Perturbation‐independent community development in low‐temperature anaerobic biological wastewater treatment bioreactors

The reproducibility and stability of low‐ temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed‐based bioreactors were used to degrade a volatile fatty acid and glucose‐based wastewater under sub‐ambient (15°C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations. Biotechnol. Bioeng. 2010;105: 79–87. © 2009 Wiley Periodicals, Inc.     

Phylogenetic and proteomic analysis of an anaerobic toluene-degrading community

Aims: This study intended to unravel the physiological interplay in an anaerobic microbial community that degrades toluene under sulfate‐reducing conditions combining proteomic and genetic techniques. Methods and Results: An enriched toluene‐degrading community (Zz5‐7) growing in batch cultures was investigated by DNA‐ and protein‐based analyses. The affiliation and diversity of the community were analysed using 16S ribosomal RNA (rRNA) genes as a phylogenetic marker as well as bssA and dsrAB genes as functional markers. Metaproteome analysis was carried out by a global protein extraction and a subsequent protein separation by two‐dimensional gel electrophoresis (2‐DE). About 85% of the proteins in the spots were identified by nano‐liquid chromatography coupled with electrospray mass spectrometry (nano‐LC–ESI‐MS/MS) analysis. DNA sequencing of bssA and the most abundant dsrAB amplicons revealed high similarities to a member of the Desulfobulbaceae, which was also predominant according to 16S rRNA gene amplicons. Metaproteome analysis provided 202 unambiguous protein identifications derived from 236 unique protein spots. The proteins involved in anaerobic toluene activation, dissimilatory sulfate reduction, hydrogen production/consumption and autotrophic carbon fixation were mainly affiliated to members of the Desulfobulbaceae and several other Deltaproteobacteria. Conclusion: Phylogenetic and metaproteomic analyses revealed a member of the Desulfobulbaceae as the key player of anaerobic toluene degradation in a sulfate‐reducing consortium. Significance and Impact of the Study: This is the first study that combines genetic and proteomic analyses to indicate the interactions in an anaerobic toluene‐degrading microbial consortium.     

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.
     

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.     

Norovirus‐binding proteins recovered from activated sludge micro‐organisms with an affinity to a noroviral capsid peptide

Aims: Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro‐organisms could be totally different from that of those particles moving freely, micro‐organisms with natural affinity ligands such as virus‐binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus‐binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. Methods and Results: NoVBPs were recovered from activated sludge micro‐organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus‐like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two‐dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N‐terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. Conclusions: NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro‐organisms. Significance and Impact of the Study: NoVBPs could be natural viral ligands and play an important role in the NoV transmission.     

Microbial activity and community structure in a lake sediment used for psychrophilic anaerobic wastewater treatment

Aims: The aim of the study was to investigate the feasibility of a continuous reactor for psychrophilic anaerobic wastewater treatment by using the sludge from cold natural environment. Methods and Results: Six sludge samples (S1–S6) were collected from different cold natural locations to select sludge with high anaerobic microbial activity under low temperatures. After a 225‐day incubation, the maximum specific methane production rate of a waterfowl lake sediment (S1) at 15°C (70·5 mLCH₄ gVSS^?1 day^?1) was much higher than all other samples. S1 was thus chosen as the seed sludge for the reactor treating synthetic brewery wastewater at 15°C, by immobilizing the micro‐organisms on polyurethane foam carriers. The chemical oxygen demand (COD) removal efficiency reached over 80% after 240‐day operation at an organic loading rate of 5·3 kg m^?3 day^?1, and significant enrichment of biomass was observed. Clone libraries of the microbial communities in the inoculum had high diversities for both archaea and bacteria. Along with a decrease in microbial community diversities, the dominant bacteria (79·5%) at the end of the operation represented the phylum Firmicutes, while the dominant archaeon (41·5%) showed a similarity of 98% with the psychrotolerant methanogen Methanosarcina lacustris. Conclusions: The possibility of using anaerobic micro‐organisms from cold environments in anaerobic wastewater treatment under psychrophilic conditions is supported by these findings. Significance and Impact of the Study: This study enriches the theory on microbial community and the application on anaerobic treatment of sludge from cold natural environments.     

