following perturbations such as an acidic shock which caused an increase in activity of two species. After cloning the 16S rDNA, the species corresponding to the peaks of interest, such as the archaeal species, could be identified by screening the clones according to their SSCP patterns and sequencing the 16S rDNA.

Effect of solid hold-up on nitrite accumulation in a biofilm reactor--molecular characterization of nitrifying communities.

Biological ammonium oxidation was carried out in two inverse turbulent bed reactors fed with synthetic mineral wastewater containing a high ammonium concentration (100 mg N-NH4+/L). Both reactors were started-up and operated in the same conditions except for the solid carrier concentration: the solid hold-up ratios applied, defined as the ratios of static to expanded bed height, were 0.1 and 0.3 in reactors R10 and R30 respectively. These two solid hold-up ratios generate different particle-to-particle collision frequencies and, therefore, detachment forces. The influence of solid hold-up on biofilm growth and nitrifying performance was studied from a macroscopic (i.e. nitrate and/or nitrite production) and microbiological point of view. After 60 days of operation, both reactors contained the same amount of biomass. However, R10 produced only nitrate while nitrite accumulated in R30. A comparison of microbial populations in the reactors showed that R10 contained both ammonium and nitrite oxidizing populations such as Nitrosomonas and Nitrospira, whereas in R30, ammonium oxidizing populations were much greater than those of nitrite oxidizers. The major ammonium-oxidizing organism was not the same in both reactors.

Monitoring of the microbial community of a sequencing batch reactor bioaugmented to improve its phosphorus removal capabilities.

The acclimatisation of an activated sludge to enhanced biological phosphorus removal conditions was followed after and without bioaugmentation with a low amount of phosphorus-accumulating sludge. Phosphorus removal yields were monitored by conventional analytical methods and microbial communities evolutions were followed by a finger printing molecular technique (PCR-SSCP). While the benefit of the bioaugmentation seems real at the level of the reactor parameters, bioaugmentation speeded up the installation of good and stable phosphorus removal yield, the establishment of the inoculated microbial community in the bioaugmented reactor is still unclear. Both the bioaugmented and the control microbial communities evolved in a similar way to end up with apparently comparable populations. At the time of the experiment, the results suggest that the microbial community inoculated for the bioaugmentation did not establish in the reactor but compensated for phosphorus accumulation until the acclimatisation of an endogenous microbial community arose.

Treatment of hypersaline industrial wastewater by a microbial consortium in a sequencing batch reactor.

Hypersaline effluents are produced by various industrial activities. Such wastewater, rich in both organic matter and salt (> 35 g l(-1)), is difficult to treat by conventional wastewater treatment processes. It is necessary to use halophilic bacteria. In this study, a bench-scale sequencing batch reactor (SBR) was inoculated with halophilic sediments in order to treat an agri-food effluent containing 120 g salt l(-1). The micro-organisms were able to treat carbon and nitrogen, provided the pH in the reactor was neutralised with phosphoric acid. Soluble COD and Soluble TKN removal attained 83% and 72% respectively. 16S rDNA identification of the halophilic microbial community showed high diversity.