Michael C. Nelson

A meta-analysis of the microbial diversity observed in anaerobic digesters

In this study, the collective microbial diversity in anaerobic digesters was examined using a meta-analysis approach. All 16S rRNA gene sequences recovered from anaerobic digesters available in public databases were retrieved and subjected to phylogenetic and statistical analyses. As of May 2010, 16,519 bacterial and 2869 archaeal sequences were found in GenBank. The bacterial sequences were assigned to 5926 operational taxonomic units (OTUs, based on ≥ 97% sequence identity) representing 28 known bacterial phyla, with Proteobacteria (1590 OTUs), Firmicutes (1352 OTUs), Bacteroidetes (705 OTUs), and Chloroflexi (693 OTUs) being predominant. Archaeal sequences were assigned to 296 OTUs, primarily Methanosaeta and the uncharacterized WSA2 group. Nearly 60% of all sequences could not be classified to any established genus. Rarefaction analysis indicates that approximately 60% of bacterial and 90% of archaeal diversity in anaerobic digesters has been sampled. This analysis of the global bacterial and archaeal diversity in AD systems can guide future studies to further examine the microbial diversity involved in AD and development of comprehensive analytical tools.

Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys.

The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.

Exposure to a social stressor disrupts the community structure of the colonic mucosa-associated microbiota

BackgroundThe microbiota of the mammalian gastrointestinal (GI) tract consists of diverse populations of commensal bacteria that interact with host physiological function. Dysregulating these populations, through exogenous means such as antibiotics or dietary changes, can have adverse consequences on the health of the host. Studies from laboratories such as ours have demonstrated that exposure to psychological stressors disrupts the population profile of intestinal microbiota. To date, such studies have primarily focused on prolonged stressors (repeated across several days) and have assessed fecal bacterial populations. It is not known whether shorter stressors can also impact the microbiota, and whether colonic mucosa-associated populations can also be affected. The mucosa-associated microbiota exist in close proximity to elements of the host immune system and the two are tightly interrelated. Therefore, alterations in these populations should be emphasized. Additionally, stressors can induce differential responses in anxiety-like behavior and corticosterone outputs in variant strains of mice. Thus, whether stressor exposure can have contrasting effects on the colonic microbiota in inbred C57BL/6 mice and outbred CD-1 mice was also examined.ResultsIn the present study, we used high throughput pyrosequencing to assess the effects of a single 2-hour exposure to a social stressor, called social disruption (SDR), on colonic mucosa-associated microbial profiles of C57BL/6 mice. The data indicate that exposure to the stressor significantly changed the community profile and significantly reduced the relative proportions of two genera and one family of highly abundant intestinal bacteria, including the genus Lactobacillus. This finding was confirmed using a quantitative real-time polymerase chain reaction (qPCR) technique. The use of qPCR also identified mouse strain-specific differences in bacterial abundances. L. reuteri, an immunomodulatory species, was decreased in stressor-exposed CD-1 mice, but not C57BL/6 mice.ConclusionsThese data illustrate that stressor exposure can affect microbial populations, including the lactobacilli, that are closely associated with the colonic mucosa. Because the lactobacilli can have beneficial effects on human health, stressor-induced reductions of their population could have important health implications.

Interrelations between the Microbiotas in the Litter and in the Intestines of Commercial Broiler Chickens

ABSTRACT The intestinal microbiota of broiler chickens and the microbiota in the litter have been well studied, but the interactions between these two microbiotas remain to be determined. Therefore, we examined their reciprocal effects by analyzing the intestinal microbiotas of broilers reared on fresh pine shavings versus reused litter, as well as the litter microbiota over a 6-week cycle. Composite ileal mucosal and cecal luminal samples from birds (n = 10) reared with both litter conditions (fresh versus reused) were collected at 7, 14, 21, and 42 days of age. Litter samples were also collected at days 7, 14, 21, and 42. The microbiotas were profiled and compared within sample types based on litter condition using PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The microbiotas were further analyzed using 16S rRNA gene clone libraries constructed from microbiota DNA extracted from both chick intestinal and litter samples collected at day 7. Results showed significant reciprocal effects between the microbiotas present in the litter and those in the intestines of broilers. Fresh litter had more environmental bacteria, while reused litter contained more bacteria of intestinal origin. Lactobacillus spp. dominated the ileal mucosal microbiota of fresh-litter chicks, while a group of bacteria yet to be classified within Clostridiales dominated in the ileal mucosal microbiota in the reused-litter chicks. The Litter condition (fresh versus reused) seemed to have a more profound impact on the ileal microbiota than on the cecal microbiota. The data suggest that the influence of fresh litter on ileal microbiota decreased as broilers grew, compared with temporal changes observed under reused-litter rearing conditions.

Shifts in microbial community structure of granular and liquid biomass in response to changes to infeed and digester design in anaerobic digesters receiving food-processing wastes

There have been few studies, to date, examining the effect of seed sludge on the microbial community established in a new anaerobic digestion (AD) system and whether or not the population present in the seed sludge establishes it self as the predominant population. Further, no reported studies have yet examined the differences in microbial populations that result from the formation of granular biomass in upflow anaerobic sludge blanket (UASB) systems. This study focused on examining the changes in microbial diversity between the initial seed sludge and the community that becomes established in a new digester. Using 16S rRNA clone libraries the diversity of microbes in both the granular and liquid biomass fractions from 3 AD sludge samples was examined and compared. Results showed that each sample had unique microbial community, with the distribution of sequences at the phylum level highly variable. This suggests that the feedstock had an effect of enriching microbial populations that are uniquely suited to a particular feedstock. Differences between the granular and liquid biomass fractions of each sample were less pronounced than differences attributable to the change in feedstock, however the results suggest that there are different functional groups in each fraction.

