V4 Region Of 16S rDNA Research Articles

Microenvironment Mediated Metabolic-Reprogramming Regulate Self-renewal and Chemo-resistance in CD133+ Tumor Initiating Cells

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas with loss of exocrine/endocrine functions as well as development of fibrosis. Dysbiosis of gut microbiome has been shown to be involved in the pathogenesis of many disease processes. Therefore, we aim to investigate the alteration in gut microbiome associated with CP in caerulein-induced mouse model.CP was induced in C57Bl/6 by using caerulein injections (50 μg/kg/h, i.p., x7, twice weekly for 10 weeks). Stool samples were collected either one week after end of injection (10-week CP) or 6 weeks (16-week CP). DNA was extracted from stool samples and V4 region of 16S rDNA was sequenced for microbiome analysis.CP was strongly associated with the alteration in the composition of the gut microbiome, evidenced by differences in α and β diversity. When β diversity was measured using both weighted and unweighted UniFrac distances, stool from control mice is significantly different from mice on 10-week or 16-week CP (q < 0.01). The α-diversity measured by Faith's phylogenetic diversity was lowest in stool from healthy control and highest in stool from mice with 16-week CP (p < 0.001). Bacteria taxa differentially enriched in CP samples were detected using linear discriminant analysis. Bacteria from genera Bifidobacterium, Akkermansia, and Desulfovibrio were enriched in samples from 10-week CP mice. Bacteria from genera Allobaculum, Prevotella, and Bacteroides were enriched in samples from 16-week CP mice.Together, these analyses reveal pronounced alteration in the gut microbiome composition, diversity, and function when mice develop CP.

Intake of total saponins and polysaccharides from Polygonatum kingianum affects the gut microbiota in diabetic rats

BackgroundThe gut microbiota has been reported to play a critical role in metabolic diseases, including in diabetes. Polygonatum kingianum has been used in the treatment of diabetes and related diseases in China for centuries. Total saponins (TSPK) and total polysaccharides (PSPK) were reported to be major types of active constituents of P. kingianum. This research aims at investigation of their therapeutical mechanisms on diabetes based on the regulation of gut microbiota. Study designType 2 diabetes (T2D) rats were induced by high-fat diet (HFD) and streptozotocin-injection from male Sprague-Dawley (SD) rats. The blood biochemical indicators were measured. Intestinal microbial diversities and the overall structural changes in gut microbiota and the contents of the short chain fatty acids (SCFAs) were discussed. MethodsT2D rats were treated with TSPK (0.025 and 0.1g/kg) and PSPK (0.1g/kg) for 56 days. Major biochemical indexes, such as fasting blood glucose (FBG), fasting insulin (FINS) and lipopolysaccharide (LPS), were measured. Intestinal microbial diversities and the overall structural changes in gut microbiota were discussed based on the sequencing results on V4 region of 16S rDNA. Moreover, the contents of the SCFAs in faeces, which were fermentation products produced from gut microbiota were determined by gas chromatography (GC). ResultsOral administration of TSPK and PSPK prevented the increase of FBG. TSPK (0.025g/kg) enhancing the content of FINS at the end of research. Furthermore, TSPK and PSPK improved the intestinal microecology by decreasing the abundances of Bacteroidetes and Proteobacteria, and increasing that of Firmicutes. However, TSPK.L, PSPK and TSPK.H displayed discrepant regulation roles on Firmicutes. TSPK.L and PSPK significantly increased the abundance of Ruminococcaceae family and Ruminococcus genus in Firmicutes phylum, however, TSPK.H increased the abundances of Veillonellaceae family and Anaerovibrio genus. 57 Key variables, altered after treated by TSPK and PSPK, correlated to the alternations of FBG, FINS, LPS and body weight were identified. In addition, TSPK.L, PSPK and TSPK.H showed different adjustment on the contents of SCFAs. ConclusionThese results suggested that, compared to the normal rats, the structure of gut microbiota was significantly changed in diabetic rats. Oral administration with TSPK and PSPK could prevent T2D by its regulation role on the gut microbiota.

