Microbial Community Metabolic Activity Research Articles

Critical steps in an environmental metaproteomics workflow.

Environmental metaproteomics is a rapidly advancing field that provides insights into the structure, dynamics, and metabolic activity of microbial communities. As the field is still maturing, it lacks consistent workflows, making it challenging for non-expert researchers to navigate. This review aims to introduce the workflow of environmental metaproteomics. It outlines the standard practices for sample collection, processing, and analysis, and offers strategies to overcome the unique challenges presented by common environmental matrices such as soil, freshwater, marine environments, biofilms, sludge, and symbionts. The review also highlights the bottlenecks in data analysis that are specific to metaproteomics samples and provides suggestions for researchers to obtain high-quality datasets. It includes recent benchmarking studies and descriptions of software packages specifically built for metaproteomics analysis. The article is written without assuming the reader's familiarity with single-organism proteomic workflows, making it accessible to those new to proteomics or mass spectrometry in general. This primer for environmental metaproteomics aims to improve accessibility to this exciting technology and empower researchers to tackle challenging and ambitious research questions. While it is primarily a resource for those new to the field, it should also be useful for established researchers looking to streamline or troubleshoot their metaproteomics experiments.

Selective trace elements significantly enhanced methane production in coal bed methane systems by stimulating microbial activity.

Trace elements are associated with the microbial degradation of organic matter and methanogenesis, as enzymes in metabolic pathways often employ trace elements as essential cofactors. However, only a few studies investigated the effects of trace elements on the metabolic activity of microbial communities associated with biogenic coalbed methane production. We aimed to determine the effects of strategically selected trace elements on structure and function of active bacterial and methanogenic communities to stimulate methane production in subsurface coalbeds. Microcosms were established with produced water and coal from coalbed methane wells located in the Powder River Basin, Wyoming, USA. In initial pilot experiments with eight different trace elements, individual amendments of Co, Cu, and Mo lead to significantly higher methane production. Transcript levels of mcrA, the key marker gene for methanogenesis, positively correlated with increased methane production. Phylogenetic analysis of the mcrA cDNA library demonstrated compositional shifts of the active methanogenic community and increase of their diversity, particularly of hydrogenotrophic methanogens. High-throughput sequencing of cDNA obtained from 16S rRNA demonstrated active and abundant bacterial groups in response to trace element amendments. Active Acetobacterium members increased in response to Co, Cu, and Mo additions. The findings of this study yield new insights into the importance of essential trace elements on the metabolic activity of microbial communities involved in subsurface coalbed methane and provide a better understanding of how microbial community composition is shaped by trace elements.IMPORTANCEMicrobial life in the deep subsurface of coal beds is limited by nutrient replenishment. While coal bed microbial communities are surrounded by carbon sources, we hypothesized that other nutrients such as trace elements needed as cofactors for enzymes are missing. Amendment of selected trace elements resulted in compositional shifts of the active methanogenic and bacterial communities and correlated with higher transcript levels of mcrA. The findings of this study yield new insights to not only identify possible limitations of microbes by replenishment of trace elements within their specific hydrological placement but also into the importance of essential trace elements for the metabolic activity of microbial communities involved in subsurface coalbed methane production and provides a better understanding of how microbial community composition is shaped by trace elements. Furthermore, this finding might help to revive already spent coal bed methane well systems with the ultimate goal to stimulate methane production.

Statistical prediction of microbial metabolic traits from genomes.

