Assessment of microbial diversity associated with CH4 emission from sugarcane vinasse storage and transportation systems.

Sugarcane bioethanol has favorable energy and greenhouse gas balance, although the production process generates several residues including vinasse, which deserves attention because of its significant methane (CH4) emission during storage and transportation stages. Considering that CH4 emissions are dependent on the structure and abundance of microbial communities, we hypothesized that different vinasse transportation systems would harbor different microbial community composition, resulting in distinct CH4 patterns. To test this hypothesis, we used high-throughput 16S rRNA sequencing with real-time PCR to evaluate the composition and abundance of microorganisms in the two main systems of vinasse storage and transportation (i.e. open channels and tanks systems) in Brazil. Our results showed higher microbial diversity and CH4 emissions in channel system, especially in the uncoated section. Significant differences in microbial community structure, diversity, and abundance between the uncoated/coated open channel and tanks indicated a clear selection at taxonomic and functional levels, especially in relation to CH4 production. These responses included higher methanogens diversity in the uncoated section of the channel and are in agreement with the methanogen abundance determined by mcrA and mba genes copy number (1.5 × 107 and 4.3 × 1010) and subsequent positive correlation with CH4 emissions (R2 = 0.8). The most representative methanogen genus across the samples was Methanobrevibacter. The results observed herein shows that the use of the coating in the bottom of channels and tanks prevent the growth and development of a methanogen-related community. We concluded that the improvements in vinasse storage and transportation systems would significantly change the microbial community and reduce CH4 emissions, thereby making bioethanol a greener biofuel.

Quenching by sodium thiosulfate does not influence 16S rRNA gene sequencing profiles of reclaimed water from three sites in the Mid-Atlantic, United States.

A quenching agent is commonly added to chlorinated, reclaimed water during sample collection to prevent chlorine-mediated die-off of viable microbiota. However, the effect of quenching on downstream 16S rRNA-based bacterial community analyses is unclear. We conducted a side-by-side comparison of 16S rRNA sequencing data from reclaimed water samples quenched with sodium thiosulfate and non-quenched samples. Our data showed that 16 S rRNA processing and sequencing methods, and resulting bacterial profiles, were not negatively impacted by quenching.

Smokeless tobacco products harbor diverse bacterial microbiota that differ across products and brands.

Smokeless tobacco products contain numerous chemical compounds, including known human carcinogens. Other smokeless tobacco constituents, including bacteria, may also contribute to adverse health effects among smokeless tobacco users. However, there is a lack of data regarding the microbial constituents of smokeless tobacco. Our goal was to characterize the bacterial microbiota of different smokeless tobacco products and evaluate differences across product types and brands. DNA was extracted from 15 brands of smokeless tobacco products (including dry snuff, moist snuff, snus, and Swedish snus) and 6 handmade products (e.g., toombak) using an enzymatic and mechanical lysis approach. Bacterial community profiling was performed using PCR amplification of the V1-V2 hypervariable region of the 16S rRNA gene, followed by 454 pyrosequencing of the resulting amplicons and sequence analysis using the QIIME package. Total viable counts were also determined to estimate the number of viable bacteria present in each product. Average total viable counts ranged from 0 to 9.35 × 107 CFU g-1. Analysis of the 16S rRNA gene sequences revealed high bacterial diversity across the majority of products tested: dry snuff products where characterized by the highest diversity indices compared to other products. The most dominant bacterial phyla across all products were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Significant differences in both bacterial community composition and in silico predicted gene content were observed between smokeless tobacco product types and between brands of specific smokeless tobacco products. These data are useful in order to comprehensively address potential health risks associated with the use of smokeless tobacco products.

Oral microbiome dysbiosis among cigarette smokers and smokeless tobacco users compared to non-users.

Tobacco use significantly influences the oral microbiome. However, less is known about how different tobacco products specifically impact the oral microbiome over time. To address this knowledge gap, we characterized the oral microbiome of cigarette users, smokeless tobacco users, and non-users over 4 months (four time points). Buccal swab and saliva samples (n = 611) were collected from 85 participants. DNA was extracted from all samples and sequencing was carried out on an Illumina MiSeq, targeting the V3-V4 region of the 16S rRNA gene. Cigarette and smokeless tobacco users had more diverse oral bacterial communities, including a higher relative abundance of Firmicutes and a lower relative abundance of Proteobacteria, when compared to non-users. Non-users had a higher relative abundance of Actinomyces, Granulicatella, Haemophilus, Neisseria, Oribacterium, Prevotella, Pseudomonas, Rothia, and Veillonella in buccal swab samples, compared to tobacco users. While the most abundant bacterial genera were relatively constant over time, some species demonstrated significant shifts in relative abundance between the first and last time points. In addition, some opportunistic pathogens were detected among tobacco users including Neisseria subflava, Bulleidia moorei and Porphyromonas endodontalis. Overall, our results provide a more holistic understanding of the structure of oral bacterial communities in tobacco users compared to non-users.