Who is who in litter decomposition? Metaproteomics reveals major microbial players and their biogeochemical functions

Leaf-litter decomposition is a central process in carbon cycling; however, our knowledge about the microbial regulation of this process is still scarce. Metaproteomics allows us to link the abundance and activity of enzymes during nutrient cycling to their phylogenetic origin based on proteins, the ‘active building blocks'' in the system. Moreover, we employed metaproteomics to investigate the influence of environmental factors and nutrients on the decomposer structure and function during beech litter decomposition. Litter was collected at forest sites in Austria with different litter nutrient content. Proteins were analyzed by 1-D-SDS-PAGE followed by liquid-chromatography and tandem mass-spectrometry. Mass spectra were assigned to phylogenetic and functional groups by a newly developed bioinformatics workflow, assignments being validated by complementary approaches. We provide evidence that the litter nutrient content and the stoichiometry of C:N:P affect the decomposer community structure and activity. Fungi were found to be the main producers of extracellular hydrolytic enzymes, with no bacterial hydrolases being detected by our metaproteomics approach. Detailed investigation of microbial succession suggests that it is influenced by litter nutrient content. Microbial activity was stimulated at higher litter nutrient contents via a higher abundance and activity of extracellular enzymes.     

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。     

Metaproteomic Survey of Six Aquatic Habitats: Discovering the Identities of Microbial Populations Active in Biogeochemical Cycling

Our goal is to strengthen the foundations of metaproteomics as a microbial community analysis tool that links the functional identity of actively expressed gene products with host phylogeny. We used shotgun metaproteomics to survey waters in six disparate aquatic habitats (Cayuga Lake, NY; Oneida Lake, NY; Gulf of Maine; Chesapeake Bay, MD; Gulf of Mexico; and the South Pacific). Peptide pools prepared from filter-gathered microbial biomass, analyzed by nano-liquid chromatography–mass spectrometry (MS/MS) generating 9,693?±?1,073 mass spectra identified 326?±?107 bacterial proteins per sample. Distribution of proteobacterial (Alpha and Beta) and cyanobacterial (Prochlorococcus and Synechococcus spp.) protein hosts across all six samples was consistent with the previously published biogeography for these microorganisms. Marine samples were enriched in transport proteins (TRAP-type for dicarboxylates and ATP binding cassette (ABC)-type for amino acids and carbohydrates) compared with the freshwater samples. We were able to match in situ expression of many key proteins catalyzing C-, N-, and S-cycle processes with their bacterial hosts across all six habitats. Pelagibacter was identified as the host of ABC-type sugar-, organic polyanion-, and glycine betaine-transport proteins; this extends previously published studies of Pelagibacter's in situ biogeochemical role in marine C- and N-metabolism. Proteins matched to Ruegeria confirmed these organism's role in marine waters oxidizing both carbon monoxide and sulfide. By documenting both processes expressed in situ and the identity of host cells, metaproteomics tested several existing hypotheses about ecophysiological processes and provided fodder for new ones.     

Microbial consortia involved in the anaerobicdegradation of hydrocarbons

In this review, we examine the energetics of well-characterized biodegradation pathways and explore the possibilities for these to support growth of multiple organisms interacting in consortia. The relevant phenotypic and/or phylogenetic characteristics of isolates and consortia mediating hydrocarbon degradation coupled with different terminal electron-acceptingprocesses (TEAP) are also reviewed. While the information on metabolic pathways has been gained from the analysis of individual isolates, the energetic framework presented here demonstrates that microbial consortia could be readily postulated for hydrocarbon degradation coupled to any TEAP. Several specialized reactions occur within these pathways, and the organisms mediating these are likely to play a key role in defining the hydrocarbon degradation characteristics of the community under a given TEAP. Comparing these processes within and between TEAPs reveals biological unity in that divergent phylotypes display similar degradation mechanisms and biological diversity in that hydrocarbon-degraders closely related as phylotypes differ in the type and variety of hydrocarbon degradation pathways they possess. Analysis of microcosms and of field samples suggests that we have only begun to reveal the diversity of organisms mediating anaerobic hydrocarbon degradation. Advancements in the understanding of how hydrocarbon-degrading communities function will be significantly affected by the extent to which organisms mediating specialized reactions can be identified, and tools developed to allow their study in situ.     