The metabolic activity of microbial communities is central to their role in biogeochemical cycles, human health, and biotechnology. Despite the abundance of sequencing data characterizing these consortia, it remains a serious challenge to predict microbial metabolic traits from sequencing data alone. Here we culture 96 bacterial isolates individually and assay their ability to grow on 10 distinct compounds as a sole carbon source. Using these data as well as two existing datasets, we show that statistical approaches can accurately predict bacterial carbon utilization traits from genomes. First, we show that classifiers trained on gene content can accurately predict bacterial carbon utilization phenotypes by encoding phylogenetic information. These models substantially outperform predictions made by constraint-based metabolic models automatically constructed from genomes. This result solidifies our current knowledge about the strong connection between phylogeny and metabolic traits. However, phylogeny-based predictions fail to predict traits for taxa that are phylogenetically distant from any strains in the training set. To overcome this we train improved models on gene presence/absence to predict carbon utilization traits from gene content. We show that models that predict carbon utilization traits from gene presence/absence can generalize to taxa that are phylogenetically distant from the training set either by exploiting biochemical information for feature selection or by having sufficiently large datasets. In the latter case, we provide evidence that a statistical approach can identify putatively mechanistic genes involved in metabolic traits. Our study demonstrates the potential power for predicting microbial phenotypes from genotypes using statistical approaches.

Season shapes the functional diversity of microbial carbon metabolism in mangrove soils of Hainan Island, China

Carbon storage in mangroves is considered a natural solution to mitigate climate change as an essential coastal blue carbon ecosystem service for climate change. The magnitude of carbon storage in soils depends on the carbon metabolic activities of the microbial community, and these dynamics are subject to the influence of climate conditions, including seasonal changes. Despite mangroves being one of the world's highest in soil carbon density and carbon sequestration rates, our understanding of this aspect remains limited. Here, we investigated the seasonal changes in the carbon metabolic profile of microbial communities in mangrove soils along the seashore of the whole Hainan Island (with the highest diversity of mangrove species in China). There was a clear season dependence in the metabolic activity and functional diversity of mangrove soil microbial community on Hainan Island, showing the trend of the rainy season > the dry season. The carbon metabolic activity in the rainy season is three times higher than in the dry season. The season plays a critical role in shaping the carbon functional diversity of microbial communities, which that by changing biotic interactions and soil properties, particularly soil TN, NO2−–N, plant richness and mean DBH. This study provides important insights into comprehend the carbon metabolic functional diversity of microbial communities in mangrove soils and provides basic data support for predicting the blue carbon feedback of mangrove ecosystems to global climate change.

Changes in interactions over ecological time scales influence single-cell growth dynamics in a metabolically coupled marine microbial community

Microbial communities thrive in almost all habitats on earth. Within these communities, cells interact through the release and uptake of metabolites. These interactions can have synergistic or antagonistic effects on individual community members. The collective metabolic activity of microbial communities leads to changes in their local environment. As the environment changes over time, the nature of the interactions between cells can change. We currently lack understanding of how such dynamic feedbacks affect the growth dynamics of individual microbes and of the community as a whole. Here we study how interactions mediated by the exchange of metabolites through the environment change over time within a simple marine microbial community. We used a microfluidic-based approach that allows us to disentangle the effect cells have on their environment from how they respond to their environment. We found that the interactions between two species—a degrader of chitin and a cross-feeder that consumes metabolic by-products—changes dynamically over time as cells modify their environment. Cells initially interact positively and then start to compete at later stages of growth. Our results demonstrate that interactions between microorganisms are not static and depend on the state of the environment, emphasizing the importance of disentangling how modifications of the environment affects species interactions. This experimental approach can shed new light on how interspecies interactions scale up to community level processes in natural environments.

НАНОБИОТЕХНОЛОГИЧЕСКИЙ ПОДХОД К ВЫЯВЛЕНИЮ И ИССЛЕДОВАНИЮ МЕТАБОЛИЧЕСКИ АКТИВНЫХ ПРИРОДНЫХ МИКРОБНЫХ СООБЩЕСТВ КАК НОВЫЙ ВИД ЭКОЛОГИЧЕСКОГО МОНИТОРИНГА

An innovative nanobiotechnological express method for the detection of metabolically active microorganisms in natural water samples based on the ability of microbial cells to generate metal nanoparticles during the reduction reaction of cations introduced into the test samples as precursor (Detection of Biogenic Nanoparticles Growth/Generation ― DBNG method) is proposed. Metallic nanoparticles generated de novo form a new solid crystalline phase and can be detected with high accuracy by various physical methods. The DBNG method was tested on model psychrotolerant bacteria and proved to be effective in studying the metabolic activity of microbial communities in samples from various cold Arctic and Antarctic ecosystems. This approach can be used in ecology for monitoring changes in the ecological state of natural ecosystems.