Bacterial symbioses of the medicinal leech Hirudo verbana

Gastrointestinal microbiomes play important roles in the health and nutrition of animals and humans. The medicinal leech, Hirudo verbana, serves as a powerful model for the study of microbial symbioses of the gut, due to its naturally limited microbiome compared with other popular models, the ability to cultivate the most abundant microbes, and genetically manipulate one of them, Aeromonas veronii. This review covers the relevance and application of leeches in modern medicine as well as recent discoveries detailing the nature of the gut microbiome. Additionally, the dual life-style of A. veronii allows one to do direct comparisons between colonization factors for beneficial and pathogenic associations, and relevant findings are detailed with respect to their role within the host and pathogenicity to other animals.

Hydrogen and volatile fatty acid production during fermentation of cellulosic substrates by a thermophilic consortium at 50 and 60 °C

The purpose of this study was to characterize the effect of temperature and cellulosic substrates on fermentative metabolites, H(2) production, and community successions in an anaerobic, cellulolytic consortium, TC60. Pyrosequencing analysis indicated that the consortium was predominated by Thermoanaerobacter and Clostridium spp. Metabolite production was analyzed with four cellulosic substrates at 4 kg/m(3). Triplicate cultures of each substrate were incubated at 50 or 60 °C. The main fermentation products (H(2), CO(2), ethanol, and acetate) were monitored over time. The ANOVA model for production rates showed a significant temperature effect (P<0.05) on all products. Increased temperature promoted higher H(2), CO(2), and ethanol yields while acetate yields were only affected prior to 24h of incubation. In addition to individual effects discerned in the model, ANOVA indicated significant interactions between the substrate and temperature. These interactions have not been previously recognized in the literature for cellulolytic and hydrogen-producing microorganisms.

Draft Genome Sequence of Aeromonas veronii Hm21, a Symbiotic Isolate from the Medicinal Leech Digestive Tract

ABSTRACT Aeromonas veronii strain Hm21 was isolated from the digestive tract of the medicinal leech Hirudo verbana and has been used to identify genes that are important for host colonization. This species is also a symbiont in the gut of zebrafish and is a pathogen of mammals and fish. We present here a 4.68-Mbp draft genome sequence for Hm21.

Impact of different ratios of feedstock to liquid anaerobic digestion effluent on the performance and microbiome of solid-state anaerobic digesters digesting corn stover

The objective of this study was to understand how the non-microbial factors of L-AD effluent affected the microbiome composition and successions in the SS-AD digesters using both Illumina sequencing and qPCR quantification of major genera of methanogens. The SS-AD digesters started with a feedstock/total effluent (F/Et) ratio 2.2 (half of the effluent was autoclaved) performed stably, while the SS-AD digesters started with a 4.4 F/Et ratio (no autoclaved effluent) suffered from digester acidification, accumulation of volatile fatty acids, and ceased biogas production two weeks after startup. Some bacteria and methanogens were affected by non-microbial factors of the L-AD fluent. Alkalinity, the main difference between the two F/Et ratios, may be the crucial factor when SS-AD digesters were started using L-AD effluent.

Mucinivorans hirudinis gen. nov., sp. nov., an anaerobic, mucin-degrading bacterium isolated from the digestive tract of the medicinal leech Hirudo verbana

Three anaerobic bacterial strains were isolated from the digestive tract of the medicinal leech Hirudo verbana, using mucin as the primary carbon and energy source. These strains, designated M3(T), M4 and M6, were Gram-stain-negative, non-spore-forming and non-motile. Cells were elongated bacilli approximately 2.4 µm long and 0.6 µm wide. Growth only occurred anaerobically under mesophilic and neutral pH conditions. All three strains could utilize multiple simple and complex sugars as carbon sources, with glucose fermented to acid by-products. The DNA G+C contents of strains M3(T), M4 and M6 were 44.9, 44.8 and 44.8 mol%, respectively. The major cellular fatty acid of strain M3(T) was iso-C15 : 0. Phylogenetic analysis of full-length 16S rRNA gene sequences revealed that the three strains shared >99 % similarity with each other and represent a new lineage within the family Rikenellaceae of the order Bacteroidales, phylum Bacteroidetes. The most closely related bacteria to strain M3(T) based on 16S rRNA gene sequences were Rikenella microfusus DSM 15922(T) (87.3 % similarity) and Alistipes finegoldii AHN 2437(T) (87.4 %). On the basis of phenotypic, genotypic and physiological evidence, strains M3(T), M4 and M6 are proposed as representing a novel species of a new genus within the family Rikenellaceae, for which the name Mucinivorans hirudinis gen. nov., sp. nov. is proposed. The type strain of Mucinivorans hirudinis is M3(T) ( = ATCC BAA-2553(T) = DSM 27344(T)).