Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

Sugiyamaella mastotermitis sp. nov. and Papiliotrema odontotermitis f.a., sp. nov. from the gut of the termites Mastotermes darwiniensis and Odontotermes obesus.

Two novel yeast species were isolated from the guts of two different termite species. A new member of the genus Sugiyamaella was isolated from the hindgut and nest material of the lower Australian termite Mastotermes darwiniensis. The second novel yeast species, isolated from the higher termite Odontotermes obesus, was identified as a member of the genus Papiliotrema. Both yeast species were able to hydrolyse xylan, methylumbelliferyl ß-xylobiose and methylumbelliferyl ß-xylotriose. The ability to debranch different hemicellulose side chains and growth without the addition of external vitamins was observed. A symbiotic role of the novel yeast species is indicated, especially in respect to xylan degradation and the production of vitamins. Here, we describe these species as Sugiyamaella mastotermitis sp. nov., MycoBank 816574 (type strain MD39VT=DSM 100793T=CBS 14182T), and Papiliotrema odontotermitis f.a., sp. nov., MycoBank 816575 (type strain OO5T=DSM 100791T=CBS 14181T). Additionally, we transfer Candida qingdaonensis to the genus Sugiyamaella and propose the following combination: Sugiyamaella qingdaonensis f.a., comb. nov., MycoBank 816576.

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids.

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast.

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines.

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been proved in physiological test systems. Furthermore, only a few data exist on the antioxidant capacities of the enzymatic oxidation products of phenolic compounds generated by PPO. We report here first results about the antioxidant effects of phenolic substances, before and after oxidation by fungal model tyrosinase and laccase. In general, the common chemical 2,2-diphenyl-1-picrylhydrazyl assay and the biological tests using two different types of cell cultures (monocytes and endothelial cells) delivered similar results. The phenols tested showed significant differences with respect to their antioxidant activity in all test systems. Their antioxidant capacities after enzymatic conversion decreased or increased depending on the individual PPO used.

The yeast Wickerhamomyces anomalus AS1 secretes a multifunctional exo-ß-1,3-glucanase with implications for winemaking.

A multifunctional exo-ß-1,3-glucanase (WaExg2) was purified from the culture supernatant of the yeast Wickerhamomyces anomalus AS1. The enzyme was identified by mass spectroscopic analysis of tryptic peptide fragments and the encoding gene WaEXG2 was sequenced. The latter codes for a protein of 427 amino acids, beginning with a probable signal peptide (17 aa) for secretion. The mature protein has a molecular mass of 47 456 Da with a calculated pI of 4.84. The somewhat higher mass of the protein in SDS-PAGE might be due to bound carbohydrates. Presumptive disulphide bridges confer a high compactness to the molecule. This explains the apparent smaller molecular mass (35 kDa) of the native enzyme determined by electrophoresis, whereas the unfolded form is consistent with the theoretical mass. Enzymatic hydrolysis of selected glycosides and glycans by WaExg2 was proved by TLC analysis of cleavage products. Glucose was detected as the sole hydrolysis product from laminarin, underlining that the enzyme acts as an exoglucanase. In addition, the enzyme efficiently hydrolysed small ß-linked glycosides (arbutin, esculin, polydatin, salicin) and disaccharides (cellobiose, gentiobiose). WaExg2 was active under typical wine-related conditions, such as low pH (3.5-4.0), high sugar concentrations (up to 20% w/v), high ethanol concentrations (10-15% v/v), presence of sulphites (up to 2 mm) and various cations. Therefore, the characterized enzyme might have multiple uses in winemaking, to increase concentrations of sensory and bioactive compounds by splitting glycosylated precursors or to reduce viscosity by hydrolysis of glycan slimes.

The cellulolytic system of the termite gut.

The demand for the usage of natural renewable polymeric material is increasing in order to satisfy the future needs for energy and chemical precursors. Important steps in the hydrolysis of polymeric material and bioconversion can be performed by microorganisms. Over about 150 million years, termites have optimized their intestinal polysaccharide-degrading symbiosis. In the ecosystem of the "termite gut," polysaccharides are degraded from lignocellulose, such as cellulose and hemicelluloses, in 1 day, while lignin is only weakly attacked. The understanding of the principles of cellulose degradation in this natural polymer-degrading ecosystem could be helpful for the improvement of the biotechnological hydrolysis and conversion of cellulose, e.g., in the case of biogas production from natural renewable plant material in biogas plants. This review focuses on the present knowledge of the cellulose degradation in the termite gut.