Gut microbial diversity analysis using Illumina sequencing for functional dyspepsia with liver depression-spleen deficiency syndrome and the interventional Xiaoyaosan in a rat model.

AIMTo investigate gut microbial diversity and the interventional effect of Xiaoyaosan (XYS) in a rat model of functional dyspepsia (FD) with liver depression-spleen deficiency syndrome.METHODSThe FD with liver depression-spleen deficiency syndrome rat model was established through classic chronic mild unpredictable stimulation every day. XYS group rats received XYS 1 h before the stimulation. The models were assessed by parameters including state of the rat, weight, sucrose test result and open-field test result. After 3 wk, the stools of rats were collected and genomic DNA was extracted. PCR products of the V4 region of 16S rDNA were sequenced using a barcoded Illumina paired-end sequencing technique. The primary composition of the microbiome in the stool samples was determined and analyzed by cluster analysis.RESULTSRat models were successfully established, per data from rat state, weight and open-field test. The microbiomes contained 20 phyla from all samples. Firmicutes, Bacteroidetes, Proteobacteria, Cyanobacteria and Tenericutes were the most abundant taxonomic groups. The relative abundance of Firmicutes, Proteobacteria and Cyanobacteria in the model group was higher than that in the normal group. On the contrary, the relative abundance of Bacteroidetes in the model group was lower than that in the normal group. Upon XYS treatment, the relative abundance of all dysregulated phyla was restored to levels similar to those observed in the normal group. Abundance clustering heat map of phyla corroborated the taxonomic distribution.CONCLUSIONThe microbiome relative abundance of FD rats with liver depression-spleen deficiency syndrome was significantly different from the normal cohort. XYS intervention may effectively adjust the gut dysbacteriosis in FD.

Assessment of bacterial community composition in response to uranium levels in sediment samples of sacred Cauvery River.

Global industrialization is a major cause of effluent discharge from industries up to alarming concentrations. Especially, uranium concentrations in water bodies are of great concern, as its radioactivity significantly affects the persistent diversity of microbiota. Recently, continuous application of pesticides in the agricultural lands and accumulation of quartz that enter the Cauvery River has significantly increased the concentration of uranium (U) and other heavy metals. To perceive the impact of uranium on bacterial diversity in Cauvery River, sediment samples collected from polluted (UP) site with 32.4Bq/K of U concentration and control (UNP) site were scrutinized for bacterial diversity through metagenomic analysis of the V3 region of 16S rDNA by Illumina sequencing. Taxonomic assignment revealed that the unpolluted sample was dominated by Bacteroidetes (27.7%), and Firmicutes (25.9%), while sediment sample from the highly polluted site revealed abundance of Proteobacteria (47.5%) followed by Bacteroidetes (22.4%) and Firmicutes (14.6%). Among Proteobacteria, Gammaproteobacteria was the most prevalent group followed by alpha, delta, epsilon, and beta in the uranium-polluted sample. Rare and abundant species analysis revealed that species like Idiomarina loihiensis was abundant in the pollutant sample; however, it was rare (<0.1%) in the sample from pristine environment. Similarly, the species distribution in both the samples varied, with the bacteria potentially active in redox activity and biosorption potential dominating in the polluted sample. Outcomes of the present study demonstrated the impact of uranium and metal accumulation on the bacterial communities and further confirmed the promising candidature of specific bacterial species as bioindicators of contamination.

