Due to the uncultivated status of the majority of microorganisms in nature, little is known about their genetic properties, biochemical functions, and metabolic characteristics. Although sequence determination of the microbial community 'genome' is now possible with high throughput sequencing technology, the complexity and magnitude of most microbial communities make meaningful data acquisition and interpretation difficult. Therefore, we are sequencing groundwater microbial communities with manageable diversity and complexity (-10-400 phylotypes) at the U.S Department of Energy's Environmental Remediation Science Program (ERSP)-Field Research Center (FRC), Oak Ridge, TN. The microbial community has been sequenced from a groundwater sample (FW106) contaminated with very high levels of nitrate, uranium and other heavy metals and pH -3.7. Consistent with trends expected in stressed ecosystems, the metagenome reveals a community of low species and strain diversity dominated by a single Frateuria-like y-proteobacteria with other y-and J3-proteobacteria present at low proportions. Metabolic reconstruction reveals specific adaptations to the geochemical conditions of FWI06 including genes encoding metal resistance (czcABC, czcD, cadA, merA, arsB), denitrification, and solvent resistance (1,2-dichloroethene, acetone, butanol). In addition to the presence of these specific genes, certain resistance genes also appear to be overrepresented in the metagenome including genes from nitrate/nitrite transport (narK) and metal translocation (czcABC, czcD, cadA), likely due to a combination of gene duplication and lateral gene transfer. A screen for positive selection shows most of these genes to be under strong negative selection, suggesting that in the short term at least, the overabundance of these transporters provide a positive fitness benefit to the cell by increasing the rate of ion transport. SNP analysis revealed a low level of polymorphism with the overwhelming majority of S

Understanding the mechanisms controlling community diversity, functions, succession, and biogeography is a central, but poorly understood, topic in ecology, particularly in microbial ecology. Although stochastic processes are believed to play nonnegligible roles in shaping community structure, their importance relative to deterministic processes is hotly debated. The importance of ecological stochasticity in shaping microbial community structure is far less appreciated. Some of the main reasons for such heavy debates are the difficulty in defining stochasticity and the diverse methods used for delineating stochasticity. Here, we provide a critical review and synthesis of data from the most recent studies on stochastic community assembly in microbial ecology. We then describe both stochastic and deterministic components embedded in various ecological processes, including selection, dispersal, diversification, and drift. We also describe different approaches for inferring stochasticity from observational diversity patterns and highlight experimental approaches for delineating ecological stochasticity in microbial communities. In addition, we highlight research challenges, gaps, and future directions for microbial community assembly research.

Hexavalent chromium [Cr(VI)] is a common environmental pollutant. However, little is known about the genetic basis of microbial evolution under Cr(VI) stress and the influence of the prior evolution histories on the subsequent evolution under Cr(VI) stress. In this study, Desulfovibrio vulgaris Hildenborough (DvH), a model sulfate-reducing bacterium, was experimentally evolved for 600 generations. By evolving the replicate populations of three genetically diverse DvH clones, including ancestor (AN, without prior experimental evolution history), non-stress-evolved EC3-10, and salt stress-evolved ES9-11, the contributions of adaptation, chance, and pre-existing genetic divergence to the evolution under Cr(VI) stress were able to be dissected. Significantly decreased lag phases under Cr(VI) stress were observed in most evolved populations, while increased Cr(VI) reduction rates were primarily observed in populations evolved from EC3-10 and ES9-11. The pre-existing genetic divergence in the starting clones showed strong influences on the changes in lag phases, growth rates, and Cr(VI) reduction rates. Additionally, the genomic mutation spectra in populations evolved from different starting clones were significantly different. A total of 14 newly mutated genes obtained mutations in at least two evolved populations, suggesting their importance in Cr(VI) adaptation. An in-frame deletion mutation of one of these genes, the chromate transporter gene DVU0426, demonstrated that it played an important role in Cr(VI) tolerance. Overall, our study identified potential key functional genes for Cr(VI) tolerance and demonstrated the important role of pre-existing genetic divergence in evolution under Cr(VI) stress conditions.

Background

Wastewater treatment plants (WWTPs) are one of the largest biotechnology applications in the world and are of critical importance to modern urban societies. An accurate evaluation of the microbial dark matter (MDM, microorganisms whose genomes remain uncharacterized) proportions in WWTPs is of great value, while there is no such research yet. This study conducted a global meta-analysis of MDM in WWTPs with 317,542 prokaryotic genomes from the Genome Taxonomy Database and proposed a "wanted list" for priority targets in further investigations of activated sludge.

Results

Compared with the Earth Microbiome Project data, WWTPs had relatively lower genome-sequenced proportions of prokaryotes than other ecosystems, such as the animal related environments. Analysis showed that the median proportions of the genome-sequenced cells and taxa (100% identity and 100% coverage in 16S rRNA gene region) in WWTPs reached 56.3% and 34.5% for activated sludge, 48.6% and 28.5% for aerobic biofilm, and 48.3% and 28.5% for anaerobic digestion sludge, respectively. This result meant MDM had high proportions in WWTPs. Besides, all of the samples were occupied by a few predominant taxa, and the majority of the sequenced genomes were from pure cultures. The global-scale "wanted list" for activated sludge contained four phyla that have few representatives and 71 operational taxonomic units with the majority of them having no genome or isolate yet. Finally, several genome mining methods were verified to successfully recover genomes from activated sludge such as hybrid assembly of the second- and third-generation sequencing.

