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Background: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether., Results: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner., Conclusions: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupus-associated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.

Background: Rotavirus vaccines have poor efficacy in infants from low- and middle-income countries. Gut microbiota is thought to influence the immune response to oral vaccines. Thus, we developed a gnotobiotic (Gn) pig model of enteric dysbiosis to study the effects of human gut microbiota (HGM) on immune responses to rotavirus vaccination, and the effects of rotavirus challenge on the HGM by colonizing Gn pigs with healthy HGM (HHGM) or unhealthy HGM (UHGM). The UHGM was from a Nicaraguan infant with a high enteropathy score (ES) and no seroconversion following administration of oral rotavirus vaccine, while the converse was characteristic of the HHGM. Pigs were vaccinated, a subset was challenged, and immune responses and gut microbiota were evaluated., Results: Significantly more rotavirus-specific IFN-γ producing T cells were in the ileum, spleen, and blood of HHGM than those in UHGM pigs after three vaccine doses, suggesting HHGM induces stronger cell-mediated immunity than UHGM. There were significant correlations between multiple Operational Taxonomic Units (OTUs) and frequencies of IFN-γ producing T cells at the time of challenge. There were significant positive correlations between Collinsella and CD8+ T cells in blood and ileum, as well as CD4+ T cells in blood, whereas significant negative correlations between Clostridium and Anaerococcus , and ileal CD8+ and CD4+ T cells. Differences in alpha diversity and relative abundances of OTUs were detected between the groups both before and after rotavirus challenge., Conclusion: Alterations in microbiome diversity and composition along with correlations between certain microbial taxa and T cell responses warrant further investigation into the role of the gut microbiota and certain microbial species on enteric immunity. Our results support the use of HGM transplanted Gn pigs as a model of human dysbiosis during enteric infection, and oral vaccine responses.

Background: Clostridium difficile is the most common known cause of antibiotic-associated diarrhea. Upon the disturbance of gut microbiota by antibiotics, C. difficile establishes growth and releases toxins A and B, which cause tissue damage in the host. The symptoms of C. difficile infection disease range from mild diarrhea to pseudomembranous colitis and toxic megacolon. Interestingly, 10-50 % of infants are asymptomatic carriers of C. difficile. This longitudinal study of the C. difficile colonization in an infant revealed the dynamics of C. difficile presence in gut microbiota., Methods: Fifty fecal samples, collected weekly between 5.5 and 17 months of age from a female infant who was an asymptomatic carrier of C. difficile, were analyzed by 16S rRNA gene sequencing., Results: Colonization switching between toxigenic and non-toxigenic C. difficile strains as well as more than 100,000-fold fluctuations of C. difficile counts were observed. C. difficile toxins were detected during the testing period in some infant stool samples, but the infant never had diarrhea. Although fecal microbiota was stable during breast feeding, a dramatic and permanent change of microbiota composition was observed within 5 days of the transition from human milk to cow milk. A rapid decline and eventual disappearance of C. difficile coincided with weaning at 12.5 months. An increase in the relative abundance of Bacteroides spp., Blautia spp., Parabacteroides spp., Coprococcus spp., Ruminococcus spp., and Oscillospira spp. and a decrease of Bifidobacterium spp., Lactobacillus spp., Escherichia spp., and Clostridium spp. were observed during weaning. The change in microbiome composition was accompanied by a gradual increase of fecal pH from 5.5 to 7., Conclusions: The bacterial groups that are less abundant in early infancy, and that increase in relative abundance after weaning, likely are responsible for the expulsion of C. difficile.