Effect of cinnamon essential oil on bacterial diversity and shelf-life in vacuum-packaged common carp (Cyprinus carpio) during refrigerated storage

The present study investigated the effect of cinnamon essential oil on the quality of vacuum-packaged common carp (Cyprinus carpio) fillets stored at 4±1°C in terms of sensory scores, physicochemical characteristics (total volatile basic nitrogen (TVB-N), biogenic amines, and color), and presence of spoilage microbiota. A total of 290,753 bacterial sequences and 162 different genera belonging to 14 phyla were observed by a high-throughput sequencing technique targeting the V3–V4 region of 16S rDNA, which showed a more comprehensive estimate of microbial diversity in carp samples compared with microbial enumeration. Before storage, Macrococcus and Aeromonas were the prevalent populations in the control samples, but cinnamon essential oil decreased the relative abundance of Macrococcus in the treated samples. Variability in the predominant microbiota in different samples during chilled storage was observed. Aeromonas followed by Lactococcus were the major contaminants in the spoiled control samples. Microbial enumeration also observed relatively higher counts of Aeromonas than other spoilage microorganisms. Compared with the control samples, cinnamon essential oil inhibited the growth of Aeromonas and Lactococcus were the predominant components in the treated samples on day 10; plate counts also revealed a relatively high level of lactic acid bacteria during refrigerated storage. However, there were no significant differences (P>0.05) in the composition of dominant microbiota between these two treatments at the end of the shelf-life. Furthermore, cinnamon essential oil treatment was more effective in inhibiting the increase of TVB-N and the accumulation of biogenic amines (especially for putrescine and cadaverine levels). Based primarily on sensory analysis, the use of cinnamon essential oil extended the shelf-life of vacuum-packaged common carp fillets by about 2days.

A comparison of microbial profiles of different regions of the equine hindgut

The microbial profiles of the luminal content of five hindgut segments of one healthy horse were compared with rectal sample to elucidate the effect of anatomical region on bacterial and archaeal community structure and to evaluate the use of faeces as a representative model of large intestine. The qualitative and quantitative changes of the microbial community composition of caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and faeces were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR using universal primers amplifying the V3 region of 16S rDNA. DGGE fingerprints revealed extensive bacterial as well as archaeal diversity in all studied samples and reflected shifts in the community structure among the caecum, the different segments of the colon and the faeces. Archaeal DGGE pattern of the caecum differed from all the other parts of the hindgut. Microbial profile similarities were found between the left and the right dorsal colon and between the left ventral colon and the faeces. The excised DGGE bands were related to uncultured bacteria and methanogens, the dominant archaeal bands of caecum and faeces were related to Methanocorpusculum sp. Diversity indices indicated the higher diversity for bacteria than for archaea and the dominance of some methanogenic species. The real-time PCR revealed the differences in the microbial quantitative composition of each segment, showing the highest number of total bacteria and archaea in the right ventral colon. The analyses of bacterial and archaeal composition along the one equine hindgut indicate that the faecal sample is similar to that of the left ventral colon, but does not represent the microbial community of the caecum and other parts of the colon.

Patterns of gut bacterial colonization in three primate species.

Host fitness is impacted by trillions of bacteria in the gastrointestinal tract that facilitate development and are inextricably tied to life history. During development, microbial colonization primes the gut metabolism and physiology, thereby setting the stage for adult nutrition and health. However, the ecological rules governing microbial succession are poorly understood. In this study, we examined the relationship between host lineage, captive diet, and life stage and gut microbiota characteristics in three primate species (infraorder, Lemuriformes). Fecal samples were collected from captive lemur mothers and their infants, from birth to weaning. Microbial DNA was extracted and the v4 region of 16S rDNA was sequenced on the Illumina platform using protocols from the Earth Microbiome Project. Here, we show that colonization proceeds along different successional trajectories in developing infants from species with differing dietary regimes and ecological profiles: frugivorous (fruit-eating) Varecia variegata, generalist Lemur catta, and folivorous (leaf-eating) Propithecus coquereli. Our analyses reveal community membership and succession patterns consistent with previous studies of human infants, suggesting that lemurs may serve as a useful model of microbial ecology in the primate gut. Each lemur species exhibits distinct species-specific bacterial diversity signatures correlating to life stages and life history traits, implying that gut microbial community assembly primes developing infants at species-specific rates for their respective adult feeding strategies.