Potential energetic return on investment positively correlated with overall soil microbial activity

Microbial communities are a critical component of the soil carbon (C) cycle as they are responsible for the decomposition of both organic inputs from plants and of soil organic C. However, there is still no consensus about how to explicitly represent their role in terrestrial C cycling. The objective of the study was to determine how the molecular and energetic properties of readily available organic matter affect the metabolic activity of the resident microbial communities in soils. This was achieved by cross-amending six soils, taken from woodland and grassland sites along an urban pressure gradient, with organic matter extracted from the same six soils and measuring heat dissipated due to the increase in microbial metabolic activity. The energetic properties of the organic matter were used to estimate a potential energetic return on investment (ROI) that microbial communities could obtain from the transformation of the organic matter. Specifically, the ROI was calculated as the ratio between the total net energy available (ΔE) and the weighted average standard state Gibbs energies of oxidation half reactions of organic C (ΔG°Cox). ΔE was measured as the heat of combustion using bomb calorimetry. ΔG°Cox was estimated using the average nominal oxidation state of C (NOSC) of the molecular species in the organic matter. The overall metabolic activity of microbial communities was positively related to the potential energetic return on investment but no significant relationship was found with the molecular diversity of organic matter. The temporal differences in metabolism across soils indicate that bacterial communities do not exploit the potential energetic return on investment in the same way: the suburban grassland communities responded more rapidly and the suburban woodland communities more slowly to the organic matter additions than the other communities. The urban gradient did not affect the properties of the molecular or energetic properties of the organic matter nor the response of the microbial communities to the organic matter additions. However, the organic matter from the grassland soils caused soils to dissipate 36.4% more heat than organic matter from the woodland soils. The metabolic response was also more rapid after the addition of grassland organic matter: the time taken for half the heat to be dissipated was 6.4 h after the addition of grassland organic matter and 6.1 h after the addition of woodland organic matter. Overall, our results suggest that microbial communities preferentially use organic matter with a high potential energetic return on investment, i.e. organic molecules that do not require high cost associated with catalysis whilst yielding a high net energetic benefit.

Taxonomic diversity and metabolic activity of microbial communities in rivers and estuarine waters of Southern Baikal in summer

We analyzed the diversity of bacterial communities, metabolic activity, pH, and anthropogenic impact in the water of the estuaries and estuarine waters of the rivers in Southern Baikal in 2018 with different chemical composition. The chemical composition of waters and values of total microbial count (TMC), organotrophic bacteria (OB), and bacterial production (BP) depended on the geographical position, length of the rivers, and their water quality. In rivers with water acidification, we did not detect an effect of pH on TMC, abundance of OB, or total amount of mesophilic aerobic and facultative anaerobic microorganisms (PC). In rivers with a discharge of untreated domestic sewage, there was an increase in the content of dissolved organic carbon (DOC), concentration of nutrients, number of fecal and mesophilic bacteria, and high metabolic activity of microorganisms. The value of bacterioplankton production reached 0.26 to 12.41 per day−1; TMC – 0.155 ± 0.002 to 3.474 ± 0.044 million cells/mL. Using the massive parallel sequencing of fragments encoding the V2–V3 region of the 16S rRNA gene, we studied the diversity of bacteria and estimated the influence of chemical composition on the diversity of communities. As in other freshwater ecosystems and the pelagic zone of Lake Baikal, the members of freshwater Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, and Verrucomicrobia predominated in the estuaries and estuarine waters of the rivers. The obtained data on the number and diversity of hygiene indicator bacteria suggested fecal pollution of the rivers of Southern Baikal.

Functional Profiling Reveals Altered Metabolic Activity in Divers' Oral Microbiota During Commercial Heliox Saturation Diving.

