Dynamics of the composition of a microbial consortium during start-up of a single-stage constant flow laboratory nitritation/anammox setup

Dynamics of the composition of the microbial community was studied during start-up of a single-stage completely mixed constant flow laboratory setup for ammonium removal by the nitritation/anammox process from the filtrate of digested sludge of the Kuryanovo wastewater treatment plant (KWTP), Moscow. To decrease the period of the start-up, the setup was initially inoculated with two types of activated sludge (nitrifying sludge from a KWTP aeration tank and sludge from a sequencing batch reactor enriched with anammox bacteria). The start-up and adjustment stage was therefore decreased to 35–40 days, and nitrogen removal efficiency reached 80% after 120 days of the setup operation. Taxonomic analysis of the composition of the microbial community was carried out by pyrosequencing of the 16S rRNA fragments obtained using the universal and planctomycetes-specific primers. In the course of adaptation of activated sludge to increasing nitrogen load, microbial community of the setup became less diverse and more specialized. The contribution of anammox bacteria of the family Brocadiaceae, closely related to Candidatus “Brocadia caroliniensis,” increased gradually. Members of the order Nitrosomonadales were involved in ammonium oxidation to nitrite. While nitrite-oxidizing bacteria of the genus Nitrospira were also detected, their share decreased with accumulation of the activated sludge. The contribution of other bacteria varied as well: the shares of the phyla Ignavibacteria, Chloroflexi, and Acidobacteria increased significantly (up to 13, 12, and 10%, respectively of the total number of reads), while relative abundance of the Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Synergistetes, Aminicenantes, Thermotogae, and Cloacimonetes decreased. Thus, application of pyrosequencing made it possible to monitor succession of the bacterial community involved in nitrogen removal by nitritation/anammox process.     

The metagenomic approach to characterization of the microbial community shift during the long-term cultivation of anammox-enriched granular sludge

A metagenomic approach was used to investigate how the microbial community composition changes when an anammox-based granular sludge reactor is seeded with nitritation-anammox biomass from a wastewater treatment plant. In the seed sample, the abundance of Candidatus Kuenenia stuttgartiensis was similar to Candidatus Jettenia caeni (12.63 vs. 11.68%). This biomass was typical in terms of microbial nitrogen conversion; both ammonia (Nitrosomonas sp.) and nitrite (Nitrospira sp.) oxidizing bacteria were detected. In the lab-scale reactor, Candidatus Kuenenia stuttgartiensis and Candidatus Jettenia caeni bacteria were also present in equal proportions (18.57 vs. 20.89%). On the contrary, Candidatus Nitrospira defluvii bacteria were highly abundant in this reactor, but no known ammonia-oxidizing bacteria were detected. In light of recent studies showing that Nitrospira sp. are capable of complete nitrification, the results presented here may well indicate that both stages of nitrification in the anammox-based granular sludge reactor were performed by this bacteria.     

Microorganisms of low-temperature heavy oil reservoirs (Russia) and their possible application for enhanced oil recovery

Physicochemical and microbiological characteristics of formation waters low-temperature heavy oil reservoirs (Russia) were investigated. The Chernoozerskoe, Yuzhno-Suncheleevskoe, and Severo-Bogemskoe oilfields, which were exploited without water-flooding, were shown to harbor scant microbial communities, while microbial numbers in the water-flooded strata of the Vostochno-Anzirskoe and Cheremukhovskoe oilfields was as high as 10⁶ cells/mL. The rates of sulfate reduction and methanogenesis were low, not exceeding 1982 ng S^2–/(L day) and 9045 nL СН₄/(L day), respectively, in the samples from water-flooded strata. High-throughput sequencing of microbial 16S rRNA gene fragments in the community of injection water revealed the sequences of the Proteobacteria (74.7%), including Betaproteobacteria (40.2%), Alphaproteobacteria (20.7%), Gammaproteobacteria (10.1%), Deltaproteobacteria (2.0%), and Epsilonproteobacteria (1.6%), as well as Firmicutes (7.9%), Bacteroidetes (4.1%), and Archaea (0.2%). DGGE analysis of microbial mcrA genes in the community of injection water revealed methanogens of the genera Methanothrix, Methanospirillum, Methanobacterium, Methanoregula, Methanosarcina, and Methanoculleus, as well as unidentified Thermoplasmata. Pure cultures of bacteria of the genera Rhodococcus, Pseudomonas, Gordonia, Cellulomonas, etc., capable of biosurfactant production when grown on heavy oil, were isolated. Enrichment cultures of fermentative bacteria producing significant amounts of volatile organic acids (acetic, propionic, and butyric) from sacchariferous substrates were obtained. These acids dissolve the carbonates of oil-bearing rock efficiently. Selection of the efficient microbial technology for enhanced recovery of heavy oil from terrigenous and carbonate strata requires model experiments with microbial isolates and the cores of oil-bearing rocks.     

Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity—throughout the different stages of the feeding process with methanol and acetate—was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH₄, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.     

Dynamics and diversity of a microbial community during the fermentation of industrialized <Emphasis Type="Italic">Qingcai</Emphasis> paocai,a traditional Chinese fermented vegetable food,as assessed by Illumina MiSeq sequencing,DGGE and qPCR assay

Paocai is a traditional Chinese fermented food and typically produced via spontaneous fermentation. We have investigated the microbial community utilized for the fermentation of industrialized Qingcai paocai using the combination of Illumina MiSeq sequencing, PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) assay. Three main phyla, namely Firmicutes, Proteobacteria and Bacteroidetes, were identified by both MiSeq sequencing and PCR-DGGE. The dominant genera observed in the fermentation were Lactobacillus, Pseudomonas, Vibrio and Halomonas. Most genera affiliated with Proteobacteria or Bacteroidetes were detected more often during the earlier part of the fermentation, while Lactobacillus (affiliated with Firmicutes) was dominant during the later fermentation stages. Fungal community analysis revealed that Debaryomyces, Pichia and Kazachstania were the main fungal genera present in industrialized Qingcai paocai, with Debaryomyces being the most dominant during the fermentation process. The quantities of dominant genera Lactobacillus and Debaryomyces were monitored using qPCR and shown to be 10⁹–10¹² and 10⁶–10¹⁰ copies/mL, respectively. During the later fermentation process of industrialized Qingcai paocai, Lactobacillus and Debaryomyces were present at 10¹¹ and 10⁸ copies/mL, respectively. These results facilitate further understanding of the unique microbial ecosystem during the fermentation of industrialized Qingcai paocai and guide future improvement of the fermentation process.     

Vertical profiles of microbial communities in perfluoroalkyl substance-contaminated soils

Poly- and perfluoroalkyl compounds (PFASs) are ubiquitous in the environment, but their influences on microbial community remain poorly known. The present study investigated the depth-related changes of archaeal and bacterial communities in PFAS-contaminated soils. The abundance and structure of microbial community were characterized using quantitative PCR and high-throughput sequencing, respectively. Microbial abundance changed considerably with soil depth. The richness and diversity of both bacterial and archaeal communities increased with soil depth. At each depth, bacterial community was more abundant and had higher richness and diversity than archaeal community. The structure of either bacterial or archaeal community displayed distinct vertical variations. Moreover, a higher content of perfluorooctane sulfonate (PFOS) could have a negative impact on bacterial richness and diversity. The rise of soil organic carbon content could increase bacterial abundance but lower the richness and diversity of both bacterial and archaeal communities. In addition, Proteobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Acidobacteria were the major bacterial groups, while Thaumarchaeota, Euryarchaeota, and unclassified Archaea dominated in soil archaeal communities. PFASs could influence soil microbial community.     

Surface-attached and suspended bacterial community structure as affected by C/N ratios: relationship between bacteria and fish production