The Bacterial Communities of Little Cigars and Cigarillos Are Dynamic Over Time and Varying Storage Conditions.

Despite their potential importance with regard to tobacco-related health outcomes, as well as their hypothesized role in the production of tobacco-specific N-nitrosamines, bacterial constituents of tobacco products lack characterization. Specifically, to our knowledge, there has been no comprehensive characterization of the effects of storage conditions on the bacterial communities associated with little cigars and cigarillos. To address this knowledge gap, we characterized the bacterial community composition of the tobacco and wrapper components of the following four products: Swisher Sweets Original; Swisher Sweets, Sweet Cherry; Cheyenne Cigars Full Flavor 100's; and Cheyenne Menthol Box. Each product was stored under three different conditions of temperature and relative humidity to mimic different user storage conditions: room (20°C 50% RH), refrigerator (5°C 18% RH) and pocket (25°C 30% RH). On days 0, 5, 9 and 14, subsamples were collected, the wrapper and tobacco were separated, and their total DNA was extracted separately and purified. Resulting DNA was then used in PCR assays targeting the V3 V4 region of the bacterial 16S rRNA gene, followed by sequencing using Illumina HiSeq 300bp PE. Resulting sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package, followed by analyses in R using the Phyloseq and Vegan packages. A single bacterial phylum, Firmicutes, dominated in the wrapper subsamples whereas the tobacco subsamples were dominated by Proteobacteria. Cheyenne Menthol Box (CMB) samples were characterized by significant differential abundances for 23 bacterial operational taxonomic units (OTUs) in tobacco subsamples and 27 OTUs in the wrapper subsamples between day 0 and day 14 under all conditions. OTUs from the genera Acinetobacter and Bacillus significantly increased in the CMB tobacco subsamples, and OTUs from Bacillus, Streptococcus, Lactobacillus, and Enterococcus significantly increased in the CMB wrapper subsamples over time. These initial results suggest that the bacterial communities of little cigars and cigarillos are dynamic over time and varying storage conditions.

Little cigars and cigarillos harbor diverse bacterial communities that differ between the tobacco and the wrapper.

Despite their potential importance with regard to infectious and chronic diseases among tobacco users, microbial constituents of tobacco products lack characterization. Specifically, to our knowledge, there are no data describing the bacterial diversity of little cigars or cigarillos. To address this knowledge gap, we tested four brands of little cigars and cigarillos. Tobacco and wrapper subsamples (n = 132) were separately subjected to DNA extraction, followed by PCR amplification of the V3V4 hypervariable region of the 16S rRNA gene, and sequencing using Illumina HiSeq. Sequences were analyzed using QIIME and Phyloseq implemented in R. We identified 2,681 operational taxonomic units across all products. Significant differences in alpha and beta diversity were observed between Swisher Sweets and Cheyenne products. Alpha and beta diversity was also significantly different between tobacco and wrapper subsamples within the same product. Beta diversity analyses of only tobacco samples identified no significant differences in the bacterial microbiota of different lots of the same products; however, the microbiota in the wrapper differed significantly across lots for all brands. Overall, Firmicutes were found to dominate in the wrapper, whereas Proteobacteria were most abundant in the tobacco. At the genus level, Bacillus and Lactobacillus dominated in the wrappers, and Staphylococcus and Pseudomonas dominated in the tobacco. Our findings suggest that the bacterial microbiota of little cigars and cigarillos is diverse and differs significantly between the tobacco and the wrapper, and across brands. Future work is necessary to evaluate the potential public health implications of these findings.

Gut microbiota-dependent modulation of innate immunity and lymph node remodeling affects cardiac allograft outcomes.

We hypothesized that the gut microbiota influences survival of murine cardiac allografts through modulation of immunity. Antibiotic pretreated mice received vascularized cardiac allografts and fecal microbiota transfer (FMT), along with tacrolimus immunosuppression. FMT source samples were from normal, pregnant (immune suppressed), or spontaneously colitic (inflammation) mice. Bifidobacterium pseudolongum (B. pseudolongum) in pregnant FMT recipients was associated with prolonged allograft survival and lower inflammation and fibrosis, while normal or colitic FMT resulted in inferior survival and worse histology. Transfer of B. pseudolongum alone resulted in reduced inflammation and fibrosis. Stimulation of DC and macrophage lines with B. pseudolongum induced the antiinflammatory cytokine IL-10 and homeostatic chemokine CCL19 but induced lesser amounts of the proinflammatory cytokines TNFα and IL-6. In contrast, LPS and Desulfovibrio desulfuricans (D. desulfuricans), more abundant in colitic FMT, induced a more inflammatory cytokine response. Analysis of mesenteric and peripheral lymph node structure showed that B. pseudolongum gavage resulted in a higher laminin α4/α5 ratio in the lymph node cortical ridge, indicative of a suppressive environment, while D. desulfuricans resulted in a lower laminin α4/α5 ratio, supportive of inflammation. Discrete gut bacterial species alter immunity and may predict graft outcomes through stimulation of myeloid cells and shifts in lymph node structure and permissiveness.