Effect of virgin and refined olive oil consumption on gut microbiota. Comparison to butter

There is increasing evidence of the health benefits of olive oil consumption in the diet. Some authors have studied the effect of high fat/high calorie diets and have detected changes on the microbiota. However, these studies are mainly based on saturated fats. Here we present a study on the specific effect on gut bacterial populations of extra virgin olive oil, rich in monounsaturated fatty acids and phenolic compounds, in comparison to refined olive oil, rich in monounsaturated fatty acids but low in phenolic compounds, and to butter, rich in saturated fatty acids and cholesterol. Four groups of animals were studied: one group of mice received a standard chow diet, and the other received three high fat diets, rich in extra virgin olive oil, refined olive oil or butter. Evolution of symbiont population in feces was studied using culture-dependent and culture-independent methods. In the latter, the V3 region of 16S rDNA was amplified and separated by denaturing gradient gel electrophoresis; followed by sequencing of the most representative bands. Culture-dependent studies and comparison of the different DGGE profiles throughout the experiment demonstrated that different dietary fats had different effects on gut microbial composition. Butter-induced changes in the microbial counts resembled those previously described in obese individuals. Interestingly, a different behavior between extra virgin and refined olive oil was also observed, extra virgin olive oil being most different from butter. To our knowledge, no studies have analyzed gut microbiota depending on diets with different fatty acid saturations including different types of olive oil. This may offer new data supporting the benefits for health of extra virgin olive oil, so important in the Mediterranean diet.

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase-positive cocci (GCC) population in sucuk, a traditional Turkish dry-fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindrome-PCR (rep-PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real-time PCR DNA melting curve analysis and high-resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species-specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep-PCR and/or PCR-RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.

Evaluation of the biodegradability of petroleum in microcosm systems by using mangrove sediments from Camamu Bay, Bahia, Brazil.

We investigated the biodegradability of oil in mangrove sediment from Camamu Bay and measured its effect on the bacterial community. Microcosms of mangrove sediment were contaminated with 0.1, 0.5, 1, 2, and 5% (w/v) oil, and the microbial activity was compared to that in uncontaminated sediment. The evolution of CO2 and gas chromatography showed the mineralization of oil compounds, which could reach 100%. Bacterial diversity was determined by polymerase chain reaction using a set of primers for the V3 and V6-V8 regions of 16S rDNA. The band profile obtained by denaturing gradient gel electrophoresis of the amplicons that were obtained for the V3 region showed a negative correlation between band number and oil concentration, whereas that of the V6-V8 region showed a positive correlation between band numbers and oil concentration. The latter also gave similar results for microcosms that were contaminated with 2 and 5% oil. These results demonstrate the mangrove sediment's capacity to recover from oil contamination (in vitro) and suggest that native mangrove microorganisms contain enzymes necessary for the catabolism of oil.

Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1-1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE

Novel specific 16S rDNA-targeted primers were successfully designed and applied to the characterization of endophytic diversity in Dendrobium officinale. Using the popular universal bacterial primers 27f/1492r, the fragments of chloroplast and mitochondrion 16S/18S rDNA were amplified from D. officinale. They shared high nucleotide identity with the chloroplast 16S rDNAs (99-100%) and with the mitochondrion 18S rDNAs (93-100%) from various plants, respectively, and both shared 73-86% identities with the bacterial 16S rDNA sequences in GenBank. The current bacterial universal primers, including 27f/1492r, match well with the chloroplast and mitochondrion 16S/18S rDNAs, which accordingly renders these primers not useful for endophytic diversity analysis. Novel 16S rDNA-targeted primers fM1 (5'-CCGCGTGNRBGAHGAAGGYYYT-3') and rC5 (5'-TAATCCTGTTTGCTCC CCAC-3') were designed, which show good specificity compared to the 16S/18S rDNAs of D. officinale, and perfect universality within bacteria except for Cyanobacteria. The primers fM1/rC5, together with 515f-GC/rC5, which overlaps the whole V4 region of 16S rDNA, were subjected to nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) to analyze the diversity of endophytic bacteria in D. officinale from three different sources in China. The results showed diversities in roots and stems of the plants from all three locations. Altogether, 29 bands were identified as bacteria, with the dominant group being Proteobacteria and the dominant genus being Burkholderia, some of which commonly has the function of nitrogen fixation and thus may play potentially important roles in D. officinale. Therefore, the nested PCR-DGGE method based on the novel primers provides a good alternative for investigating the communities and roles of endophytes in D. officinale.

