Proteotyping of laboratory-scale biogas plants reveals multiple steady-states in community composition.

Complex microbial communities are the functional core of anaerobic digestion processes taking place in biogas plants (BGP). So far, however, a comprehensive characterization of the microbiomes involved in methane formation is technically challenging. As an alternative, enriched communities from laboratory-scale experiments can be investigated that have a reduced number of organisms and are easier to characterize by state of the art mass spectrometric-based (MS) metaproteomic workflows. Six parallel laboratory digesters were inoculated with sludge from a full-scale BGP to study the development of enriched microbial communities under defined conditions. During the first three month of cultivation, all reactors (R1-R6) were functionally comparable regarding biogas productions (375-625 NL Lreactor volume-1 d-1), methane yields (50-60%), pH values (7.1-7.3), and volatile fatty acids (VFA, <5 mM). Nevertheless, a clear impact of the temperature (R3, R4) and ammonia (R5, R6) shifts was observed for the respective reactors. In both reactors operated under thermophilic regime, acetic and propionic acid (10-20 mM) began to accumulate. While R4 recovered quickly from acidification, the levels of VFA remained to be high in R3 resulting in low pH values of 6.5-6.9. The digesters R5 and R6 operated under the high ammonia regime (>1 gNH3 L-1) showed an increase to pH 7.5-8.0, accumulation of acetate (>10 mM), and decreasing biogas production (<125 NL Lreactor volume-1 d-1). Tandem MS (MS/MS)-based proteotyping allowed the identification of taxonomic abundances and biological processes. Although all reactors showed similar performances, proteotyping and terminal restriction fragment length polymorphisms (T-RFLP) fingerprinting revealed significant differences in the composition of individual microbial communities, indicating multiple steady-states. Furthermore, cellulolytic enzymes and cellulosomal proteins of Clostridium thermocellum were identified to be specific markers for the thermophilic reactors (R3, R4). Metaproteins found in R3 indicated hydrogenothrophic methanogenesis, whereas metaproteins of acetoclastic methanogenesis were identified in R4. This suggests not only an individual evolution of microbial communities even for the case that BGPs are started at the same initial conditions under well controlled environmental conditions, but also a high compositional variance of microbiomes under extreme conditions.

Sample prefractionation with liquid isoelectric focusing enables in depth microbial metaproteome analysis of mesophilic and thermophilic biogas plants.

Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution.

Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram.

We previously showed that opdA from Sphingomonas sp. PWE1 encodes a putative flavin monooxygenase capable of transforming octylphenol (OP) via type II ipso substitution. Here, we demonstrate that an opdA homolog is responsible for OP and related alkyl/alkoxyphenol degradation in the nonylphenol degrader Sphingomonas sp. TTNP3. PCR and Southern blot analyses revealed that TTNP3 contained an opdA homolog, while a TTNP3 derivative unable to grow on nonylphenol (TTNP3d) did not. OpdA expression was confirmed in wild-type TTNP3 via two dimensional gel electrophoresis. Activity was restored to TTNP3d following complementation with opdA. Sequence analysis of an opdA homolog from another nonylphenol degrader, Sphingobium xenophagum Bayram, revealed that the predicted protein sequences from PWE1 and Bayram were identical, but differed from TTNP3 by four amino acids. In order to assess differences, we heterologously expressed the two unique opdA homologs and compared their effect on the disappearance of five alkyl/alkoxyphenol substrates and subsequent appearance of hydroquinone. For all substrates, except OP, the levels of substrate disappearance and hydroquinone appearance were significantly lower in cultures expressing odpA (TTNP3) than those expressing opdA (PWE1/Bayram). These differences in substrate specificity were consistent with an in silico model which predicted that two of the amino acid differences between odpA (TTNP3) and opdA (PWE1/Bayram) lay in a putative substrate binding pocket. While these strains are known to use the same type II ipso substitution mechanism for alkylphenol degradation, this work provides the first preliminary evidence that opdA homologs also encode the type I ipso substitution activity responsible for the degradation of alkoxyphenols.

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.

Metaproteomics of fecal samples of Crohn's disease and Ulcerative Colitis.

Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis. Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used. Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase. SIGNIFICANCE: Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases.

Metaproteome analysis reveals that syntrophy, competition, and phage-host interaction shape microbial communities in biogas plants.