Bacteria play crucial roles in the combined system of substrate addition and C/N control, which has been demonstrated to improve aquaculture production. However, the complexity of surface-attached bacteria on substrates and suspended bacteria in the water column hamper further application of this system. This study firstly applied this combined system into the culture of grass carp, and then explored the relationship between microbial complexes from surface-attached and suspended bacteria in this system and the production of grass carp. In addition, this study investigated bacterial community structures as affected by four C/N ratios using Illumina sequencing technology. The results demonstrated that the weight gain rate and specific growth rate of grass carp in the CN20 group (C/N ratio 20:1) were the highest (P < 0.05), and dietary supplementation of the microbial complex had positive effects on the growth of grass carp (P < 0.05). Sequencing data revealed that, (1) the proportions of Verrucomicrobiae and Rhodobacter (surface-attached), sediminibacterium (suspended), and emticicia (surface-attached and suspended) were much higher in the CN20 group compared with those in the other groups (P < 0.05); (2) Rhodobacter, Flavobacterium, Acinetobacter, Pseudomonas, Planctomyces, and Cloacibacterium might be important for the microbial colonization on substrates; (3) as the C/N ratio increased, proportions of Hydrogenophaga (surface-attached and suspended), Zoogloea, and Flectobacillus (suspended) increased, but proportions of Bacillus, Clavibacter, and Cellvibro (surface-attached and suspended) decreased. In summary, a combined system of substrate addition and C/N control increased the production of grass carp, and Verrucomicrobiae and Rhodobacter in the surface-attached bacterial community were potential probiotic bacteria that contributed to the enhanced growth of grass carp.     

Deep sequencing analysis of bacterial community structure of Soldhar hot spring,India

We investigated the bacterial community structure of Soldhar hot spring with extreme high temperature 95°C located in Uttarakhand, India using high throughput sequencing. Bacterial phyla Proteobacteria (88.8%), Deinococcus-Thermus (7.5%), Actinobacteria (2.3%), and Firmicutes (1.07%) were predominated in the sequencing survey, whereas Bacteroidetes, Verrucomicrobia, Aquificae and Acidobacteria were detected in relatively lower abundance in Soldhar hot spring. At the family level, Comamonadaceae (52.5%), Alteromonadaceae (15.9%), and Thermaceae (7.5%) were mostly dominated in the ecosystem followed by Chromatiaceae, Microbacteriaceae, and Cyclobacteriaceae. Besides, members of Rhodobacteraceae, Moraxellaceae, Xanthomonadaceae, Aquificaceae, Enterobacteriaceae, Bacillaceae, Methylophilaceae, etc. were detected as a relatively lower abundance. In the present study we discuss the overall microbial community structure and their relevance to the ecology of the Soldhar hot spring environment.     

Microbial community of freshwater sponges in Lake Baikal

The microbial community of Baikal sponges has been studied in five species belonging to the genera Swartschewskia, Baicalospongia, and Lubomirskia of the endemic family Lubomirskiidae. The results show that the total numbers of bacteria and bacterioplankton production have an effect on the growth of L. baicalensis body. Bacteria of the genera Pseudomonas, Bacillus, Micrococcus, Sarcina, Flavobacterium, Arthrobacter, and Acinetobacter living in the sponges are representatives of the Baikal bacterioplankton. Actinomycetes of the genera Streptomyces and Micromonospora are a permanent component of the cultivable sponge microbial community. The numbers and enzyme activities of heterotrophic, oligotrophic, and psychrophilic bacteria isolated from different sponge species and from the ambient water in autumn and in winter have been estimated.     

Diversity and dynamics of the bacterial community involved in pig manure biodegradation in a microbial fermentation bed system

Overproduction of livestock manures with unpleasant odors causes significant environmental problems. The microbial fermentation bed (MFB) system is considered an effective approach to recycling utilization of agricultural byproducts and pig manure (PM). To gain a better understanding of bacterial communities present during the degradation of PM in MFB, the PM bacterial community was evaluated at different fermentation stages using 16S rRNA high throughput sequencing technology. The heatmap plot clustered five samples into short-term fermentation stage of 0–10 days and long-term fermentation stage of 15–20 days. The most abundant OTUs at the phylum level were Firmicutes, Actinobacteria and Proteobacteria in the long-term fermentation stage of PM, whereas Firmicutes, Bacteroidetes, and Proteobacteria predominated in the short-term fermentation stage of PM. At the genus level, organic degradation strains, such as Corynebacterium, Bacillus, Virgibacillus, Pseudomonas, Actinobacteria, Lactobacillus, Pediococcus were the predominate genera at the long-term fermentation stage, but were found only rarely in the short-term fermentation stage. C/N ratios increased and the concentration of the unpleasant odor substance 3-hydroxy-5-methylisoxazole (3-MI) decreased with prolonged period of fermentation. Redundancy analysis (RDA) demonstrated that the relative abundance of Firmicutes, Actinobacteria, Acidobacteria and Proteobacteria had a close relationship with degradation of 3-MI and increasing C/N ratio. These results provide valuable additional information about bacterial community composition during PM biodegradation in animal husbandry.     