The nitritation-anammox process has been a promising nitrogen removal technology towards sustainable wastewater treatment, but its application in treating domestic wastewater with relatively low ammonium concentrations (mainstream) remains a great challenge. In this study, an innovative lab-scale upflow membrane-aerated biofilm reactor (UMABR) was employed to treat a synthetic wastewater containing 70 mg N L(-1) ammonium. With a DO level at 0.6 ± 0.1 mg O2 L(-1) and HRT of 32 h, the effluent ammonium concentration was 4.8 ± 2.0 mg N L(-1). Increasing the nitrogen loading rate from 52.4 to 104.8 g N m(-3) d(-1) with stepwise decreasing HRT from 32 to 16 h resulted in an average TN removal efficiency of 81% without nitrite accumulation. The average observed NO3(-)-N (residue)/NH4(+)-N (consumed) ratio of 8% was below the "theoretical ratio" of 13% and further reduction of nitrate residue needs to be addressed. Fluorescence in situ hybridization (FISH) and high-throughput sequencing analyses showed the coexistence of anammox bacteria and ammonium-oxidizing bacteria (AOB) in both biofilm and granular samples. Anammox bacteria accounted for up to 63.3% of the microbial community of the granules, with Candidatus Jettenia being the distinctly dominant anammox genus. In contrast, the biofilm contained abundant Nitrosomonadaceae (AOB, 33.1%). In addition, the brown-yellow granules exhibited a more balanced community structure with anammox bacteria and AOB accounting for 33.7% and 18.2%, respectively, which may contribute to the long-term operation of single-stage nitritation-anammox process. These results demonstrate that the nitritation-anammox UMABR could potentially be used for nitrogen removal from mainstream in some specific regions with relatively warm temperature., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Crude (4-methylcyclohexyl)methanol (MCHM) caused extensive contamination of drinking water, wastewater, and the environment during the 2014 West Virginia Chemical Spill. However, information related to the environmental degradation of cis- and trans-4-MCHM, the main components of the crude 4-MCHM mixture, remains largely unknown. This study is among the first to investigate the degradation kinetics and transformation of 4-MCHM isomers in activated sludge. The 4-MCHM loss was mainly due to biodegradation to form carbon dioxide (CO2), plus acetic, propionic, isobutyric, and isovaleric acids with little contribution from adsorption. The biodegradation of 4-MCHM isomers followed the first-order kinetic model with half-lives higher than 0.50 days. Nitrate augmented the degradation of 4-MCHM isomers, while glucose and acetate decreased their degradation. One 4-MCHM-degrading bacterium isolated from activated sludge was identified as Acinetobacter bouvetii strain EU40 based on 16S rRNA gene sequences. This study will enhance the prediction of the environmental fate of 4-MCHM in water treatment systems., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cyclohexane and some of its derivatives have been a major concern because of their significant adverse human health effects and widespread occurrence in the environment. The 2014 West Virginia chemical spill has raised public attention to (4-methylcyclohexyl)methanol (4-MCHM), one cyclohexane derivative, which is widely used in coal processing but largely ignored. In particular, the environmental fate of its primary components, cis- and trans-4-MCHM, remains largely unexplored. This study aimed to investigate the degradation kinetics and mineralization of cis- and trans-4-MCHM by sediment microorganisms under aerobic and anaerobic conditions. We found the removal of cis- and trans-4-MCHM was mainly attributed to biodegradation with little contribution from sorption. A nearly complete aerobic degradation of 4-MCHM occurred within 14 days, whereas the anaerobic degradation was reluctant with residual percentages of 62.6% of cis-4-MCHM and 85.0% of trans-4-MCHM after 16-day incubation. The cis-4-MCHM was degraded faster than the trans under both aerobic and anaerobic conditions, indicating an isomer-specific degradation could occur during the 4-MCHM degradation. Nitrate addition enhanced 4-MCHM mineralization by about 50% under both aerobic and anaerobic conditions. Both cis- and trans-4-MCHM fit well with the first-order kinetic model with respective degradation rates of 0.46-0.52 and 0.19-0.31 day(-)(1) under aerobic condition. Respective degradation rates of 0.041-0.095 and 0.013-0.052 day(-)(1) occurred under anaerobic condition. One bacterial strain capable of effectively degrading 4-MCHM isomers was isolated from river sediments and identified as Bacillus pumilus at the species level based on 16S rRNA gene sequence and 97% identity. Our findings will provide critical information for improving the prediction of the environmental fate of 4-MCHM and other cyclohexane derivatives with similar structure as well as enhancing the development of feasible treatment technologies to mitigate these compounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. Despite years of study, the etiology of SLE is still unclear. Both genetic and environmental factors have been implicated in the disease mechanisms. In the past decade, a growing body of evidence has indicated an important role of gut microbes in the development of autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and multiple sclerosis. However, such knowledge on SLE is little, though we have already known that environmental factors can trigger the development of lupus. Several recent studies have suggested that alterations of the gut microbial composition may be correlated with SLE disease manifestations, while the exact roles of either symbiotic or pathogenic microbes in this disease remain to be explored. Elucidation of the roles of gut microbes - as well as the roles of diet that can modulate the composition of gut microbes - in SLE will shed light on how this autoimmune disorder develops, and provide opportunities for improved biomarkers of the disease and the potential to probe new therapies. In this review, we aim to compile the available evidence on the contributions of diet and gut microbes to SLE occurrence and pathogenesis.

