Linwei Wu

Responses of the functional structure of soil microbial community to livestock grazing in the Tibetan alpine grassland

Microbes play key roles in various biogeochemical processes, including carbon (C) and nitrogen (N) cycling. However, changes of microbial community at the functional gene level by livestock grazing, which is a global land-use activity, remain unclear. Here we use a functional gene array, GeoChip 4.0, to examine the effects of free livestock grazing on the microbial community at an experimental site of Tibet, a region known to be very sensitive to anthropogenic perturbation and global warming. Our results showed that grazing changed microbial community functional structure, in addition to aboveground vegetation and soil geochemical properties. Further statistical tests showed that microbial community functional structures were closely correlated with environmental variables, and variations in microbial community functional structures were mainly controlled by aboveground vegetation, soil C/N ratio, and NH4 (+) -N. In-depth examination of N cycling genes showed that abundances of N mineralization and nitrification genes were increased at grazed sites, but denitrification and N-reduction genes were decreased, suggesting that functional potentials of relevant bioprocesses were changed. Meanwhile, abundances of genes involved in methane cycling, C fixation, and degradation were decreased, which might be caused by vegetation removal and hence decrease in litter accumulation at grazed sites. In contrast, abundances of virulence, stress, and antibiotics resistance genes were increased because of the presence of livestock. In conclusion, these results indicated that soil microbial community functional structure was very sensitive to the impact of livestock grazing and revealed microbial functional potentials in regulating soil N and C cycling, supporting the necessity to include microbial components in evaluating the consequence of land-use and/or climate changes.

The microbial gene diversity along an elevation gradient of the Tibetan grassland.

Tibet is one of the most threatened regions by climate warming, thus understanding how its microbial communities function may be of high importance for predicting microbial responses to climate changes. Here, we report a study to profile soil microbial structural genes, which infers functional roles of microbial communities, along four sites/elevations of a Tibetan mountainous grassland, aiming to explore the potential microbial responses to climate changes via a strategy of space-for-time substitution. Using a microarray-based metagenomics tool named GeoChip 4.0, we showed that microbial communities were distinct for most but not all of the sites. Substantial variations were apparent in stress, N and C-cycling genes, but they were in line with the functional roles of these genes. Cold shock genes were more abundant at higher elevations. Also, gdh converting ammonium into urea was more abundant at higher elevations, whereas ureC converting urea into ammonium was less abundant, which was consistent with soil ammonium contents. Significant correlations were observed between N-cycling genes (ureC, gdh and amoA) and nitrous oxide flux, suggesting that they contributed to community metabolism. Lastly, we found by Canonical correspondence analysis, Mantel tests and the similarity tests that soil pH, temperature, NH4+–N and vegetation diversity accounted for the majority (81.4%) of microbial community variations, suggesting that these four attributes were major factors affecting soil microbial communities. On the basis of these observations, we predict that climate changes in the Tibetan grasslands are very likely to change soil microbial community functional structure, with particular impacts on microbial N-cycling genes and consequently microbe-mediated soil N dynamics.

Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

Illumina’s MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1–3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility.

Zonal Soil Type Determines Soil Microbial Responses to Maize Cropping and Fertilization

Microbial communities are essential drivers of soil functional processes such as nitrification and heterotrophic respiration. Although there is initial evidence revealing the importance of soil type in shaping microbial communities, there has been no in-depth, comprehensive survey to robustly establish it as a major determinant of microbial community composition, functional gene structure, or ecosystem functioning. We examined bacterial and fungal community structures using Illumina sequencing, microbial functional genes using GeoChip, microbial biomass using phospholipid fatty acid analysis, as well as functional processes of soil nitrification potential and CO2 efflux. We demonstrated the critical role of soil type in determining microbial responses to land use changes at the continental level. Our findings underscore the inherent difficulty in generalizing ecosystem responses across landscapes and suggest that assessments of community feedback must take soil types into consideration. ABSTRACT Soil types heavily influence ecological dynamics. It remains controversial to what extent soil types shape microbial responses to land management changes, largely due to lack of in-depth comparison across various soil types. Here, we collected samples from three major zonal soil types spanning from cold temperate to subtropical climate zones. We examined bacterial and fungal community structures, as well as microbial functional genes. Different soil types had distinct microbial biomass levels and community compositions. Five years of maize cropping (growing corn or maize) changed the bacterial community composition of the Ultisol soil type and the fungal composition of the Mollisol soil type but had little effect on the microbial composition of the Inceptisol soil type. Meanwhile, 5 years of fertilization resulted in soil acidification. Microbial compositions of the Mollisol and Ultisol, but not the Inceptisol, were changed and correlated (P < 0.05) with soil pH. These results demonstrated the critical role of soil type in determining microbial responses to land management changes. We also found that soil nitrification potentials correlated with the total abundance of nitrifiers and that soil heterotrophic respiration correlated with the total abundance of carbon degradation genes, suggesting that changes in microbial community structure had altered ecosystem processes. IMPORTANCE Microbial communities are essential drivers of soil functional processes such as nitrification and heterotrophic respiration. Although there is initial evidence revealing the importance of soil type in shaping microbial communities, there has been no in-depth, comprehensive survey to robustly establish it as a major determinant of microbial community composition, functional gene structure, or ecosystem functioning. We examined bacterial and fungal community structures using Illumina sequencing, microbial functional genes using GeoChip, microbial biomass using phospholipid fatty acid analysis, as well as functional processes of soil nitrification potential and CO2 efflux. We demonstrated the critical role of soil type in determining microbial responses to land use changes at the continental level. Our findings underscore the inherent difficulty in generalizing ecosystem responses across landscapes and suggest that assessments of community feedback must take soil types into consideration. Author Video: An author video summary of this article is available.