Background: The extreme environment in saturation diving affects all life forms, including the bacteria that reside on human skin and mucosa. The oral cavity alone is home to hundreds of different bacteria. In this study, we examined the metabolic activity of oral bacteria from healthy males during commercial heliox saturation diving. We focused on environmentally induced changes that might affect the divers’ health and fitness.Methods: We performed pathway abundance analysis using PICRUSt2, a bioinformatics software package that uses marker gene data to compute the metabolic activity of microbial communities. The analysis is based on 16S rRNA metagenomic data generated from the oral microbiota of 23 male divers before, during, and after 4weeks of commercial heliox saturation diving. Environmentally induced changes in bacterial metabolism were computed from differences in predicted pathway abundances at baseline before, versus during, and immediately after saturation diving.Results and Conclusion: The analysis predicted transient changes that were primarily associated with the survival and growth of bacteria in oxygenated environments. There was a relative increase in the abundance of aerobic metabolic pathways and a concomitant decrease in anaerobic metabolic pathways, primarily comprising of energy metabolism, oxidative stress responses, and adenosylcobalamin biosynthesis. Adenosylcobalamin is a bioactive form of vitamin B12 (vitB12), and a reduction in vitB12 biosynthesis may hypothetically affect the divers’ physiology. While host effects of oral bacterial vitamin metabolism are uncertain, this is a finding that concurs with the existing recommendations for vitB12 supplements as part of the divers’ diet, whether to boost antioxidant defenses in bacteria or their host or to improve oxygen transport during saturation diving.

Response of microbial community composition and function to emergent plant rhizosphere of a constructed wetland in northern China

To understand the mechanisms by which emergent plants influence the microbial communities in wetland sediments, we analyzed the response of microbial composition, abundance, metabolic activity, and metabolic genes to three emergent plant species [Cyperus alternifolius L. (Cyp), Typha angustifolia L. (Typ), and Phragmites australis (Cav.) Trin. ex Steud. (Phr)], and determined the important physicochemical properties of the soils and root exudates in Zhaoniu River Constructed Wetland in northern China. We found the composition of both the microbial communities and the metabolic genes differed between rhizosphere and non-rhizosphere sediments. The rhizosphere microbial abundance in Phr in summer was significantly higher than in the other two plants and in the non-rhizosphere. The rates of microbial respiration and ammonia oxidation in rhizosphere sediments were significantly higher than those in non-rhizosphere sediments. Our statistical analyses showed that the total organic carbon (TOC) concentration in root exudates and the oxidation–reduction potential (ORP) significantly influenced the differences in the composition, abundance, and metabolic activities of the microbial community and metabolic genes between rhizosphere and non-rhizosphere sediments, suggesting that the secretion of root exudates and oxygen by emergent plants was the main mechanism affecting the composition and function of the microbial community. As the result of the high levels of TOC and the ORP, the rhizosphere sediments showed relatively high abundances of the genes related to biodegradation of xenobiotic compounds. By contrast, non-rhizosphere sediments showed relatively high abundances of genes related to carbon fixation and sulfate reduction, possibly due to the low levels of TOC and ORP. Plants with an enhanced ability to secrete root exudates and oxygen, such as Phr, should be preferred in constructed wetlands to increase the metabolic activity of sediment microbial communities. This study extends our understanding of the mechanisms by which the plant rhizosphere affects the ecological functions of wetlands.

On the Possibility of Aerobic Methane Production by Pelagic Microbial Communities of the Laptev Sea

—The taxonomic diversity and metabolic activity of microbial communities in the Laptev Sea water column above and outside the methane seep field were studied. The concentrations of dissolved methane in the water column at both stations were comparable until the depth of the pycnocline (25 m). At this depth, local methane maxima were recorded, with the highest concentration (116 nM CH4) found at the station outside the methane seep field. Results of the 16S rRNA gene sequencing and measurements of the rates of hydrogenotrophic methanogenesis indicated the absence of methanogenesis caused by the methanogenic archaea in the pycnocline and in other horizons of the water column. The 16S rRNA-based analysis of microbial phylogenetic diversity, as well as radiotracer analysis of the rates of primary production (PP), dark CO2 assimilation (DCA), and methane oxidation (MO), indicated the functioning of a diverse community of pelagic microorganisms capable of transforming a wide range of organic compounds under oligotrophic conditions of the Arctic basin. Hydrochemical prerequisites and possible microbial agents of aerobic methane production via demethylation of methylphosphonate and decomposition of dimethylsulfoniopropionate using dissolved organic matter synthesized in the PP, DCA, and MO processes are discussed.