Plasmacytoid dendritic cells (pDCs) are professional type I IFN producers believed to promote lupus. However, questions exist about whether they function at the same level throughout the course of lupus disease. We analyzed high-purity pDCs sorted from lupus mice. Although pDCs produced a large amount of IFN-α during disease initiation, those sorted from late-stage lupus mice were found to be defective in producing IFN-α. These pDCs expressed an increased level of MHC, suggesting a functional drift to Ag presentation. We examined the potential mechanism behind the defect and identified a novel transcriptional factor, Foxj2, which repressed the expression of several genes in pDCs, but not IFN-α. Dysregulation in pDCs appears to be predisposed, because they exhibited an altered transcriptional profile before the onset of clinical signs. Our results suggest that pDCs do not function the same throughout the disease course and lose the ability to produce IFN-α in late-stage lupus mice., (Copyright © 2015 by The American Association of Immunologists, Inc.)

It has long been recognized that the mammalian gut microbiota has a role in the development and activation of the host immune system. Much less is known on how host immunity regulates the gut microbiota. Here we investigated the role of adaptive immunity on the mouse distal gut microbial composition by sequencing 16 S rRNA genes from microbiota of immunodeficient Rag1(-/-) mice, versus wild-type mice, under the same housing environment. To detect possible interactions among immunological status, age and variability from anatomical sites, we analyzed samples from the cecum, colon, colonic mucus and feces before and after weaning. High-throughput sequencing showed that Firmicutes, Bacteroidetes and Verrucomicrobia dominated mouse gut bacterial communities. Rag1(-) mice had a distinct microbiota that was phylogenetically different from wild-type mice. In particular, the bacterium Akkermansia muciniphila was highly enriched in Rag1(-/-) mice compared with the wild type. This enrichment was suppressed when Rag1(-/-) mice received bone marrows from wild-type mice. The microbial community diversity increased with age, albeit the magnitude depended on Rag1 status. In addition, Rag1(-/-) mice had a higher gain in microbiota richness and evenness with increase in age compared with wild-type mice, possibly due to the lack of pressure from the adaptive immune system. Our results suggest that adaptive immunity has a pervasive role in regulating gut microbiota's composition and diversity.

Lead (Pb) is a prominent toxic metal in natural and engineered systems. Current knowledge on Pb toxicity to the activated sludge has been limited to short-term (≤24 h) toxicity. The effect of extended Pb exposure on process performance, bacterial viability, and community compositions remains unknown. We quantified the 24-h and 7-day Pb toxicity to chemical oxygen demand (COD) and NH3–N removal, bacterial viability, and community compositions using lab-scale experiments. Our results showed that 7-day toxicity was significantly higher than the short-term 24-h toxicity. Ammonia-oxidizing bacteria were more susceptible than the heterotrophs to Pb toxicity. The specific oxygen uptake rate responded quickly to Pb addition and could serve as a rapid indicator for detecting Pb pollutions. Microbial viability decreased linearly with the amount of added Pb at extended exposure. The bacterial community diversity was markedly reduced with elevated Pb concentrations. Surface analysis suggested that the adsorbed form of Pb could have contributed to its toxicity along with the dissolved form. Our study provides for the first time a systematic investigation of the effect of extended exposure of Pb on the performance and microbiology of aerobic treatment processes, and it indicates that long-term Pb toxicity has been underappreciated by previous studies.