Temporal Variations in Cigarette Tobacco Bacterial Community Composition and Tobacco-Specific Nitrosamine Content Are Influenced by Brand and Storage Conditions.

Tobacco products, specifically cigarettes, are home to microbial ecosystems that may play an important role in the generation of carcinogenic tobacco-specific nitrosamines (TSNAs), as well as the onset of multiple adverse human health effects associated with the use of these products. Therefore, we conducted time-series experiments with five commercially available brands of cigarettes that were either commercially mentholated, custom-mentholated, user-mentholated, or non-mentholated. To mimic user storage conditions, the cigarettes were incubated for 14 days under three different temperatures and relative humidities (i.e., pocket, refrigerator, and room). Overall, 360 samples were collected over the course of 2 weeks and total DNA was extracted, PCR amplified for the V3V4 hypervariable region of the 16S rRNA gene and sequenced using Illumina MiSeq. A subset of samples (n = 32) was also analyzed via liquid chromatography with tandem mass spectrometry for two TSNAs: N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Comparative analyses of the five tobacco brands revealed bacterial communities dominated by Pseudomonas, Pantoea, and Bacillus, with Pseudomonas relatively stable in abundance regardless of storage condition. In addition, core bacterial operational taxonomic units (OTUs) were identified in all samples and included Bacillus pumilus, Rhizobium sp., Sphingomonas sp., unknown Enterobacteriaceae, Pantoea sp., Pseudomonas sp., Pseudomonas oryzihabitans, and P. putida. Additional OTUs were identified that significantly changed in relative abundance between day 0 and day 14, influenced by brand and storage condition. In addition, small but statistically significant increases in NNN levels were observed in user- and commercially mentholated brands between day 0 and day 14 at pocket conditions. These data suggest that manufacturing and user manipulations, such as mentholation and storage conditions, may directly impact the microbiome of cigarette tobacco as well as the levels of carcinogens.

Mentholation affects the cigarette microbiota by selecting for bacteria resistant to harsh environmental conditions and selecting against potential bacterial pathogens.

BACKGROUND: There is a paucity of data regarding the microbial constituents of tobacco products and their impacts on public health. Moreover, there has been no comparative characterization performed on the bacterial microbiota associated with the addition of menthol, an additive that has been used by tobacco manufacturers for nearly a century. To address this knowledge gap, we conducted bacterial community profiling on tobacco from user- and custom-mentholated/non-mentholated cigarette pairs, as well as a commercially-mentholated product. Total genomic DNA was extracted using a multi-step enzymatic and mechanical lysis protocol followed by PCR amplification of the V3-V4 hypervariable regions of the 16S rRNA gene from five cigarette products (18 cigarettes per product for a total of 90 samples): Camel Crush, user-mentholated Camel Crush, Camel Kings, custom-mentholated Camel Kings, and Newport Menthols. Sequencing was performed on the Illumina MiSeq platform and sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package. RESULTS: In all products, Pseudomonas was the most abundant genera and included Pseudomonas oryzihabitans and Pseudomonas putida, regardless of mentholation status. However, further comparative analysis of the five products revealed significant differences in the bacterial compositions across products. Bacterial community richness was higher among non-mentholated products compared to those that were mentholated, particularly those that were custom-mentholated. In addition, mentholation appeared to be correlated with a reduction in potential human bacterial pathogens and an increase in bacterial species resistant to harsh environmental conditions. CONCLUSIONS: Taken together, these data provide preliminary evidence that the mentholation of commercially available cigarettes can impact the bacterial community of these products.

Enrichment of specific bacterial and eukaryotic microbes in the rhizosphere of switchgrass (Panicum virgatum†L.) through root exudates.

Identification of microbes that actively utilize root exudates is essential to understand plant-microbe interactions. To identify active root exudate-utilizing microorganisms associated with switchgrass - a potential bioenergy crop - plants were labelled in situ with (13) CO2 , and 16S and 18S rRNA genes in the (13) C-labelled rhizosphere DNA were pyrosequenced. Multi-pulse labelling for 5 days produced detectable (13) C-DNA, which was well separated from unlabelled DNA. Methylibium from the order Burkholderiales were the most heavily labelled bacteria. Pythium, Auricularia and Galerina were the most heavily labelled eukaryotic microbes. We also identified a Glomus intraradices-like species; Glomus members are arbuscular mycorrhizal fungi that are able to colonize the switchgrass root. All of these heavily labelled microorganisms were also among the most abundant species in the rhizosphere. Species belonging to Methylibium and Pythium were the most heavily labelled and the most abundant bacteria and eukaryotes in the rhizosphere of switchgrass. Our results revealed that nearly all of the dominant rhizosphere bacterial and eukaryotic microbes were able to utilize root exudates. The enrichment of microbial species in the rhizosphere is selective and mostly due to root exudation, which functions as a nutrition source, promoting the growth of these microbes.