元蛋白质组学在微生物功能研究中的应用

后基因组时代,仅依靠基因组方法来研究原位微生物群落的功能已远远不够,在这种背景下元蛋白质组学研究逐渐兴起。应用元蛋白质组学技术可大规模研究原位微生物群落的蛋白质表达,分析生态系统中微生物的功能,寻找新的功能基因和代谢通路,为微生物群体的基因和功能多样性研究提供数据。同时,还可鉴定与微生物功能相关的蛋白质,这些蛋白质未来可以作为生物标记物为环境可持续发展铺路。综述了元蛋白质组学的发展概况及其在微生物功能研究中的重大作用,强调了元蛋白质组学方法在分析新功能基因及其相关基因,揭示微生物多样性与微生物群体功能之间的关系等方面起到的作用,并对其应用前景进行了展望。     

Metaproteomics: studying functional gene expression in microbial ecosystems

The recent availability of extensive metagenomic sequences from various environmental microbial communities has extended the postgenomic era to the field of environmental microbiology. Although still restricted to a small number of studies, metaproteomic investigations have revealed interesting aspects of functional gene expression within microbial habitats that contain limited microbial diversity. These studies highlight the potential of proteomics for the study of microbial consortia. However, the application of proteomic investigations to complex microbial assemblages such as seawater and soil still presents considerable challenges. Nonetheless, metaproteomics will enhance the understanding of the microbial world and link microbial community composition to function.     

Effects of glucose overloading on microbial community structure and biogas production in a laboratory-scale anaerobic digester

This study characterizes the response of the microbial communities of a laboratory-scale mesophilic biogas process, fed with a synthetic substrate based on cellulose and egg albumin, to single pulses of glucose overloading (15 or 25 times the daily feed based on VS). The microbial biomass and community structure were determined from analyses of membrane phospholipids. The ratio between phospholipid fatty acids (PLFAs; eubacteria and eucaryotes) and di-ethers (PLEL; archaea) suggested that methanogens constituted 4-8% of the microbial biomass. The glucose addition resulted in transient increases in the total biomass of eubacteria while there were only small changes in community structure. The total gas production rate increased, while the relative methane content of the biogas and the alkalinity decreased. However, the biomass of methanogens was not affected by the glucose addition. The results show that the microbial communities of biogas processes can respond quickly to changes in the feeding rate. The glucose overload resulted in a transient general stimulation of degradation rates and almost a doubling of eubacterial biomass, although the biomass increase corresponded to only 7% of the glucose C added.     

宏蛋白质组学技术在废水生物处理工艺研究领域中的应用

随着后基因组时代的到来,宏蛋白质组学逐渐兴起并在生命科学基础领域和临床医药领域成功运用,宏蛋白质组学技术现已成为各研究领域炙手可热的方法之一.宏蛋白质组学技术在废水生物处理研究领域中的应用刚起步,但已展示其强大功能.本文主要综述近年来国内外宏蛋白质组学在废水生物处理研究领域的研究进展,回顾及总结了宏蛋白质组学的研究策略及应用,如鉴定功能性蛋白质/酶、揭示污染物的微生物降解途径、推断废水生物处理系统的关键代谢途径、及探讨不同污泥微生物群落微生态变化等.
     

Proteomics in environmental and technical microbiology

Nowadays, the field of proteomics encompasses various techniques for the analysis of the entirety of proteins in biological samples. Not only 2D electrophoresis as the primary method, but also MS‐based workflows and bioinformatic tools are being increasingly applied. In particular, research in microbiology was significantly influenced by proteomics during the last few decades. Hence, this review presents results of proteomic studies carried out in areas, such as fundamental microbiological research and biotechnology. In addition, the emerging field of metaproteomics is addressed because high‐throughput genome sequencing and high‐performance MS facilitate the access to such complex samples from microbial communities as found in sludge from wastewater treatment plants and biogas plants. Both current technical limitations and new concepts in this growing and important area are discussed. Moreover, it was convincingly shown that future prospective applications of proteomics in technical and environmental microbiology might also be closely connected with other Omics approaches as well as bioinformatics for systems biology studies.     