BACKGROUND: In biogas plants, complex microbial communities produce methane and carbon dioxide by anaerobic digestion of biomass. For the characterization of the microbial functional networks, samples of 11 reactors were analyzed using a high-resolution metaproteomics pipeline. RESULTS: Examined methanogenesis archaeal communities were either mixotrophic or strictly hydrogenotrophic in syntrophy with bacterial acetate oxidizers. Mapping of identified metaproteins with process steps described by the Anaerobic Digestion Model 1 confirmed its main assumptions and also proposed some extensions such as syntrophic acetate oxidation or fermentation of alcohols. Results indicate that the microbial communities were shaped by syntrophy as well as competition and phage-host interactions causing cell lysis. For the families Bacillaceae, Enterobacteriaceae, and Clostridiaceae, the number of phages exceeded up to 20-fold the number of host cells. CONCLUSION: Phage-induced cell lysis might slow down the conversion of substrates to biogas, though, it could support the growth of auxotrophic microbes by cycling of nutrients.

Identification of spore allergens from the indoor mould Aspergillus versicolor.

BACKGROUND: Indoor mould growth and dampness are associated with respiratory health effects and allergies and several studies demonstrated that mainly Aspergillus versicolor and Penicillium expansum are responsible for indoor mould exposure. In contrast, commercialized test systems to diagnose allergic reactions to this mould species are not available. In this study, allergenic proteins from spores of the indoor relevant species A. versicolor and P. expansum should get detected and identified. METHODS: We used two-dimensional (2D)-gel electrophoresis of spore proteins and immunoblotting with sera from patients participating in an epidemiologic study about indoor exposure of moulds and their influence on the development of allergies (ESTERSPEGA). Sera were screened for IgE antibodies specific for proteins from A. versicolor, A. fumigatus and P. expansum in one-dimensional blots and in 2D immunoblots. From the 2D gels, the corresponding spots were picked and identified by mass spectrometry. RESULTS: More than 20 allergens from A. versicolor were identified; in particular, seven major allergens were selected, which were detected by more than 90% of the positive sera. The most abundant allergen was glyceraldehyde-3-phosphate dehydrogenase, followed by an unnamed protein, which displays a high homology to sobitol/xylose reductase. The other allergens were identified as catalase A, hypothetical protein AN6918.2, enolase, hypothetical protein AN0297.2 and a protein with homology to a fungal malate dehydrogenase. CONCLUSIONS: The results indicate an important role of spore proteins from A. versicolor for sensitization against indoor moulds and identification of the major allergens might enable species-specific diagnosis of allergic reactions.

Proteotyping of biogas plant microbiomes separates biogas plants according to process temperature and reactor type.

BACKGROUND: Methane yield and biogas productivity of biogas plants (BGPs) depend on microbial community structure and function, substrate supply, and general biogas process parameters. So far, however, relatively little is known about correlations between microbial community function and process parameters. To close this knowledge gap, microbial communities of 40 samples from 35 different industrial biogas plants were evaluated by a metaproteomics approach in this study. RESULTS: Liquid chromatography coupled to tandem mass spectrometry (Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer) of all 40 samples as triplicate enabled the identification of 3138 different metaproteins belonging to 162 biological processes and 75 different taxonomic orders. The respective database searches were performed against UniProtKB/Swiss-Prot and seven metagenome databases. Subsequent clustering and principal component analysis of these data allowed for the identification of four main clusters associated with mesophile and thermophile process conditions, the use of upflow anaerobic sludge blanket reactors and BGP feeding with sewage sludge. Observations confirm a previous phylogenetic study of the same BGP samples that was based on 16S rRNA gene sequencing by De Vrieze et al. (Water Res 75:312-323, 2015). In particular, we identified similar microbial key players of biogas processes, namely Bacillales, Enterobacteriales, Bacteriodales, Clostridiales, Rhizobiales and Thermoanaerobacteriales as well as Methanobacteriales, Methanosarcinales and Methanococcales. For the elucidation of the main biomass degradation pathways, the most abundant 1 % of metaproteins was assigned to the KEGG map 1200 representing the central carbon metabolism. Additionally, the effect of the process parameters (i) temperature, (ii) organic loading rate (OLR), (iii) total ammonia nitrogen (TAN), and (iv) sludge retention time (SRT) on these pathways was investigated. For example, high TAN correlated with hydrogenotrophic methanogens and bacterial one-carbon metabolism, indicating syntrophic acetate oxidation. CONCLUSIONS: This is the first large-scale metaproteome study of BGPs. Proteotyping of BGPs reveals general correlations between the microbial community structure and its function with process parameters. The monitoring of changes on the level of microbial key functions or even of the microbial community represents a well-directed tool for the identification of process problems and disturbances.Graphical abstractCorrelation between the different orders and process parameter, as well as principle component analysis of all investigated biogas plants based on the identified metaproteins.