Comparative faecal microbiota of dogs with and without calcium oxalate stones

The absence of enteric oxalate-metabolizing bacterial species (OMBS) increases the likelihood of calcium oxalate (CaOx) urolithiasis in humans and dogs. The goal of this study was to compare the gut microbiota of healthy dogs and CaOx stone formed dogs (CaOx-dogs), especially with respect to OMBS. Faecal samples from healthy and CaOx-dogs were obtained to analyse the hindgut microbiota by sequencing the V3 region of bacterial 16S rDNA. In total, 1223 operational taxonomic units (OTUs) were identified at 97% identity. Only 38% of these OTUs were shared by both groups. Significant differences in the relative abundance of 152OTUs and 36 genera were observed between the two groups of dogs. The faecal microbiota of healthy dogs is distinct from that of CaOx-dogs, indicating that the microbiota is altered in CaOx-dogs. This is the first study that has compared the gut microbial diversity in healthy and CaOx-dogs. Results of this study indicate the future need for functional and comparative analyses of the total array of oxalate-metabolizing genes between healthy and CaOx stone formers, rather than focusing on specific bacterial species, to understand the critical role of OMBS in CaOx urolithiasis.

Determination of cheese origin by using 16S rDNA fingerprinting of bacteria communities by PCR–DGGE: Preliminary application to traditional Minas cheese

The bacterial diversity occurring in traditional Minas cheese, made from raw milk, produced in four different regions of Minas Gerais state in Brazil, namely Cerrado, Araxa, Serro and Serra da Canastra, were evaluated by PCR–DGGE analysis of the V3 region of the bacterial 16S rDNA using primers previously described in the literature. Samples of Minas cheese made with pasteurized milk from Serro region were used as control. Bacterial DNA templates for PCR were directly extracted from cheese samples in a unique step. The PCR–DGGE revealed 1 band for the cheese samples made from pasteurized milk and 7 to 13 bands for those from traditional cheeses. Some bands were common for all cheeses and some were unique for each region. The results suggest that specific band profiles from traditional cheeses from each region may be used as a biological bar code to discriminate their origins. In addition, the band profiles clearly distinguished cheese made from pasteurized and raw milk. DGGE band sequencing analysis showed that species belonging to the genera Streptococcus may predominate in the traditional Minas cheese.

Bacterial Diversity at Different Sites of the Digestive Tract of Weaned Piglets Fed Liquid Diets

Bacterial diversity was studied using PCR-DGGE, cloning and sequencing. DNA was isolated from digesta samples from stomach, ileum and colon of 28 weaned piglets (Large WhiteMong Cai) fed dry control feed, naturally fermented liquid feed (FE) and a liquid diet with inclusion of rice distiller's residue feed. General bacterial diversity was described using DGGE analysis of the V3 region of 16S rDNA. The microbial populations in the stomach and the ileum were considerably influenced by the diet, while only marginal effects were observed in the colon. There was a large variation of the microbial flora in the stomach between individuals fed non-fermented diets. In contrast, animals fed diet FE had a more uniform microbial flora in the stomach and the ileum compared to the other diets. In total 47 bands from the DGGE profiles were cloned. In stomach, most frequently lactic acid bacteria were found. Feeding diet FE resulted in the occurrence of Pediococcus species in stomach and ileum. In pigs fed the other diets, Lactobacillus gallinarum, Lactobacillus johnsonii and Lactobacillus fermentum were found in stomach and ileum. Most of the sequences of bands isolated from colon samples and several from ileum matched to unknown bacteria, which often grouped within Prevotellaceae, Enterobacteriaceae, Bacteroidaceae and Erysipelotrichaceae. This study demonstrates that fermented liquid feed affects bacterial diversity and the specific microflora in stomach and ileum, which provides a potential to modulate the gut microflora with dietary means to increase the abundance of beneficial bacteria and improve piglets' health.