Characterization of a biogas-producing microbial community by short-read next generation DNA sequencing

ABSTRACT: BACKGROUND: Renewable energy production is currently a major issue worldwide. Biogas is a promising renewable energy carrier as the technology of its production combines the elimination of organic waste with the formation of a versatile energy carrier, methane. In consequence of the complexity of the microbial communities and metabolic pathways involved the biotechnology of the microbiological process leading to biogas production is poorly understood. Metagenomic approaches are suitable means of addressing related questions. In the present work a novel high-throughput technique was tested for its benefits in resolving the functional and taxonomical complexity of such microbial consortia. RESULTS: It was demonstrated that the extremely parallel SOLiDTM short-read DNA sequencing platform is capable of providing sufficient useful information to decipher the systematic and functional contexts within a biogas-producing community. Although this technology has not been employed to address such problems previously, the data obtained compare well with those from similar high-throughput approaches such as 454-pyrosequencing GS FLX or Titanium. The predominant microbes contributing to the decomposition of organic matter include members of the Eubacteria, class Clostridia, order Clostridiales, family Clostridiaceae. Bacteria belonging in other systematic groups contribute to the diversity of the microbial consortium. Archaea comprise a remarkably small minority in this community, given their crucial role in biogas production. Among the Archaea, the predominant order is the Methanomicrobiales and the most abundant species is Methanoculleus marisnigri. The Methanomicrobiales are hydrogenotrophic methanogens. Besides corroborating earlier findings on the significance of the contribution of the Clostridia to organic substrate decomposition, the results demonstrate the importance of the metabolism of hydrogen within the biogas producing microbial community. CONCLUSIONS: Both microbiological diversity and the regulatory role of the hydrogen metabolism appear to be the driving forces optimizing biogas-producing microbial communities. The findings may allow a rational design of these communities to promote greater efficacy in large-scale practical systems. The composition of an optimal biogas-producing consortium can be determined through the use of this approach, and this systematic methodology allows the design of the optimal microbial community structure for any biogas plant. In this way, metagenomic studies can contribute to significant progress in the efficacy and economic improvement of biogas production.     

Metaproteomics to understand how microbiota function: The crystal ball predicts a promising future

In the medical, environmental, and biotechnological fields, microbial communities have attracted much attention due to their roles and numerous possible applications. The study of these communities is challenging due to their diversity and complexity. Innovative methods are needed to identify the taxonomic components of individual microbiota, their changes over time, and to determine how microoorganisms interact and function. Metaproteomics is based on the identification and quantification of proteins, and can potentially provide this full picture. Due to the wide molecular panorama and functional insights it provides, metaproteomics is gaining momentum in microbiome and holobiont research. Its full potential should be unleashed in the coming years with progress in speed and cost of analyses. In this exploratory crystal ball exercise, I discuss the technical and conceptual advances in metaproteomics that I expect to drive innovative research over the next few years in microbiology. I also debate the concepts of ‘microbial dark matter’ and ‘Metaproteomics-Assembled Proteomes (MAPs)’ and present some long-term prospects for metaproteomics in clinical diagnostics and personalized medicine, environmental monitoring, agriculture, and biotechnology.     

Metaproteome analysis to determine the metabolically active part of a thermophilic microbial community producing biogas from agricultural biomass

Complex consortia of microorganisms are responsible for biogas production. A lot of information about the taxonomic structure and enzymatic potential of such communities has been collected by a variety of gene-based approaches, yet little is known about which of all the assumable metabolic pathways are active throughout the process of biogas formation. To tackle this problem, we established a protocol for the metaproteomic analysis of samples taken from biogas reactors fed with agricultural biomass. In contrast to previous studies where an anaerobic digester was fed with synthetic wastewater, the complex matrix in this study required the extraction of proteins with liquid phenol and the application of paper bridge loading for 2-dimensional gel electrophoresis. Proteins were subjected to nanoHPLC (high-performance liquid chromatography) coupled to tandem mass spectrometry for characterization. Several housekeeping proteins as well as methanogenesis-related enzymes were identified by a MASCOT search and de novo sequencing, which proved the feasibility of our approach. The establishment of such an approach is the basis for further metaproteomic studies of biogas-producing communities. In particular, the apparent status of metabolic activities within the communities can be monitored. The knowledge collected from such experiments could lead to further improvements of biogas production.     