Bacteriophage–prokaryote dynamics and interaction within anaerobic digestion processes across time and space

BackgroundBacteriophage–prokaryote dynamics and interaction are believed to be important in governing microbiome composition and ecosystem functions, yet our limited knowledge of the spatial and temporal variation in phage and prokaryotic community compositions precludes accurate assessment of their roles and impacts. Anaerobic digesters are ideal model systems to examine phage–host interaction, owing to easy access, stable operation, nutrient-rich environment, and consequently enormous numbers of phages and prokaryotic cells.ResultsEquipped with high-throughput, cutting-edge environmental genomics techniques, we examined phage and prokaryotic community composition of four anaerobic digesters in full-scale wastewater treatment plants across China. Despite the relatively stable process performance in biogas production, phage and prokaryotic groups fluctuated monthly over a year of study period, showing significant correlations between those two groups at the α- and β-diversity levels. Strikingly, phages explained 40.6% of total variations of the prokaryotic community composition, much higher than the explanatory power by abiotic factors (14.5%). Consequently, phages were significantly (P < 0.010) linked to parameters related to process performance including biogas production and volatile solid concentrations. Association network analyses showed phage–prokaryote pairs were shallowly conserved since they were detected only within small viral clades.ConclusionsThose results collectively demonstrate phages as a major biotic factor in controlling prokaryotic composition and process performance. Therefore, phages may play a larger role in shaping prokaryotic community dynamics and process performance of anaerobic digesters than currently appreciated.

Microbial Functional Gene Diversity Predicts Groundwater Contamination and Ecosystem Functioning

ABSTRACT Contamination from anthropogenic activities has significantly impacted Earth’s biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly (P < 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning. IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology. Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology.

Evaluating the lingering effect of livestock grazing on functional potentials of microbial communities in Tibetan grassland soils

Background and aimsLivestock grazing is a widely practiced land-use regime that can impose lingering effects on global biogeochemical cycles. However, elucidating the mechanisms of related eco-processes, which are largely mediated by the microbial community, remains challenging.MethodsHere, we collected soil samples from two Tibetan grassland sites subjected to grazing in winter followed by a 3-month recovery. We then evaluated functional potentials of microbial communities via a metagenomic tool known as GeoChip 4.0.ResultsSignificant alterations were detected in post-grazing grassland soils, and further analysis showed that plant diversity was the best indicator of alterations in functional potentials. Relative abundances of labile C degradation genes decreased at the 3400-m site, but those of recalcitrant C degradation genes increased, which could be explained by the higher soil recalcitrant C input owing to their being substantially more forbs species at this site. Nitrification genes decreased at both sites, probably owing to increased soil moisture conducive to oxygen-limiting conditions. Relative abundance of denitrification genes increased at the 3200-m site, concomitant with increased N2O emissions.ConclusionsThese results demonstrated that functional gene compositions of the microbial community were altered in post-grazing grassland soils, and linked to soil biogeochemical processes.

GeoChip as a metagenomics tool to analyze the microbial gene diversity along an elevation gradient

To examine microbial responses to climate change, we used a microarray-based metagenomics tool named GeoChip 4.0 to profile soil microbial functional genes along four sites/elevations of a Tibetan mountainous grassland. We found that microbial communities differed among four elevations. Soil pH, temperature, NH4+–N and vegetation diversity were four major attributes affecting soil microbial communities. Here we describe in details the experiment design, the data normalization process, soil and vegetation analyses associated with the study published on ISME Journal in 2014 [1], whose raw data have been uploaded to Gene Expression Omnibus (accession number GSM1185243).

Microbial functional trait of rRNA operon copy numbers increases with organic levels in anaerobic digesters

The ecological concept of the r-K life history strategy is widely applied in macro-ecology to characterize functional traits of taxa. However, its adoption in microbial communities is limited, owing to the lack of a measureable, convenient functional trait for classification. In this study, we performed an experiment of stepwise organic amendments in triplicate anaerobic digesters. We found that high resource availability significantly favored microbial r-strategists such as Bacillus spp. Incremental resource availability heightened average rRNA operon copy number of microbial community, resulting in a strong, positive correlation (r>0.74, P<0.008). This study quantifies how resource availability manipulations influence microbial community composition and supports the idea that rRNA operon copy number is an ecologically meaningful trait which reflects resource availability.