Acute effects of PAH contamination on microbial community of different forest soils

Polycyclic aromatic hydrocarbons (PAHs) are hazardous organic compounds with mutagenic, genotoxic and carcinogenic properties. Although PAHs in soil can cause toxicity to microorganisms, the microbial community is able to degrade these compounds. For this reason, it is important to study acute and short-term effects of PAH contamination on soil microbial community, also to shed light on its possible exploitation in soil restoration.The effects of acute PAH contamination on the structure and metabolic activity of microbial communities in three forest (beech, holm oak, black pine) soils were studied. The soils were spiked with phenanthrene, pyrene or benzo[a]pyrene and incubated in experimental mesocosms, under controlled conditions. Enzymatic activities (laccase, total peroxidase and hydrolase), as well as microbial biomass and community structure (through phospholipid fatty acid and ergosterol analyses), were evaluated in the three soil systems 4 days after contamination and compared to no-spiked soils. In soil under holm oak, there was a stimulation of Gram+ bacteria after contamination with all the 3 PAHs, whereas in soil under pine, pyrene and phenanthrene additions mainly stimulated fungi and actinomycetes.

Effects of fortification of Daqu with various yeasts on microbial community structure and flavor metabolism.

Fortification of Daqu with isolated functional strains can influence the metabolic activity of the microbial community, and thus alter the flavors of the Baijiu produced with Daqu as a fermentation starter. Here, we analyzed the microbial community dynamics of, and volatile compound production by, Daqu fortified respectively with three high-yield ethyl caproate-producing yeasts (Saccharomyces cerevisiae Y7#09, Hyphopichia burtonii F12507 and Clavispora lusitaniae YX3307), or with a mixture of these three strains, during the fermentation of Baijiu. The microbial community was investigated using Illumina HiSeq technology. Three bacterial genera (Bacillus, Lactobacillus and Enterobacter) and four fungal genera (Pichia, Clavispora, Saccharomyces and Saccharomycopsis) were dominant in the microbial communities. The volatile compounds were examined by gas chromatography-mass spectrometry. Forty-one flavor compounds were detected in all samples, including seven alcohols, 26 esters and four aldehydes. In particular, an increase in ethyl caproate content was associated with Daqu fortified with S. cerevisiae Y7#09, C. lusitaniae YX3307, or the mixed inoculum. The ester content of these fortified Daqu was higher in the later stage of the fermentation than that in unfortified Daqu, or in Daqu fortified with H. burtonii F12507. Our results show that fortification of Daqu with aroma-producing yeast strains influenced the microbial community composition in the Daqu and affected its metabolic activity. Overall, this study reveals the features of fortified Daqu microbial communities in different phases and improves understanding of the relationships between aroma-producing yeast and the metabolic activity of microbial communities in Baijiu production.

Biological Activity of Sod-Podzolic Soils in a Long-Term Experiment Involving Various Agrotechnical Methods

The effects from the multiyear use of grain–fallow–plow crop rotation schemes comprising legumes, permanent cultivation of winter rye and barley, and bare fallow regime on the ecological and physiological state of microbial communities in a sod-podzolic heavy loamy soil were examined in the course of a long-term, stationary experiment. Biological activity parameters of soil microbiocoenoses, including the potential intensity of CO2 emission, microbial biomass gross respiration rate, metabolic coefficient, and nitrogen fixation, were assessed. It is shown that the application of organic fertilizers (manure) in the crop rotation scheme with 28.6% legumes ensures the best humus state of the soil and a significant increase in the total nitrogen content. It is established that the share of microbial biomass (Cmic) in the total organic matter of the sod-podzolic heavy loamy soil is 1.50–3.24%. The application of mineral fertilizers results in a slight decrease in the microbial biomass carbon content and increase in the labile organic carbon content. The metabolic activity of microbial communities decreases in all long-term experimental scenarios regardless of the land use techniques. The permanent intensive cultivation of crops, either with or without crop rotation, disturbs the microbiocoenosis stability in soils of the Cis-Ural region.