The symbiotic relationship between the mammalian host and gut microbes has fascinated many researchers in recent years. Use of germ-free animals has contributed to our understanding of how commensal microbes affect the host. Immunodeficiency animals lacking specific components of the mammalian immune system, on the other hand, enable studying of the reciprocal function-how the host controls which microbes to allow for symbiosis. Here we review the recent advances and discuss our perspectives of how to better understand the latter, with an emphasis on the effects of adaptive immunity on the composition and diversity of gut commensal bacteria.

Gut microbiota has been recognized as an important environmental factor in health, as well as in metabolic and immunological diseases, in which perturbation of the host gut microbiota is often observed in the diseased state. However, little is known on the role of gut microbiota in systemic lupus erythematosus. We investigated the effects of host genetics, sex, age, and dietary intervention on the gut microbiome in a murine lupus model. In young, female lupus-prone mice resembling women at childbearing age, a population with the highest risk for lupus, we found marked depletion of lactobacilli, and increases in Lachnospiraceae and overall diversity compared to age-matched healthy controls. The predicted metagenomic profile in lupus-prone mice showed a significant enrichment of bacterial motility- and sporulation-related pathways. Retinoic acid as a dietary intervention restored lactobacilli that were downregulated in lupus-prone mice, and this correlated with improved symptoms. The predicted metagenomes also showed that retinoic acid reversed many lupus-associated changes in microbial functions that deviated from the control. In addition, gut microbiota of lupus-prone mice were different between sexes, and an overrepresentation of Lachnospiraceae in females was associated with an earlier onset of and/or more severe lupus symptoms. Clostridiaceae and Lachnospiraceae, both harboring butyrate-producing genera, were more abundant in the gut of lupus-prone mice at specific time points during lupus progression. Together, our results demonstrate the dynamics of gut microbiota in murine lupus and provide evidence to suggest the use of probiotic lactobacilli and retinoic acid as dietary supplements to relieve inflammatory flares in lupus patients., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Microbial fuel cells (MFCs) employ microorganisms to recover electric energy from organic matter. However, fundamental knowledge of electrochemically active bacteria is still required to maximize MFCs power output for practical applications. This review presents microbiological and electrochemical techniques to help researchers choose the appropriate methods for the MFCs study. Pre-genomic and genomic techniques such as 16S rRNA based phylogeny and metagenomics have provided important information in the structure and genetic potential of electrode-colonizing microbial communities. Post-genomic techniques such as metatranscriptomics allow functional characterizations of electrode biofilm communities by quantifying gene expression levels. Isotope-assisted phylogenetic analysis can further link taxonomic information to microbial metabolisms. A combination of electrochemical, phylogenetic, metagenomic, and post-metagenomic techniques offers opportunities to a better understanding of the extracellular electron transfer process, which in turn can lead to process optimization for power output., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

We generated a neonatal pig model with human infant gut microbiota (HGM) to study the effect of a probiotic on the composition of the transplanted microbiota following rotavirus vaccination and challenge. All the HGM-transplanted pigs received two doses of an oral attenuated rotavirus vaccine. The gut microbiota of vaccinated pigs were investigated for effects of Lactobacillus rhamnosus GG (LGG) supplement and homotypic virulent human rotavirus (HRV) challenge. High-throughput sequencing of V4 region of 16S rRNA genes demonstrated that HGM-transplanted pigs carried microbiota similar to that of the C-section delivered baby. Firmicutes and Proteobacteria represented over 98% of total bacteria in the human donor and the recipient pigs. HRV challenge caused a phylum-level shift from Firmicutes to Proteobacteria. LGG supplement prevented the changes in microbial communities caused by HRV challenge. In particular, members of Enterococcus in LGG-supplemented pigs were kept at the baseline level, while they were enriched in HRV challenged pigs. Taken together, our results suggested that HGM pigs are valuable for testing the microbiota's response to probiotic interventions for treating infantile HRV infection.