基于宏组学方法认识微生物群落及其功能

进入后基因组学时代,测序技术飞速发展,测序成本明显下降,形成了涵盖宏基因组学、宏转录组学和宏蛋白质组学的宏组学技术,推动了对微生物群落的多样性、结构及潜在基因功能方面的深入研究。最近随着整合的宏组学技术的提出及应用,全面系统分析微生物群落动态变化及其代谢功能已成为可能,这将成为微生物生态学研究的新趋势。本文综述了宏组学在研究海洋湖泊、深海热泉、人体肠道、牛瘤胃生境、森林土壤与堆肥生境等环境中微生物群落的结构和功能方面的最新进展与成功应用案例。     

Signal Processing for Metagenomics: Extracting Information from the Soup

Traditionally, studies in microbial genomics have focused on single-genomes from cultured species, thereby limiting their focus to the small percentage of species that can be cultured outside their natural environment. Fortunately, recent advances in high-throughput sequencing and computational analyses have ushered in the new field of metagenomics, which aims to decode the genomes of microbes from natural communities without the need for cultivation. Although metagenomic studies have shed a great deal of insight into bacterial diversity and coding capacity, several computational challenges remain due to the massive size and complexity of metagenomic sequence data. Current tools and techniques are reviewed in this paper which address challenges in 1) genomic fragment annotation, 2) phylogenetic reconstruction, 3) functional classification of samples, and 4) interpreting complementary metaproteomics and metametabolomics data. Also surveyed are important applications of metagenomic studies, including microbial forensics and the roles of microbial communities in shaping human health and soil ecology.     

Reprint of Organic waste conversion through anaerobic digestion: A critical insight into the metabolic pathways and microbial interactions

Anaerobic digestion is a promising method for energy recovery through conversion of organic waste to biogas and other industrial valuables. However, to tap the full potential of anaerobic digestion, deciphering the microbial metabolic pathway activities and their underlying bioenergetics is required. In addition, the behavior of organisms in consortia along with the analytical abilities to kinetically measure their metabolic interactions will allow rational optimization of the process. This review aims to explore the metabolic bottlenecks of the microbial communities adopting latest advances of profiling and ¹³C tracer-based analysis using state of the art analytical platforms (GC, GC-MS, LC-MS, NMR). The review summarizes the phases of anaerobic digestion, the role of microbial communities, key process parameters of significance, syntrophic microbial interactions and the bottlenecks that are critical for optimal bioenergetics and enhanced production of valuables. Considerations into the designing of efficient synthetic microbial communities as well as the latest advances in capturing their metabolic cross talk will be highlighted. The review further explores how the presence of additives and inhibiting factors affect the metabolic pathways. The critical insight into the reaction mechanism covered in this review may be helpful to optimize and upgrade the anaerobic digestion system.     

Organic waste conversion through anaerobic digestion: A critical insight into the metabolic pathways and microbial interactions

Anaerobic digestion is a promising method for energy recovery through conversion of organic waste to biogas and other industrial valuables. However, to tap the full potential of anaerobic digestion, deciphering the microbial metabolic pathway activities and their underlying bioenergetics is required. In addition, the behavior of organisms in consortia along with the analytical abilities to kinetically measure their metabolic interactions will allow rational optimization of the process. This review aims to explore the metabolic bottlenecks of the microbial communities adopting latest advances of profiling and ¹³C tracer-based analysis using state of the art analytical platforms (GC, GC-MS, LC-MS, NMR). The review summarizes the phases of anaerobic digestion, the role of microbial communities, key process parameters of significance, syntrophic microbial interactions and the bottlenecks that are critical for optimal bioenergetics and enhanced production of valuables. Considerations into the designing of efficient synthetic microbial communities as well as the latest advances in capturing their metabolic cross talk will be highlighted. The review further explores how the presence of additives and inhibiting factors affect the metabolic pathways. The critical insight into the reaction mechanism covered in this review may be helpful to optimize and upgrade the anaerobic digestion system.     

Biogas production from cellulose-containing substrates: A review

Anaerobic microbial conversion of organic substrates to various biofuels is one of the alternative energy sources attracting the greatest attention of scientists. The advantages of biogas production over other technologies are the ability of methanogenic communities to degrade a broad range of substrates and concomitant benefits: neutralization of organic waste, reduction of greenhouse gas emission, and fertilizer production. Cellulose-containing materials are good substrate, but their full-scale utilization encounters a number of problems, including improvement of the quality and amount of biogas produced and maintenance of the stability and high efficiency of microbial communities. We review data on microorganisms that form methanogenic cellulolytic communities, enzyme complexes of anaerobes essential for cellulose fiber degradation, and feedstock pretreatment, as biodegradation is hindered in the presence of lignin. Methods for improving biogas production by optimization of microbial growth conditions are considered on the examples of biogas formation from various types of plant and paper materials: office paper and cardboard.     

