Husen Zhang

Human gut microbiota in obesity and after gastric bypass

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 H2-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 H2-oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H2-utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H2-producing bacteria with relatively high numbers of H2-utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H2 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.

Gut Microbiota and Its Possible Relationship With Obesity

Obesity results from alterations in the bodys 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.

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria

ABSTRACT Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h−1, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h−1, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h−1, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h−1, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h−1) and exceeded that of strain KJ on perchlorate (0.14 h−1). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [n = 7]), chlorate (0.44 ± 0.05 [n = 7]), and perchlorate (0.50 ± 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

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 H2‐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. Biotechnol. Bioeng. 2010;105: 69–78.

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 106 or 109 total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/106-AODC) than in water samples (0.02 to 0.4 PRM/106-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/109-AODC) than from other sites (nondetectable to 0.77 PRM/109-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.

Full-scale application of focused-pulsed pre-treatment for improving biosolids digestion and conversion to methane

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

Effect of dechlorination and sulfate reduction on the microbial community structure in denitrifying membrane-biofilm reactors

540,000 net of electricity and other operating and maintenance costs. This represents a payback period of three years or less.

An electron-flow model can predict complex redox reactions in mixed-culture fermentative bioH2: microbial ecology evidence.

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.

Integrating High-Throughput Pyrosequencing and Quantitative Real-Time PCR to Analyze Complex Microbial Communities

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. Biotechnol. Bioeng. 2009; 104: 687–697

Photobiodegradation of phenol with ultraviolet irradiation of new ceramic biofilm carriers

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.