New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time PCR (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, we discuss the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems. We explain parallel sequencing of amplicon libraries and using bar codes to differentiate multiple samples in a pyrosequencing run. We also describe best procedures and chemistries for QPCR amplifications and address advantages of applying automation to increase accuracy. We provide three examples in which we used pyrosequencing and QPCR together to define and quantify members of microbial communities: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. We highlight our key findings in these systems and how both methods were used in concert to achieve those findings. Finally, we supply detailed methods for generating PCR amplicon libraries for pyrosequencing, pyrosequencing data analysis, QPCR methodology, instrumentation, and automation.

Activated sludge acclimated to biodegrade phenol was allowed to attach on and in light porous ceramic carriers and to function as a biofilm in a photolytic circulating-bed bioreactor (PCBBR). Phenol degradation in the PCBBR was investigated following three protocols: photolysis with ultraviolet light alone (P), biodegradation alone (B), and the two mechanisms operating simultaneously (P/B). Phenol was degraded at approximately equal rates by B and P/B, each of which was much faster than the rate by P. Furthermore, phenol was mineralized to a significantly greater extent with P/B than with either P or B. SEM showed that the biofilm survived well inside macropores that presumably shaded the microorganisms from UV irradiation, even though the UV light greatly reduced biofilm on outer surface of the carriers in the P/B experiments. Rapid biodegradation of phenol, enhanced mineralization, and survival of bacteria inside macropores demonstrated that being in a biofilm inside the porous carriers protected the bacteria from UV-light toxicity, allowing intimate coupling of photolysis and biodegradation.

The influences of fluorescence labeling on PCR amplification and T-RFLP analysis were examined by the analyses of a soil bacterial and archaeal community using both clone library and T-RFLP methods. The PCR amplification and microbial community structure patterns were compared among the primers labeled with and without fluorescent groups. PCR amplification was negatively affected by the labeling groups of the primers, which may be caused by the increment of primer molecular weight. It is known that thermodynamic movement of molecules will be slowed as molecular weight increased. Therefore it is understandable that the reaction of primer-DNA template hybridization will be inhibited with the fluorescent groups added to the primer(s). An effective "Gradient-Decreasing Annealing Time Program," in which the annealing time was initially set long and reduced cycle by cycle, can improve PCR efficiency under comparable amplification specificity with the fluorescent-labeled primers. No significant negative impact was observed in the altered conditions., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Recent studies showed that the chlorinated solvents trichloroethene (TCE), 1,1,1-trichloroethane (TCA), and chloroform (CF) were reductively dehalogenated in a H(2)-based membrane biofilm reactor (MBfR) under denitrifying conditions. Here, we describe a detailed phylogenetic characterization of MBfR biofilm communities having distinctly different metabolic functions with respect to electron-acceptor reduction. Using massively parallel pyrosequencing of the V6 region of the 16S rRNA gene, we detected 312, 592, and 639 operational taxonomic units (OTU) in biofilms of three MBfRs that reduced nitrate; nitrate and TCE; or nitrate, sulfate, and all three chlorinated solvents. Comparative community analysis revealed that 13% of the OTUs were shared by all MBfRs, regardless of the feed, but 65% were unique to one MBfR. Pyrosequencing and real-time quantitative PCR showed that Dehalococcoides were markedly enriched in the TCE+nitrate biofilm. The input of a mixture of three chlorinated compounds, which coincided with the onset of sulfate reduction, led to a more diverse community that included sulfate-reducing bacteria (Desulfovibrio) and nitrate-reducing bacteria (Geothrix and Pseudomonas). Our results suggest that chlorinated solvents, as additional electron acceptors to nitrate and sulfate, increased microbial diversity by allowing bacteria with special metabolic capabilities to grow in the biofilm.