Characterization and quantification of the nickel resistant microbial community in activated sludge using 16S rDNA and nickel resistance genes

The effect of nickel on the microbial community in the activated sludge of a sequencing batch reactor (SBR) reactor was investigated by continuously dosing nickel from 60 to 240 mg Ni(II) L−1. The diversity of the microbial community was investigated by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis of the variable V3 region of the bacterial 16S rDNA. The experimental results showed that the community structure changed significantly after dosing with nickel with a shift in dominant species, the disappearance of some original species and the emergence of some new species. The existence of a nickel resistant gene was also investigated using PCR. The obtained nickel resistance gene had a maximum homology with the plasmid pMOL30 of Ralstonia metallidurans CH34. The quantitative real‐time PCR results indicated that the quantity of the nickel resistance gene was related to the nickel concentration loaded to the reactor.

Distribution of oil‐degrading bacteria in coastal seawater, Toyama Bay, Japan

Oil-degrading bacteria are considered to play an important role in the biodegradation of spilled or released oil in the sea. The distribution of indigenous oil-degrading bacteria in the coastal seawater of Toyama Bay, Japan, was examined. Surface seawater samples with or without oil film in fishing port were analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of bacterial 16S rDNA. Sequence analysis revealed that several DGGE bands clearly detected only in samples with oil film corresponded to Cyanobacteria. Moreover, we cultured surface seawater samples with oil film in two different liquid culture media, a marine broth and an NSW medium; each culture contained 0.5% (w/v) C-heavy oil. Emulsification of the oil was observed at day 6 in the marine broth and day 9 in the NSW medium. Time-dependent changes of bacterial communities in those culture media were analyzed by DGGE. Interestingly, we found that Alcanivorax sp. became one of the dominant bacteria in each culture medium when emulsification of the oil began. Alcanivorax sp. is one of the well-known oil-degrading bacteria in seawater and is associated with the production of biosurfactants. These results suggest that Cyanobacteria and Alcanivorax play important roles in the bioremediation of oil-contaminated areas in Toyama Bay.

Effect of fermentation temperature on hydrogen production from cow waste slurry by using anaerobic microflora within the slurry

We examined hydrogen production from a dairy cow waste slurry (13.4 g of volatile solids per liter) by batch cultures in a temperature range from 37 to 85 degrees C, using microflora naturally present within the slurry. Without the addition of seed bacteria, hydrogen was produced by simply incubating the slurry, using the microflora within the slurry. Interestingly, two peaks of fermentation temperatures for hydrogen production from the slurry were observed at 60 and 75 degrees C (392 and 248 ml H2 per liter of slurry, respectively). After the termination of the hydrogen evolution, the microflora cultured at 60 degrees C displayed hydrogen-consuming activity, but hydrogen-consuming activity of the microflora cultured at 75 degrees C was not detected, at least for 24 days. At both 60 and 75 degrees C, the main by-product was acetate, and the optimum pH of the slurry for hydrogen production was around neutral. Bacteria related to hydrogen-producing moderate and extreme thermophiles, Clostridium thermocellum and Caldanaerobacter subterraneus, were detected in the slurries cultured at 60 and 75 degrees C, respectively, by denaturing gradient gel electrophoresis analyses, using the V3 region of 16S rDNA.

Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste

The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques. The temperature increased quickly in the first hours of the treatment cycles and values up to 72 degrees C were reached. Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles. A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60 degrees C. Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found. In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%). The other Bacilli could not be assigned to a known species. Schineria larvae was the second most abundant species (14%) in the clone library. Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40 degrees C and 60 degrees C. Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated.

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing.

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.