Tae Kwon Lee

Ecological Patterns of nifH Genes in Four Terrestrial Climatic Zones Explored with Targeted Metagenomics Using FrameBot, a New Informatics Tool

ABSTRACT Biological nitrogen fixation is an important component of sustainable soil fertility and a key component of the nitrogen cycle. We used targeted metagenomics to study the nitrogen fixation-capable terrestrial bacterial community by targeting the gene for nitrogenase reductase (nifH). We obtained 1.1 million nifH 454 amplicon sequences from 222 soil samples collected from 4 National Ecological Observatory Network (NEON) sites in Alaska, Hawaii, Utah, and Florida. To accurately detect and correct frameshifts caused by indel sequencing errors, we developed FrameBot, a tool for frameshift correction and nearest-neighbor classification, and compared its accuracy to that of two other rapid frameshift correction tools. We found FrameBot was, in general, more accurate as long as a reference protein sequence with 80% or greater identity to a query was available, as was the case for virtually all nifH reads for the 4 NEON sites. Frameshifts were present in 12.7% of the reads. Those nifH sequences related to the Proteobacteria phylum were most abundant, followed by those for Cyanobacteria in the Alaska and Utah sites. Predominant genera with nifH sequences similar to reads included Azospirillum, Bradyrhizobium, and Rhizobium, the latter two without obvious plant hosts at the sites. Surprisingly, 80% of the sequences had greater than 95% amino acid identity to known nifH gene sequences. These samples were grouped by site and correlated with soil environmental factors, especially drainage, light intensity, mean annual temperature, and mean annual precipitation. FrameBot was tested successfully on three ecofunctional genes but should be applicable to any. IMPORTANCE High-throughput phylogenetic analysis of microbial communities using rRNA-targeted sequencing is now commonplace; however, such data often allow little inference with respect to either the presence or the diversity of genes involved in most important ecological processes. To study the gene pool for these processes, it is more straightforward to assess the genes directly responsible for the ecological function (ecofunctional genes). However, analyzing these genes involves technical challenges beyond those seen for rRNA. In particular, frameshift errors cause garbled downstream protein translations. Our FrameBot tool described here both corrects frameshift errors in query reads and determines their closest matching protein sequences in a set of reference sequences. We validated this new tool with sequences from defined communities and demonstrated the tool’s utility on nifH gene fragments sequenced from soils in well-characterized and major terrestrial ecosystem types. High-throughput phylogenetic analysis of microbial communities using rRNA-targeted sequencing is now commonplace; however, such data often allow little inference with respect to either the presence or the diversity of genes involved in most important ecological processes. To study the gene pool for these processes, it is more straightforward to assess the genes directly responsible for the ecological function (ecofunctional genes). However, analyzing these genes involves technical challenges beyond those seen for rRNA. In particular, frameshift errors cause garbled downstream protein translations. Our FrameBot tool described here both corrects frameshift errors in query reads and determines their closest matching protein sequences in a set of reference sequences. We validated this new tool with sequences from defined communities and demonstrated the tool’s utility on nifH gene fragments sequenced from soils in well-characterized and major terrestrial ecosystem types.

Treatment of alcohol distillery wastewater using a bacteroidetes-dominant thermophilic microbial fuel cell

Simultaneous electricity generation and distillery wastewater (DWW) treatment were accomplished using a thermophilic microbial fuel cell (MFC). The results suggest that thermophilic MFCs, which require less energy for cooling the DWW, can achieve high efficiency for electricity generation and also reduce sulfate along with oxidizing complex organic substrates. The generated current density (2.3 A/m(2)) and power density (up to 1.0 W/m(2)) were higher than previous wastewater-treating MFCs. The significance of the high Coulombic efficiency (CE; up to 89%) indicated that electrical current was the most significant electron sink in thermophilic MFCs. Bacterial diversity based on pyrosequencing of the 16S rRNA gene revealed that known Deferribacteres and Firmicutes members were not dominant in the thermophilic MFC fed with DWW; instead, uncharacterized Bacteroidetes thermophiles were up to 52% of the total reads in the anode biofilm. Despite the complexity of the DWW, one single bacterial sequence (OTU D1) close to an uncultured Bacteriodetes bacterium became predominant, up to almost 40% of total reads. The proliferation of the D1 species was concurrent with high electricity generation and high Coulombic efficiency.