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate-one where methanogenesis was allowed and another in which it was completely inhibited with 2-bromoethane sulfonate. We observed a three-way syntrophy among ethanol fermenters, acetate-oxidizing anode-respiring bacteria (ARB), and a H2 scavenger. When methanogenesis was allowed, H2-oxidizing methanogens were the H2 scavengers, but when methanogenesis was inhibited, homo-acetogens became a channel for electron flow from H2 to current through acetate. We established the presence of homo-acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo-acetogens. Both methods documented the presence of the homo-acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol-fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H(2)-scavenging syntrophic partners that were either H2-oxidizing methanogens or homo-acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron-flow distribution and H2-scavenging mechanism.

We developed the first model for predicting community structure in mixed-culture fermentative biohydrogen production using electron flows and NADH2 balances. A key assumption of the model is that H2 is produced only via the pyruvate decarboxylation-ferredoxin-hydrogenase pathway, which is commonly the case for fermentation by Clostridium and Ethanoligenens species. We experimentally tested the model using clone libraries to gauge community structures with mixed cultures in which we did not pre-select for specific bacterial groups, such as spore-formers. For experiments having final pHs 3.5 and 4.0, where H2 yield and soluble end-product distribution were distinctly different, we established stoichiometric reactions for each condition by using experimentally determined electron equivalent balances. The error in electron balancing was only 3% at final pH 3.5, in which butyrate and acetate were dominant organic products and the H2 yield was 2.1 mol H2/mol glucose. Clone-library analysis showed that clones affiliated with Clostridium sp. BL-22 and Clostridium sp. HPB-16 were dominant at final pH 3.5. For final pH 4.0, the H2 yield was 0.9 mol H2/mol glucose, ethanol, and acetate were the dominant organic products, and the electron balance error was 13%. The significant error indicates that a second pathway for H2 generation was active. The most abundant clones were affiliated with Klebsiella pneumoniae, which uses the formate-cleavage pathway for H2 production. Thus, the clone-library analyses confirmed that the model predictions for when the pyruvate decarboxylation-ferredoxin-hydrogenase pathway was (final pH 3.5) or was not (final pH 4.0) dominant. With the electron-flow model, we can easily assess the main mechanisms for H2 formation and the dominant H2-producing bacteria in mixed-culture fermentative bioH2., (Copyright 2009 Wiley Periodicals, Inc.)

The low yield of methane in anaerobic digestion systems represents a loss of energy that can be captured as renewable energy when the input sludge is pre-treated to make it more bioavailable. We investigated Focused-Pulsed (FP) pre-treatment, which make complex biological solids more bioavailable by exposing them to rapid pulses of a very strong electric field. We investigated how the microbial ecology in full-scale anaerobic digesters was altered when the digesters' methane production rate was significantly increased by FP pre-treatment. Using clone libraries and quantitative PCR, we demonstrated a shift in methanogenic genera to the acetate-cleaving Methanosaeta and away from the H(2)-oxidizing Methanoculleus. In addition, the acetate concentration in the effluent was very low, probably due to the dominance of Methanosaeta, which are capable of scavenging low acetate concentrations. By analyzing 36,797 pyrosequencing tags from the V6 region of the bacterial 16S rRNA gene, along with archaeal and bacterial clone libraries and quantitative PCR, we compared the microbial community composition before and after FP treatment. The bacterial community became more diverse after FP pre-treatment and was populated more by phylotypes associated with cellulose fermentation (Ruminococcus), scavenging of biomass-derived organic carbon (Chloroflexi), and homo-acetogenesis (Treponema). We interpret that, as the overall activity of the community was stimulated by addition of more bioavailable organic matter, the bacterial community became more phylogenetically diverse to take advantage of the added input of biodegradable material and in response to the more efficient utilization of acetate by Methanosaeta.