Protein synthesis patterns in Acinetobacter calcoaceticus induced by phenol and catechol show specificities of responses to chemostress.

The proteins induced in Acinetobacter calcoaceticus by the potentially toxic growth substrates phenol and catechol were analyzed by 2D-electrophoresis of cell extracts and compared with those induced by heat shock and oxidative stress. Although both aromatic compounds are quite similar, the only difference being that catechol has an additional hydroxyl group, the responses obtained differed considerably. Phenol has greater lipophilicity and mainly induced heat shock proteins, whereas catechol, which causes the production of reactive oxygen species, predominantly induced oxidative stress proteins. Furthermore, some special proteins were induced by phenol or catechol, which might be useful as biomarkers for chemostress, and could be involved in the catalytic degradation of potentially toxic compounds.

Metaproteome analysis of the microbial communities in agricultural biogas plants.

In biogas plants agricultural waste and energy crops are converted by complex microbial communities to methane for the production of renewable energy. In Germany, this process is widely applied namely in context of agricultural production systems. However, process disturbances, are one of the major causes for economic losses. In addition, the conversion of biomass, in particular of cellulose, is in most cases incomplete and, hence, insufficient. Besides technical aspects, a more profound characterization concerning the functionality of the microbial communities involved would strongly support the improvement of yield and stability in biogas production. To monitor these communities on the functional level, metaproteome analysis was applied in this study to full-scale agricultural biogas plants. Proteins were extracted directly from sludge for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent identification with mass spectrometry. Protein profiles obtained with SDS-PAGE were specific for different biogas plants and often stable for several months. Differences of protein profiles were visualized by clustering, which allowed not only the discrimination between mesophilic and thermophilic operated biogas plants but also the detection of process disturbances such as acidification. In particular, acidification of a biogas plant was detected in advance by disappearance of major bands in SDS-PAGE. Identification of proteins from SDS-PAGE gels revealed that methyl CoM reductase, which is responsible for the release of methane during methanogenesis, from the order Methanosarcinales was significantly decreased. Hence, it is assumed that this enzyme might be a promising candidate to serve as a predictive biomarker for acidification.

Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene.

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO(4) and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k(cat)/K(m)) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s(-1) mM(-1), respectively, with K(m) values of 4.2, 67.8, and 104.9 microM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15 degrees C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.

Chemicals and heat generate different protein patterns in Acinetobacter calcoaceticus.

The effect of exposing Acinetobacter calcoaceticus 69-V to DNP-stress and heat shock was examined by two-dimensional gel electrophoresis of proteins, which were detected either by autoradiography or by silver staining. Both DNP stress and heat shock led to altered patterns of protein synthesis or concentration. About 10% of the proteins which were synthesized newly or at an increased rate and about 25% of those which were found newly or with an increased concentration after DNP treatment were identified after heat shock, too.

Induction of heat shock proteins in response to primary alcohols in Acinetobacter calcoaceticus.

Cells of Acinetobacter calcoaceticus 69-V, a species able to metabolize a range of aliphatic hydrocarbons and alcohols, were confronted with ethanol, butanol, hexanol or heat shock during growth on acetate as sole source of carbon and energy. The primary alcohols and the heat shock led to the synthesis of new proteins or amplified expression of specific, common and general proteins, which were detected by silver staining after two-dimensional gel electrophoresis. Some of the alcohol-inducible proteins were identified as heat shock proteins by comparing protein patterns of alcohol-shocked cells with those of heat-shocked cells, and by N-terminal amino acid sequencing. DnaK was found to be amplified after all treatments, but GroEI only after heat shock and ethanol treatment. The N-terminal amino acid sequence of the protein, which was considerably amplified after alcohol treatment and heat shock, shows homology to HtpG (high temperature protein G). Some of the heat shock proteins induced by ethanol differ from those induced by butanol and hexanol, suggesting there are at least two different signals for the induction of some heat shock proteins by primary alcohols. This could be due to the different localization of ethanol, butanol and hexanol in the membrane, or because higher cytoplasmic concentrations of ethanol than of butanol or hexanol were applied in these tests in order to keep concentrations of the alcohols in the membrane roughly similar. Besides heat shock proteins, a group of proteins were observed which were only induced by butanol and hexanol, possibly indicating the existence of a further defense mechanism against high concentrations of hydrophobic substrates preventing protein denaturation and membrane damage.