Changes in the microbial community during repeated anaerobic microbial dechlorination of pentachlorophenol

Pentachlorophenol (PCP) has been widely used as a pesticide in paddy fields and has imposed negative ecological effect on agricultural soil systems, which are in typically anaerobic conditions. In this study, we investigated the effect of repeated additions of PCP to paddy soil on the microbial communities under anoxic conditions. Acetate was added as the carbon source to induce and accelerate cycles of the PCP degradation. A maximum degradation rate occurred at the 11th cycle, which completely transformed 32.3 μM (8.6 mg L^?1) PCP in 5 days. Illumina high throughput sequencing of 16S rRNA gene was used to profile the diversity and abundance of microbial communities at each interval and the results showed that the phyla of Bacteroidates, Firmicutes, Proteobacteria, and Euryarchaeota had a dominant presence in the PCP-dechlorinating cultures. Methanosarcina, Syntrophobotulus, Anaeromusa, Zoogloea, Treponema, W22 (family of Cloacamonaceae), and unclassified Cloacamonales were found to be the dominant genera during PCP dechlorination with acetate. The microbial community structure became relatively stable as cycles increased. Treponema, W22, and unclassified Cloacamonales were firstly observed to be associated with PCP dechlorination in the present study. Methanosarcina that have been isolated or identified in PCP dechlorination cultures previously was apparently enriched in the PCP dechlorination cultures. Additionally, the iron-cycling bacteria Syntrophobotulus, Anaeromusa, and Zoogloea were enriched in the PCP dechlorination cultures indicated they were likely to play an important role in PCP dechlorination. These findings increase our understanding for the microbial and geochemical interactions inherent in the transformation of organic contaminants from iron rich soil, and further extend our knowledge of the PCP-transforming microbial communities in anaerobic soil conditions.     

Total and active microbial communities and <Emphasis Type="Italic">phoD</Emphasis> as affected by phosphate depletion and pH in soil

Background and Aims

Soil microbial communities contribute to organic phosphorus cycling in a variety of ways, including secretion of the PhoD alkaline phosphatase. We sampled a long-term grassland fertilization trial in Switzerland characterized by a natural pH gradient. We examined the effects of phosphate depletion and pH on total and active microbial community structures and on the structure and composition of the total and active phoD-harboring community.

Methods

Archaeal, bacterial and fungal communities were investigated using T-RFLP and phoD-harboring members of these communities were identified by 454-sequencing.

Results

Phosphate depletion decreased total, resin-extractable and organic phosphorus and changed the structure of all active microbial communities, and of the total archaeal and phoD-harboring communities. Organic carbon, nitrogen and phosphorus increased with pH, and the structures of all total and active microbial communities except the total fungal community differed between the two pH levels. phoD-harboring members were affiliated to Actinomycetales, Bacilliales, Gloeobacterales, Planctomycetales and Rhizobiales.

Conclusions

Our results suggest that pH and associated soil factors are important determinants of microbial and phoD-harboring community structures. These associated factors include organic carbon and total nitrogen, and to a lesser degree phosphorus status, and active communities are more responsive than total communities. Key players in organic P mineralization are affiliated to phyla that are known to be important in organic matter decomposition.

     

Comparison of microbial communities during the anaerobic digestion of <Emphasis Type="Italic">Gracilaria</Emphasis> under mesophilic and thermophilic conditions

Mesophilic and thermophilic anaerobic digesters (MD and TD, respectively) utilizing Gracilaria and marine sediment as the substrate and inoculum, respectively, were compared by analyzing their performances and microbial community changes. During three successive transfers, the average cumulative methane yields in the MD and TD were 222.6 ± 17.3 mL CH₄/g volatile solids (VS) and 246.1 ± 11 mL CH₄/g VS, respectively. The higher hydrolysis rate and acidogenesis in the TD resulted in a several fold greater accumulation of volatile fatty acids (acetate, propionate, and butyrate) followed by a larger pH drop with a prolonged recovery than in the MD. However, the operational stability between both digesters remained comparable. Pyrosequencing analyses revealed that the MD had more complex microbial diversity indices and microbial community changes than the TD. Interestingly, Methanomassiliicoccales, the seventh methanogen order was the predominant archaeal order in the MD along with bacterial orders of Clostridiales, Bacteriodales, and Synergistales. Meanwhile, Coprothermobacter and Methanobacteriales dominated the bacterial and archaeal community in the TD, respectively. Although the methane yield is comparable, both MD and TD show a different profile of pH, VFA and the microbial communities.     