The sulfate-methane transition zone (SMTZ) is a widespread feature of continental margins, representing a diffusion-controlled interface where there is enhanced microbial activity. SMTZ microbial activity is commonly associated with the anaerobic oxidation of methane (AOM), which is carried out by syntrophic associations between sulfate-reducing bacteria and methane-oxidizing archaea. While our understanding of the microorganisms catalyzing AOM has advanced, the diversity and ecological role of the greater microbial assemblage associated with the SMTZ have not been well characterized. In this study, the microbial diversity above, within, and beneath the Santa Barbara Basin SMTZ was described. ANME-1-related archaeal phylotypes appear to be the primary methane oxidizers in the Santa Barbara Basin SMTZ, which was independently supported by exclusive recovery of related methyl coenzyme M reductase genes (mcrA). Sulfate-reducing Deltaproteobacteria phylotypes affiliated with the Desulfobacterales and Desulfosarcina-Desulfococcus clades were also enriched in the SMTZ, as confirmed by analysis of dissimilatory sulfite reductase (dsr) gene diversity. Statistical methods demonstrated that there was a close relationship between the microbial assemblages recovered from the two horizons associated with the geochemically defined SMTZ, which could be distinguished from microbial diversity recovered from the sulfate-replete overlying horizons and methane-rich sediment beneath the transition zone. Comparison of the Santa Barbara Basin SMTZ microbial assemblage to microbial assemblages of methane seeps and other organic matter-rich sedimentary environments suggests that bacterial groups not typically associated with AOM, such as Planctomycetes and candidate division JS1, are additionally enriched within the SMTZ and may represent a common bacterial signature of many SMTZ environments worldwide.

Recent evidence suggests that the microbial community in the human intestine may play an important role in the pathogenesis of obesity. We examined 184,094 sequences of microbial 16S rRNA genes from PCR amplicons by using the 454 pyrosequencing technology to compare the microbial community structures of 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric-bypass surgery. Phylogenetic analysis demonstrated that although the Bacteria in the human intestinal community were highly diverse, they fell mainly into 6 bacterial divisions that had distinct differences in the 3 study groups. Specifically, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of Gammaproteobacteria. Numbers of the H(2)-producing Prevotellaceae were highly enriched in the obese individuals. Unlike the highly diverse Bacteria, the Archaea comprised mainly members of the order Methanobacteriales, which are H(2)-oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H(2)-utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H(2)-producing bacteria with relatively high numbers of H(2)-utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H(2) transfer between bacterial and archaeal species is an important mechanism for increasing energy uptake by the human large intestine in obese persons. The large bacterial population shift seen in the post-gastric-bypass individuals may reflect the double impact of the gut alteration caused by the surgical procedure and the consequent changes in food ingestion and digestion.

Obesity results from alterations in the body's regulation of energy intake, expenditure, and storage. Recent evidence, primarily from investigations in animal models, suggests that the gut microbiota affects nutrient acquisition and energy regulation. Its composition has also been shown to differ in lean vs obese animals and humans. In this article, we review the published evidence supporting the potential role of the gut microbiota in the development of obesity and explore the role that modifying the gut microbiota may play in its future treatment. Evidence suggests that the metabolic activities of the gut microbiota facilitate the extraction of calories from ingested dietary substances and help to store these calories in host adipose tissue for later use. Furthermore, the gut bacterial flora of obese mice and humans include fewer Bacteroidetes and correspondingly more Firmicutes than that of their lean counterparts, suggesting that differences in caloric extraction of ingested food substances may be due to the composition of the gut microbiota. Bacterial lipopolysaccharide derived from the intestinal microbiota may act as a triggering factor linking inflammation to high-fat diet-induced metabolic syndrome. Interactions among microorganisms in the gut appear to have an important role in host energy homeostasis, with hydrogen-oxidizing methanogens enhancing the metabolism of fermentative bacteria. Existing evidence warrants further investigation of the microbial ecology of the human gut and points to modification of the gut microbiota as one means to treat people who are over-weight or obese.