Soil microbial community characteristics in degraded karst tiankeng invaded by Eupatorium adenophorum.

After invading the grand negative landform karst degraded tiankeng, Eupatorium adenophorum will affect the plant diversity, community succession and natural ecological restoration of degraded tiankeng by changing soil microbial community. Taking the degraded tiankeng "Bajiaxiantang" as the research area, the soil around E. adenophorum roots in different habitats was selected to explore the microbial community characteristics of E. adenophorum roots using the Biolog-ECO micro-plate method. The results showed that the invasion degree of E. adenophorum was in order of pit slope shrub-grassland > pit bottom grassland > pit slope forest > pit head shrub-grassland. The characteristics of E. adenophorum varied among different habitats due to habitat heterogeneity. The change of microbial community metabolic activity, dominance index, richness index and evenness index of E. adenophorum roots was closely related with the invasion degree, with significant diffe-rence between inside and outside of the tiankeng. Amino acids and polymers were the main carbon sources utilized by soil microorganism. The α diversity of herbaceous plants in different habitats were correlated with that of soil microbial community. The diversity of soil microbial community around E. adenophorum roots increased significantly with the increases of invasion level, which could form a microenvironment conducive to invasion and colonization. In the future, the degraded tiankeng would eventually form the climax community of underground forests with community succession and natural ecological restoration, which might result in the disappearance of E. adenophorum in the degraded tiankeng.

Influence of exogenous ginsenosides on new forest soil microbial communities

Ginsenosides are the main active ingredient and allelochemicals of Panax ginseng, and they play an important role in ginseng growth and in ecological adaptation. To study the influence of ginsenosides on soil microbial communities, the method of given exogenous total ginsenosides of different concentrations were used to study the influence of ginsenosides on new forest soil microbial community, evaluate the change of metabolic activity of microbial community and investigate the ecological effect of ginsenosides on soil microbial community. Results showed that, exogenous total ginsenosides promoted metabolic activity of microbial community in new forest soil at different concentrations compared with the control after 10 d and 40 d treatment. After 10 d,except for the Evenness index, all of the other indices indicated that the functional diversity of the soil microbial community in the new forest firstly increased then decreased with increase of the total ginsenosides concentration. The Substrate richness for 0.01 g•L⁻¹ soil treatment was significantly different from that of the control. After 20 d, 30 d and 40 d, except for the Evenness index, all of the other indices indicated that the functional diversity of the soil microbial community in the new forest increased with total ginsenosides. These results suggested that ginsenosids can change soil microbial community and microbial metabolic activity, which alter soil microbial ecology and accordingly affect the growth of ginseng with accumulation of ginsenosides in the soil.

Functional activity of soil microbial communities in post-fire pine stands of Tolyatti, Samara oblast

The state of microbial communities in gray-humus soils (Eutric Fluvic Arenosols (Ochric)) of pine stands in the city of Tolyatti after forest fires of 2010 is analyzed. It is shown that fires exert negative effects on the structure and metabolic activity of microbial communities in the postpyrogenic soils. The content of the carbon of microbial biomass and the intensity of microbial respiration in the upper organic horizons of the post-fire plots decrease by 6.5 and 3.4 times, respectively, in comparison with those in the soils of background plots. However, the fire has not affected the studied microbiological parameters of the soils at the depths of more than 10 cm. The maximum content of the carbon of microbial biomass carbon and the maximum intensity of microbial respiration have been found in the subsurface AY2 and АС horizons two–three years the fire. An increase in the microbial metabolic quotient (the ratio of soil respiration to microbial biomass) attests to the disturbance of the ecophysiological state of soil microbial communities after the pyrogenic impact.