Broiler chicken cecal microbiocenoses depending on mixed fodder

Molecular genetic techniques (NGS sequencing and quantitative PCR) were used to determine the composition of the cecal bacterial community of broiler chickens fed with different mixed fodder. The cecal microbiome exhibited taxonomic diversity, with both typical inhabitants of avian intestine belonging to the families Clostridiaceae, Eubacteriaceae, and Lactobacillaceae and to the phylum Bacteroidetes, and new unidentified taxa, as well as bacteria of the families Lachnospiraceae and Ruminococcaceae, which were previously considered restricted to the rumen microbiota. Contrary to traditional concepts, enterococci and bifidobacteria were among the minor components of the community, lactate-fermenting species were absent, and typical avian pathogens of the genus Staphylococcus were detected but seldom. Members of the family Suterellaceae and the genus Gallibacterium, which are responsible for avian respiratory infections, were also detected. Significant fluctuations of abundance and composition of microbial groups within the cecal community and of the parameters of broiler productivity were found to occur depending on the feed allowance. Cellulose content in the feed had the most pronounced effect on the composition and structure of bacterial communities. Decreased cellulose content resulted in a decrease of bacterial abundance by an order of magnitude and in increased ratios of members of the phylum Bacteroidetes and the family Clostridiaceae, which possess the enzymes degrading starch polysaccharides. Abundance of the normal inhabitants of avian intestine belonging to the genus Lactobacillus and the order Bacillales decreased, while the share of Escherichia and members of the family Sutterellaceae increased, including some species capable of causing dysbiotic changes in the avian intestine. No significant change in the abundance of cellulolytics of the families Ruminococcaceae, Lachnospiraceae, and Eubacteriaceae was observed.     

Effect of metal concentration on the microbial community in acid mine drainage of a polysulfide ore deposit

The composition of microbial communities of acid mine drainage (AMD) in two wells drilled in the terrace of the Sherlovaya Gora open-cast polymetallic ores mine (Eastern Siberia) was studied. While drainage water filling two wells, ShG14-1 and ShG14-8, had similar values of pH (2.6), Eh (447–494 mV), and temperature (6.5°C), the water in the first well contained more metals and sulfate. The water in ShG14-1 and ShG14-8 contained, respectively, 1898 and 434 mg/L of iron, 734 and 49 mg/L of manganese, 81 and 7 mg/L of copper, 3597 and 787 mg/L of zinc, and 15990 and 3632 mg/L of sulfate. Molecular analysis of the microbial communities was performed using pyrosequencing of the 16S rRNA gene fragments. The ShG14-8 microbial community included such bacterial taxa typically found in AMD sites as Gallionella (38.8% of total 16S rRNA gene sequences), Ferrovum (4.4%), Acidiphilium (9.1%), Acidisphaera (8.2%), Acidithiobacillus (7.2%), and Leptospirillum (4.6%). In the ShG14-1 sample with higher content of metals, strict acidophiles Acidithiobacillus (16.0%) and Leptospirillum (25.4%) were more abundant, while Gallionella, Ferrovum, Acidiphilium and Acidisphaera were almost absent. Ferrimicrobium (16.8%) and Sulfobacillus (1.4%) were detected in ShG14-1 but not in ShG14-8. Thus, the increase in concentration of metals in the acid mine drainage water under the same value of total acidity substantially altered the composition of the microbial community, preventing the development of “moderate” alpha- and beta-proteobacterial acidophiles, so that the community was dominated by the bacteria characteristic of the extremely acidic drainage waters.     