Different response of bacteria,archaea and fungi to process parameters in nine full-scale anaerobic digesters

Biogas production is a biotechnological process realized by complex bacterial, archaeal and likely fungal communities. Their composition was assessed in nine full-scale biogas plants with distinctly differing feedstock input and process parameters. This study investigated the actually active microbial community members by using a comprehensive sequencing approach based on ribosomal 16S and 28S rRNA fragments. The prevailing taxonomical units of each respective community were subsequently linked to process parameters. Ribosomal rRNA of bacteria, archaea and fungi, respectively, showed different compositions with respect to process parameters and supplied feedstocks: (i) bacterial communities were affected by the key factors temperature and ammonium concentration; (ii) composition of archaea was mainly related to process temperature; and (iii) relative abundance of fungi was linked to feedstocks supplied to the digesters. Anaerobic digesters with a high methane yield showed remarkably similar bacterial communities regarding identified taxonomic families. Although archaeal communities differed strongly on genus level from each other, the respective digesters still showed high methane yields. Functional redundancy of the archaeal communities may explain this effect. 28S rRNA sequences of fungi in all nine full-scale anaerobic digesters were primarily classified as facultative anaerobic Ascomycota and Basidiomycota. Since the presence of ribosomal 28S rRNA indicates that fungi may be active in the biogas digesters, further research should be carried out to examine to which extent they are important players in anaerobic digestion processes.     

Metaproteomics reveals major microbial players and their biodegradation functions in a large-scale aerobic composting plant

Composting is an appropriate management alternative for municipal solid waste; however, our knowledge about the microbial regulation of this process is still scare. We employed metaproteomics to elucidate the main biodegradation pathways in municipal solid waste composting system across the main phases in a large-scale composting plant. The investigation of microbial succession revealed that Bacillales, Actinobacteria and Saccharomyces increased significantly with respect to abundance in composting process. The key microbiologic population for cellulose degradation in different composting stages was different. Fungi were found to be the main producers of cellulase in earlier phase. However, the cellulolytic fungal communities were gradually replaced by a purely bacterial one in active phase, which did not support the concept that the thermophilic fungi are active through the thermophilic phase. The effective decomposition of cellulose required the synergy between bacteria and fungi in the curing phase.     

Biodegradation of Cellulose-Containing Substrates by Micromycetes Followed by Bioconversion into Biogas

The ability of micromycetes Trichoderma viride and Aspergillus terreus to decompose the cellulosecontaining substrates was studied. Office paper and cardboard, as well as a paper mixture, were found to be the most hydrolyzable. The cellulolytic activity of T. viride was 2–3 times higher than that of A. terreus; the highest values of 0.80 and 0.73 U/mL were obtained from office paper and the mixture of different types of paper, respectively. The micromycete cultivation conditions (composition of culture medium, sucrose cosubstrate addition, seeding technique) and the conditions of the fungus biomass treatment for its subsequent bioconversion into biogas by anaerobic microbial communities were optimized. It was shown that pretreatment improves the efficiency of biogas production from lignocellulosic materials when inoculated with microbial community of cattle manure. After pretreatment of the Jerusalem artichoke phytomass (stems and leaves) and its subsequent bioconversion into biogas by methanogenic community, the biogas yield was increased by1.5 times.     

Anaerobic digestion of microalgal biomass for bioenergy production,removal of nutrients and microcystin: current status

Using renewable microalgal biomass as active feedstocks for biofuels and bioproducts is explored to substitute petroleum-based fuels and chemicals. In the last few years, the importance of microalgae biomass has been realized as a renewable feedstock due to several positive attributes associated with it. Biorefinery via anaerobic digestion (AD) of microalgal biomass is a promising and sustainable method to produce value-added chemicals, edible products and biofuels. Microalgal biomass pretreatment is a significant process to enhance methane production by AD. Findings on the AD microbial community’s variety and organization can give novel in turn on digester steadiness and presentation. This review presents a vital study of the existing facts on the AD microbial community and AD production. Co-digestion of microalgal biomass with different co-substrates was used in AD to enhance biogas production, and the process was economically viable with improved biodegradability. Microcystins, which are produced by toxic cyanobacterial blooms, create a severe hazard to environmental health. Anaerobic biodegradation is an effective method to degrade the microcystins and convert into nontoxic products. However, for the cost-effective conversion of biomass to energy and other beneficial byproducts, additional highly developed research is still required for large-scale AD of microalgal biomass.