The functional and taxonomic richness of wastewater treatment plant microbial communities are associated with each other and with ambient nitrogen and carbon availability.

The number of functional traits of a wastewater treatment plant (WWTP) microbial community (i.e. functional richness) is thought to be an important determinant of its overall functional performance, but the ecological factors that determine functional richness remain unclear. The number of taxa within a community (i.e. taxonomic richness) is one ecological factor that might be important. Communities that contain more taxa are more likely to have more functional traits, and a positive association is therefore expected between functional and taxonomic richness. Empirical tests for this positive association among WWTP communities, however, are lacking. We address this knowledge gap by measuring the functional and taxonomic richness of 10 independent WWTP communities. We demonstrate that functional and taxonomic richness are positively associated with each other. We further demonstrate that functional and taxonomic richness are negatively associated with the effluent NH4 -N and BOD5 concentrations. This led us to hypothesize that correlated variation in functional and taxonomic richness is likely related to variation in ambient nitrogen and carbon availability. We finally demonstrate that this hypothesis is consistent with the functional and taxonomic attributes of the WWTP communities. Together, our results improve our basic understanding of the ecology and functioning of WWTP communities.

Increased nitrous oxide accumulation by bioelectrochemical denitrification under autotrophic conditions: kinetics and expression of denitrification pathway genes.

Under autotrophic conditions, we investigated the effects of different current densities on bioelectrochemical denitrification (BED). In this study, nitrate consumption and nitrous oxide (N2O) production, microbial diversity and population dynamics, and denitrification pathway gene expressions were explored in continuous flow BED reactors at different current densities (0.2, 1, 5, 10 and 20 A/m(2)). We found that, under the autotrophic conditions, N2O accumulation was increased with increase in current density. The maximum rate of denitrification was 1.65 NO3(-)-N (g/NCCm(3).h), and approximately 70% of the reduced N was accumulated as N2O. After each current density was applied, pyrosequencing of the expressed 16S rRNA genes amplified from the cathodic biofilms revealed that that 16 genera were active and in common at all currents, and that eight of those showed a statistically significant correlation with particular current densities. The relative expression of napA and narG was highest, whereas nosZ was low relative to its level in the inoculum suggesting that this could have contributed the high N2O accumulation. Kinetic analysis of nitrate reduction and N2O accumulation followed Michaelis-Menten kinetics. The Vmax for nitrate consumption and N2O accumulation were similar, however the Km values determined as A/m(2) were not. This study provides better understanding of the community and kinetics of a current-fed, autotrophic, cathodic biofilm for evaluating its potential for scale-up and for N2O recovery.

Association of Biodiversity with the Rates of Micropollutant Biotransformations among Full-Scale Wastewater Treatment Plant Communities

ABSTRACT Biodiversities can differ substantially among different wastewater treatment plant (WWTP) communities. Whether differences in biodiversity translate into differences in the provision of particular ecosystem services, however, is under active debate. Theoretical considerations predict that WWTP communities with more biodiversity are more likely to contain strains that have positive effects on the rates of particular ecosystem functions, thus resulting in positive associations between those two variables. However, if WWTP communities were sufficiently biodiverse to nearly saturate the set of possible positive effects, then positive associations would not occur between biodiversity and the rates of particular ecosystem functions. To test these expectations, we measured the taxonomic biodiversity, functional biodiversity, and rates of 10 different micropollutant biotransformations for 10 full-scale WWTP communities. We have demonstrated that biodiversity is positively associated with the rates of specific, but not all, micropollutant biotransformations. Thus, one cannot assume whether or how biodiversity will associate with the rate of any particular micropollutant biotransformation. We have further demonstrated that the strongest positive association is between biodiversity and the collective rate of multiple micropollutant biotransformations. Thus, more biodiversity is likely required to maximize the collective rates of multiple micropollutant biotransformations than is required to maximize the rate of any individual micropollutant biotransformation. We finally provide evidence that the positive associations are stronger for rare micropollutant biotransformations than for common micropollutant biotransformations. Together, our results are consistent with the hypothesis that differences in biodiversity can indeed translate into differences in the provision of particular ecosystem services by full-scale WWTP communities.