Taxonomical and functional microbial community dynamics in an Anammox-ASBR system under different Fe (III) supplementation

In the present study, we explored the metabolic versatility of anaerobic ammonium oxidation (anammox) bacteria in a variety of Fe (III) concentrations. Specifically, we investigated the impacts of Fe (III) on anammox growth rates, on nitrogen removal performance, and on microbial community dynamics. The results from our short-term experiments revealed that Fe (III) concentrations (0.04–0.10 mM) significantly promote the specific anammox growth rate from 0.1343 to 0.1709 d^?1. In the long-term experiments, the Anammox-anaerobic sequencing batch reactor (ASBR) was operated over 120 days and achieved maximum NH₄ ⁺-N, NO₂ ^?-N, and TN efficiencies of 90.98 ± 0.35, 93.78 ± 0.29, and 83.66 ± 0.46 %, respectively. Pearson’s correlation coefficients between anammox-(narG + napA), anammox-nrfA, and anammox-FeRB all exceeded r = 0.820 (p < 0.05), confirming an interaction and ecological association among the nitrogen and iron-cycling-related microbial communities. Illumina MiSeq sequencing indicated that Chloroflexi (34.39–39.31 %) was the most abundant phylum in an Anammox-ASBR system, followed by Planctomycetes (30.73–35.31 %), Proteobacteria (15.40–18.61 %), and Chlorobi (4.78–6.58 %). Furthermore, we found that higher Fe (III) supplementation (>0.06 mM) could result in the community succession of anammox species, in which Candidatus Brocadia and Candidatus Kuenenia were the dominant anammox bacteria species. Combined analyses indicated that the coupling of anammox, dissimilatory nitrogen reduction to ammonium, and iron reduction accounted for nitrogen loss in the Anammox-ASBR system. Overall, the knowledge gained in this study provides novel insights into the microbial community dynamics and metabolic potential of anammox bacteria under Fe (III) supplementation.     

Phylogenetic Diversity of Microorganisms from the Sludge of a Biogas Reactor Processing Oil-Containing and Municipal Waste

High-throughput sequencing of the 16S rRNA gene fragments was used to determine the phylogenetic diversity of prokaryotes, including human pathogens, in the liquid phase of the sludge of a biogas reactor processing oil-containing and municipal waste. A unique microbial community was found to develop in the sludge, which comprised the microorganisms of municipal wastewater (bacteria of human feces) and specific groups of aerobic and anaerobic microorganisms, which possibly arrived with oil-containing water. In the 16S rRNA gene library, the sequences of representatives of Firmicutes prevailed (54.9%), which belonged to anaerobic bacteria of the genera Gelria (26.6%), Syntrophomonas (6.0%), Lutispora (2.0%), and uncultured Clostridia (group MBA03, 11.1%). The Proteobacteria sequences (20.7%) belonged mostly to the metabolically diverse members of the genus Pseudomonas (13.8%). The phylum Bacteroidetes (7%) was represented by uncultured bacteria (VadinBC27 wastewater-sludge group), while members of the phylum Cloacimonetes were mainly syntrophic bacteria Candidatus Cloacamonas (7.5%). The sequences of bacteria commonly occurring in oilfields (Clostridia, Anaerolinea, Bacteroidetes, sulfate-reducing Deltaproteobacteria, members of the family Syntrophaceae, and of the genera Thauera, Pseudomonas, Dechloromonas, and Petrimonas) were revealed. No sequences of bacteria known to be pathogenic to humans were found. The cultured microorganisms were aerobic organotrophic and anaerobic fermenting, denitrifying, and methanogenic prokaryotes. Fermenting and methanogenic enrichments grew on a broad range of organic substrates (sucrose, glycerol, starch), producing volatile fatty acids (acetate, n-butyrate, and propionate), gases (Н₂, СО₂, and CH₄), and decreasing pH of the medium from 7.0 to 4.5–5.0. The possible application of the biogas reactor sludge as a source of fermenting and methanogenic anaerobic prokaryotes, as well as of aerobic hydrocarbonoxidizing bacteria for oilfield introduction and for production of new preparations for enhanced oil recovery and for bioremediation of oil contamination is discussed.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

<Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the <Emphasis Type="Italic">pha</Emphasis> genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located at chromosome 1 of strain LB400. During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400 cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40?±?0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.