Conclusions

This work elucidated the proportion of MDM in WWTPs, defined the "wanted list" of activated sludge for future investigations, and certified potential genome recovery methods. The proposed methodology of this study can be applied to other ecosystems and improve understanding of ecosystem structure across diverse habitats. Video Abstract.

Many environmental sites (e.g., groundwater, seawater, contaminated sites) contain low microbial biomass, which can make it difficult to effectively analyze these samples using genomic techniques requiring large amounts of DNA. Functional gene microarrays, for example, require microgram quantities of DNA. Whole community genome amplification (WCGA) amplifies the entire community in a sample using random priming. As such, the use of WCGA would be beneficial for samples containing low concentrations of DNA or that would be difficult to resample. Two DNA polymerases are known to catalyze this reaction: phi29 and Bst. Phi29 has been previously shown to be effective for amplifying microbial community DNA with a low amplification bias. This study evaluated the use of Bst for WCGA. Initial experiments compared the amplification of Desulfovibrio vulgaris using phi29 and Bst. An aliquot of DNA (10 ng) was amplified using random heptamers containing two additional 5-nitroindole residues on the 5' end and a phosphorothioate linkage on the 3' end. Amplified DNA (1 mu g) and the same amount of unamplified DNA were labeled with Cy5-dUTP and Cy3-dUTP, respectively. The unamplified DNA was pooled and then co-hybridized with the amplified DNA to a D. vulgaris microarray at 45 oC for 10 h. All hybridizations were done in triplicate. The signal-to-noise ratio (SNR) was calculated for the unamplified sample and all genes with SNR <3 in any of the triplicates was removed and the remaining genes were used for analysis. The average amplified to unamplified signal intensity was 1.02 (+0.005) and 1.05 (+0.005) for Bst and phi29, respectively. The representational bias (DjTotal) values were similar for both the Bst and phi29 amplifications (0.181 and 0.157, respectively). The percentage of genes whose ratios were > 1.5, 2, 3, and 4 were 12.56, 2.79, 0.15, and 0.03percent, respectively, for Bst and 12.22, 3.08, 0.23, and 0.03percent, respectively, for phi29. These results suggest that Bst would be a viable substitute for phi29. Further experiments are underway to compare the amplification of other pure culture DNA and examine microbial community DNA amplification.

The origin of cellular complexity characterizing eukaryotic cells remains a central unresolved issue in the study of diversification of cellular life on Earth. The isolation by Imachi et al.¹ of a member of the Asgard archaea² - a contemporary relative of organisms thought to have given rise to eukaryotic cells about 2 billion years ago - now promises new insight. The complete genome sequence of the isolated Lokiarchaeum strain confirms that the eukaryotic signature proteins (ESPs) previously identified in the Lokiarchaeota³ and other Asgard archaea² are indeed encoded by these archaeal genomes and do not represent contamination from eukaryotes. These ESPs encode homologs of eukaryotic actins, small GTPases and the ESCRT complex proteins and are required for the functioning of complex eukaryotic cells. The new, slowly growing, anaerobic laboratory strain allows a first direct look at these organisms and provides key insights into the morphology and metabolism of an Asgard archaeal organism. The work has provided valuable information for other laboratories that aim to isolate and characterize related organisms from other environments.

Understanding the community assembly mechanisms controlling biodiversity patterns is a central issue in ecology. Although it is generally accepted that both deterministic and stochastic processes play important roles in community assembly, quantifying their relative importance is challenging. Here we propose a general mathematical framework to quantify ecological stochasticity under different situations in which deterministic factors drive the communities more similar or dissimilar than null expectation. An index, normalized stochasticity ratio (NST), was developed with 50% as the boundary point between more deterministic (<50%) and more stochastic (>50%) assembly. NST was tested with simulated communities by considering abiotic filtering, competition, environmental noise, and spatial scales. All tested approaches showed limited performance at large spatial scales or under very high environmental noise. However, in all of the other simulated scenarios, NST showed high accuracy (0.90 to 1.00) and precision (0.91 to 0.99), with averages of 0.37 higher accuracy (0.1 to 0.7) and 0.33 higher precision (0.0 to 1.8) than previous approaches. NST was also applied to estimate stochasticity in the succession of a groundwater microbial community in response to organic carbon (vegetable oil) injection. Our results showed that community assembly was shifted from more deterministic (NST = 21%) to more stochastic (NST = 70%) right after organic carbon input. As the vegetable oil was consumed, the community gradually returned to be more deterministic (NST = 27%). In addition, our results demonstrated that null model algorithms and community similarity metrics had strong effects on quantifying ecological stochasticity.

Increased amount of nano-silver will be released into domestic and industrial waste streams due to its extensive application. However, great controversy still exists on the effects of silver nanoparticle (Ag-NP) on biological wastewater treatment processes and a toxicology model has not been built yet. Four sequencing batch reactors with activated sludge has been run for over three months with different silver species at a concentration of 1mg Ag/L in influent. Both freshly prepared Ag-NPs and aged Ag-NPs were tested with released silver ion as control. Results in this study showed that Ag-NPs, especially freshly prepared Ag-NPs, can help to maintain or even increase the diversity of microbial community in activated sludge and the biomass concentration even under long-term treatment. It indicates that the hormesis model need to be considered for the toxicology of Ag-NPs.