Metagenomic Analysis of Chicken Gut Microbiota for Improving Metabolism and Health of Chickens — A Review

Chicken is a major food source for humans, hence it is important to understand the mechanisms involved in nutrient absorption in chicken. In the gastrointestinal tract (GIT), the microbiota plays a central role in enhancing nutrient absorption and strengthening the immune system, thereby affecting both growth and health of chicken. There is little information on the diversity and functions of chicken GIT microbiota, its impact on the host, and the interactions between the microbiota and host. Here, we review the recent metagenomic strategies to analyze the chicken GIT microbiota composition and its functions related to improving metabolism and health. We summarize methodology of metagenomics in order to obtain bacterial taxonomy and functional inferences of the GIT microbiota and suggest a set of indicator genes for monitoring and manipulating the microbiota to promote host health in future.

Molecular approaches for the detection and monitoring of microbial communities in bioaerosols: A review.

Bioaerosols significantly affect atmospheric processes while they undergo long-range vertical and horizontal transport and influence atmospheric chemistry and physics and climate change. Accumulating evidence suggests that exposure to bioaerosols may cause adverse health effects, including severe disease. Studies of bioaerosols have primarily focused on their chemical composition and largely neglected their biological composition and the negative effects of biological composition on ecosystems and human health. Here, current molecular methods for the identification, quantification, and distribution of bioaerosol agents are reviewed. Modern developments in environmental microbiology technology would be favorable in elucidation of microbial temporal and spatial distribution in the atmosphere at high resolution. In addition, these provide additional supports for growing evidence that microbial diversity or composition in the bioaerosol is an indispensable environmental aspect linking with public health.

Novel Biphenyl-Oxidizing Bacteria and Dioxygenase Genes from a Korean Tidal Mudflat

ABSTRACT Gene-targeted FLX titanium pyrosequencing integrated with stable isotope probing (SIP) using [13C]biphenyl substrate revealed that tidal mudflat sediments harbor novel aromatic ring hydroxylating dioxygenases (ARHD). More than 80% of the detected ARHD genes comprise four clades (0.5 distance) with 49 to 70% amino acid identity to sequences in public databases. The 16S rRNA sequences enriched in the 13C fraction were from the Betaproteobacteria, bacilli (primarily Paenibacillus-like), and unclassified phyla.

A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates

The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific micropollutants. This work is an important step towards developing tools to predict biotransformation rates in WWTPs based on taxonomic composition.

Improved detection of microbial risk of releasing genetically modified bacteria in soil by using massive sequencing and antibiotic resistance selection

High-throughput 16S rRNA gene-targeted pyrosequencing was used with commonly used risk assessment techniques to evaluate the potential microbial risk in soil after inoculating genetically modified (GM) Corynebacterium glutamicum. To verify the risk, reference experiments were conducted in parallel using well-defined and frequently used GM Escherichia coli and wild-type strains. The viable cell count showed that the number of GM bacteria in the soil was reduced to below the detection limit within 10 days, while the molecular indicator for GM plasmids was detected throughout the experiment by using quantitative real-time polymerase chain reactions. Subsequent pyrosequencing showed an insignificant influence of the GM bacteria and/or their GM plasmids on the structure of the soil bacterial community this was similar to non-GM wild-type strains. However, pyrosequencing combined with kanamycin-resistant bacteria selection uncovered a potential risk of GM bacteria on the soil bacterial community and pathogens. The results of the improved methodology showed that the microbial risk attributable to GM C. glutamicum was relatively lower than that attributable to the reference GM E. coli.