We tested at full-scale the innovative Focused Pulsed (FP) technology for pre-treating waste sludge in order to improve methane gas production and biosolids reduction in sludge digestion, but without incurring problems of odors, toxicity, and high costs for chemical or energy consumption. FP pre-treatment of a mixture of primary and secondary sludge increased the soluble COD by 160% and DOC 120% over the control. FP pre-treatment of 63% of the input waste sludge increased biogas production by over 40% and reduced biosolids requiring disposal by 30% when compared to the plant baseline. FP pre-treatment also correlated with a shift of the bacterial and archaeal communities. The most significant change was that the acetate-cleaving Methanosaeta became the dominant methanogen. Full FP pre-treatment should increase biogas production and biosolids removal by 60% and 40%, respectively. Full FP pre-treatment should generate energy benefits of at least 2.7 times and as high as 18 times the FP energy input, depending on heat recovery from FP treatment. For a plant treating 76,000 m3/d of wastewater (380 m3-sludge/d), FP treatment should generate an annual economic benefit of approximately $540,000 net of electricity and other operating and maintenance costs. This represents a payback period of three years or less., (Copyright (c) IWA Publishing 2008.)

A mesophilic unsaturated flow (trickle bed) reactor was designed and tested for H2 production via fermentation of glucose. The reactor consisted of a column packed with glass beads and inoculated with a pure culture (Clostridium acetobutylicum ATCC 824). A defined medium containing glucose was fed at a flow rate of 1.6 mL/min (0.096 L/h) into the capped reactor, producing a hydraulic retention time of 2.1 min. Gas-phase H2 concentrations were constant, averaging 74 +/- 3% for all conditions tested. H2 production rates increased from 89 to 220 mL/hL of reactor when influent glucose concentrations were varied from 1.0 to 10.5 g/L. Specific H2 production rate ranged from 680 to 1270 mL/g glucose per liter of reactor (total volume). The H2 yield was 15-27%, based on a theoretical limit by fermentation of 4 moles of H2 from 1 mole of glucose. The major fermentation by-products in the liquid effluent were acetate and butyrate. The reactor rapidly (within 60-72 h) became clogged with biomass, requiring manual cleaning of the system. In order to make long-term operation of the reactor feasible, biofilm accumulation in the reactor will need to be controlled through some process such as backwashing. These tests using an unsaturated flow reactor demonstrate the feasibility of the process to produce high H2 gas concentrations in a trickle-bed type of reactor. A likely application of this reactor technology could be H2 gas recovery from pre-treatment of high carbohydrate-containing wastewaters.

Hydrogen gas (approximately 60% H(2)) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l(-1)). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H(2) by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30 degrees C and 37 degrees C). At 30-h HRT, the H(2) production rate was 80 ml h(-1) and yield was 0.91 mol H(2)/mol glucose. At 10-h HRT, the H(2) production rate was more than 5 times higher at 436 ml h(-1), and yield was 1.61 mol H(2)/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30 degrees C to 37 degrees C, the steady-state H(2) production rate increased slightly to 463 ml h(-1) and yield was 1.8 mol H(2)/mol glucose. Compared to 30 degrees C, RISA fingerprints at 37 degrees C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).

Water treatment technologies are needed that can remove perchlorate from drinking water without introducing organic chemicals that stimulate bacterial growth in water distribution systems. Hydrogen is an ideal energy source for bacterial degradation of perchlorate as it leaves no organic residue and is sparingly soluble. We describe here the isolation of a perchlorate-respiring, hydrogen-oxidizing bacterium (Dechloromonas sp. strain HZ) that grows with carbon dioxide as sole carbon source. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that was isolated from a gas-phase anaerobic packed-bed biofilm reactor treating perchlorate-contaminated groundwater. The ability of strain HZ to grow autotrophically with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from CO2. Chemolithotrophic growth with hydrogen was coupled with complete reduction of perchlorate (10 mM) to chloride with a maximum doubling time of 8.9 h. Strain HZ also grew using acetate as the electron donor and chlorate, nitrate, or oxygen (but not sulphate) as an electron acceptor. Phylogenetic analysis of the 16S rRNA sequence placed strain HZ in the genus Dechloromonas within the beta subgroup of the Proteobacteria. The study of this and other novel perchlorate-reducing bacteria may lead to new, safe technologies for removing perchlorate and other chemical pollutants from drinking water.