Use of 16S rRNA Gene Based Clone Libraries to Assess Microbial Communities Potentially Involved in Anaerobic Methane Oxidation in a Mediterranean Cold Seep

This study provides data on the diversities of bacterial and archaeal communities in an active methane seep at the Kazan mud volcano in the deep Eastern Mediterranean sea. Layers of varying depths in the Kazan sediments were investigated in terms of (1) chemical parameters and (2) DNA-based microbial population structures. The latter was accomplished by analyzing the sequences of directly amplified 16S rRNA genes, resulting in the phylogenetic analysis of the prokaryotic communities. Sequences of organisms potentially associated with processes such as anaerobic methane oxidation and sulfate reduction were thus identified. Overall, the sediment layers revealed the presence of sequences of quite diverse bacterial and archaeal communities, which varied considerably with depth. Dominant types revealed in these communities are known as key organisms involved in the following processes: (1) anaerobic methane oxidation and sulfate reduction, (2) sulfide oxidation, and (3) a range of (aerobic) heterotrophic processes. In the communities in the lowest sediment layer sampled (22–34 cm), sulfate-reducing bacteria and archaea of the ANME-2 cluster (likely involved in anaerobic methane oxidation) were prevalent, whereas heterotrophic organisms abounded in the top sediment layer (0–6 cm). Communities in the middle layer (6–22 cm) contained organisms that could be linked to either of the aforementioned processes. We discuss how these phylogeny (sequence)-based findings can support the ongoing molecular work aimed at unraveling both the functioning and the functional diversities of the communities under study.     

On the energetics of chemolithotrophy in nonequilibrium systems: case studies of geothermal springs in Yellowstone National Park

Chemolithotrophic micro‐organisms are important primary producers in high‐temperature geothermal environments and may catalyse a number of different energetically favourable redox reactions as a primary energy source. Analysis of geochemical constituents followed by chemical speciation and subsequent calculation of reaction free energies (ΔG_rxn) is a useful tool for evaluating the thermodynamic favourability and potential energy available for microbial metabolism. The primary goal of this study was to examine relationships among geochemical gradients and microbial population distribution, and to evaluate the utility of energetic approaches for predicting microbial metabolism from free‐energy calculations, utilizing as examples, several geothermal habitats in Yellowstone National Park where thorough geochemical and phylogenetic analyses have been performed. Acidic (pH ～ 3) and near‐neutral (pH ～ 6–7) geothermal springs were chosen for their range in geochemical properties. Aqueous and solid phase samples obtained from the source pools and the outflow channels of each spring were characterized for all major chemical constituents using laboratory and field methods to accurately measure the concentrations of predominant oxidized and reduced species. Reaction free energies (ΔG_rxn) for 33 oxidation–reduction reactions potentially important to chemolithotrophic micro‐organisms were calculated at relevant spring temperatures after calculating ion activities using an aqueous equilibrium model. Free‐energy values exhibit significant variation among sites for reactions with pH dependence. For example, free‐energy values for reactions involving Fe³⁺ are especially variable across sites due in large part to the pH dependence of Fe³⁺ activity, and exhibit changes of up to 40 kJ mol^?1 electron from acidic to near neutral geothermal springs. Many of the detected 16S rRNA gene sequences represent organisms whose metabolisms are consistent with exergonic processes. However, sensitivity analyses demonstrated that reaction free energies do not generally represent the steep gradients in local geochemical conditions resulting from air–water gas exchange and solid phase deposition that are important in defining microbial habitats and 16S rRNA gene sequence distribution within geothermal outflow channels.     

Synergetic effect of pH and biochemical components on bacterial diversity during mesophilic anaerobic fermentation of biomass-origin waste

Aims: To investigate the synergetic effect of pH and biochemical components on bacterial community structure during mesophilic anaerobic degradation of solid wastes with different origins, and under acidic or neutral conditions. Methods and Results: The bacterial community in 16 samples of solid wastes with different biochemical compositions and origins was evaluated during mesophilic anaerobic degradation at acidic and neutral pH. Denaturing gradient gel electrophoresis (DGGE) and single‐strand conformation polymorphism (SSCP) were used to compare the communities. Multivariate analysis of the DGGE and SSCP results revealed that most of the dominant microbes were dependent on the content of easily degradable carbohydrates in the samples. Furthermore, the dominant microbes were divided into two types, those that preferred an acid environment and those that preferred a neutral environment. A shift in pH was found to change their preference for medium substrates. Although most of the substrates with similar origin and biochemical composition had similar microbial diversity during fermentation, some microbes were found only in substrates with specific origins. For example, two microbes were only found in substrate that contained lignocellulose and animal protein without starch. These microbes were related to micro‐organisms that are found in swine manure, as well as in other intestinal or oral niches. In addition, the distribution of fermentation products was less sensitive to the changes in pH and biochemical components than the microbial community. Conclusions: Bacterial diversity during anaerobic degradation of organic wastes was affected by both pH and biochemical components; however, pH exerted a greater effect. Significance and Impact of the Study: The results of this study reveal that control of pH may be an effective method to produce a stable bacterial community and relatively similar product distribution during anaerobic digestion of waste, regardless of variation in the waste feedstocks.     

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.