Impact of glyphosate-resistant corn, glyphosate applications and tillage on soil nutrient ratios, exoenzyme activities and nutrient acquisition ratios.

We report results of the last two years of a 7 year field experiment designed to test the null hypothesis: applications of glyphosate on glyphosate-resistant (GR) and non-resistant (non-GR) corn (Zea mays L.) under conventional tillage and no-till would have no effect on soil exoenzymes and microbial activity. Bulk soil (BS) and rhizosphere soil (RS) macronutrient ratios were not affected by either GR or non-GR corn, or glyphosate applications. Differences observed between exoenzyme activities were associated with tillage rather than glyphosate applications. In 2013, nutrient acquisition ratios for bulk and rhizosphere soils indicated P limitations, but sufficient assimilable N. In 2014, P limitations were observed for bulk and rhizosphere soils, in contrast to balanced C and N acquisition ratios in rhizosphere soils. Stoichiometric relationships indicated few differences between glyphosate and non-glyphosate treatments. Negative correlations between C:P and N:P nutrient ratios and nutrient acquisition ratios underscored the inverse relation between soil nutrient status and microbial community exoenzyme activities. Inconsistent relationships between microbial community metabolic activity and exoenzyme activity indicated an ephemeral effect of glyphosate on BS exoenzyme activity. Except for ephemeral effects, glyphosate applications appeared not to affect the function of the BS and RS exoenzymes under conventional tillage or no-till. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Fractionation of biogas plant sludge material improves metaproteomic characterization to investigate metabolic activity of microbial communities.

With the development of high resolving mass spectrometers, metaproteomics evolved as a powerful tool to elucidate metabolic activity of microbial communities derived from full-scale biogas plants. Due to the vast complexity of these microbiomes, application of suitable fractionation methods are indispensable, but often turn out to be time and cost intense, depending on the method used for protein separation. In this study, centrifugal fractionation has been applied for fractionation of two biogas sludge samples to analyze proteins extracted from (i) crude fibers, (ii) suspended microorganisms, and (iii) secreted proteins in the supernatant using a gel-based approach followed by LC-MS/MS identification. This fast and easy method turned out to be beneficial to both the quality of SDS-PAGE and the identification of peptides and proteins compared to untreated samples. Additionally, a high functional metabolic pathway coverage was achieved by combining protein hits found exclusively in distinct fractions. Sample preparation using centrifugal fractionation influenced significantly the number and the types of proteins identified in the microbial metaproteomes. Thereby, comparing results from different proteomic or genomic studies, the impact of sample preparation should be considered. All MS data have been deposited in the ProteomeXchange with identifier PXD001508 (http://proteomecentral.proteomexchange.org/dataset/PXD001508).

Metaproteomics of complex microbial communities in biogas plants.

Production of biogas from agricultural biomass or organic wastes is an important source of renewable energy. Although thousands of biogas plants (BGPs) are operating in Germany, there is still a significant potential to improve yields, e.g. from fibrous substrates. In addition, process stability should be optimized. Besides evaluating technical measures, improving our understanding of microbial communities involved into the biogas process is considered as key issue to achieve both goals. Microscopic and genetic approaches to analyse community composition provide valuable experimental data, but fail to detect presence of enzymes and overall metabolic activity of microbial communities. Therefore, metaproteomics can significantly contribute to elucidate critical steps in the conversion of biomass to methane as it delivers combined functional and phylogenetic data. Although metaproteomics analyses are challenged by sample impurities, sample complexity and redundant protein identification, and are still limited by the availability of genome sequences, recent studies have shown promising results. In the following, the workflow and potential pitfalls for metaproteomics of samples from full-scale BGP are discussed. In addition, the value of metaproteomics to contribute to the further advancement of microbial ecology is evaluated. Finally, synergistic effects expected when metaproteomics is combined with advanced imaging techniques, metagenomics, metatranscriptomics and metabolomics are addressed.