Complete genome sequence of the hydrogenotrophic, methanogenic archaeon Methanoculleus bourgensis strain MS2(T), Isolated from a sewage sludge digester.

Methanoculleus bourgensis, of the order Methanomicrobiales, is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2(T), isolated from a sewage sludge digester.

Molecular and biochemical properties of the S-layer protein from the wine bacterium Lactobacillus hilgardii B706.

Different strains of the genus Lactobacillus can be regularly isolated from must and wine samples. By various physiological activities, they can improve or reduce the wine quality. Lactobacillus hilgardii that is known to survive under harsh wine conditions is classified as a spoilage bacterium, e.g. due to the production of histamine. Many lactobacilli form an S-layer as the outermost cell wall component which has been found to facilitate the colonization of special ecological niches. A detailed understanding of the properties related to their S-layer proteins is necessary to improve the knowledge of the interactions between different bacterial cells and with the surrounding environments. The S-layer protein from the wine-related L. hilgardii strain B706 has been isolated and its gene sequence determined. The deduced amino acid sequence corresponds to a 41 kDa protein with an isoelectric point of 9.6 without additional posttranslational modifications after splitting off the leader peptide. The complete protein is organized in a 32 amino acids signal sequence for membrane translocation, a positively charged N-terminal domain that binds to the cell wall and a negatively charged C-terminal domain. When the S-layer was removed, the corresponding L. hilgardii B706 cells became more sensitive to bacteriolytic enzymes and some wine-related stress conditions. From a practical point of view, the S-layer may be considered as a target for the inhibition of food-spoiling lactobacilli.

Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany.

Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no distinct region specificity of O. oeni populations.

Methane oxidation in industrial biogas plants-Insights in a novel methanotrophic environment evidenced by pmoA gene analyses and stable isotope labelling studies.

A broad methanotrophic community consisting of 16 different operational taxonomic units (OTUs) was detected by particulate methane monooxygenase A (pmoA) gene analyses of reactor sludge samples obtained from an industrial biogas plant. Using a cloning-sequencing approach, 75% of the OTUs were affiliated to the group of type I methanotrophs (γ-Proteobacteria) and 25% to type II methanotrophs (α-Proteobacteria) with a distinct predominance of the genus Methylobacter. By database matching, half of the total OTUs may constitute entirely novel species. For evaluation of process conditions that support growth of methanotrophic bacteria, qPCR analyses of pmoA gene copy numbers were performed during a sampling period of 70 days at varying reactor feeding scenarios. During the investigation period, methanotrophic cell counts estimated by qPCR fluctuated between 3.4 × 104 and 2 × 105 cells/mL with no distinct correlation to the organic loading rate, the amount of CH4, O2 and NH4-N. Methanotrophic activity was proofed even at low O2 levels (1%) by using stable carbon isotope labelling experiments of CH4 in batch experiments inoculated with reactor sludge. Supplementation of 13C labelled CH4 in the headspace of the reaction vials unambiguously confirmed the formation of 13C labelled CO2. Thus, industrial biogas reactors can be considered as a further methanotrophic habitat that exhibits a unique methanotrophic community which is specifically adapted to high CH4 and low O2 concentrations. To the best of our knowledge, our study is the first accurate detection and quantification of methanotrophic bacteria in industrial biogas reactors.

Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps.

Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC 33499). The DNA-DNA hybridization revealed a similarity of 66% with E. adhaerens (NCIMB12342(T)). In contrast to the type strain the isolate M3A possesses the capacity to nodulate plant roots. This is the first report on the detailed identification of a rhizobia-related strain from the intestinal tract of animals. Strain M3A has been deposited with two culture collections (DSM10169; ATCC BAA-396).

TNT transformation products are affected by the growth conditions of Raoultella terrigena.

High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [(14)C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.

Isolation of lactic acid-forming bacteria from biogas plants.

Direct molecular approaches provide hints that lactic acid bacteria play an important role in the degradation process of organic material to methanogenetic substrates in biogas plants. However, their diversity in biogas fermenter samples has not been analyzed in detail yet. For that reason, five different biogas fermenters, which were fed mainly with maize silage and manure from cattle or pigs, were examined for the occurrence of lactic acid-forming bacteria. A total of 197 lactic acid-forming bacterial strains were isolated, which we assigned to 21 species, belonging to the genera Bacillus, Clostridium, Lactobacillus, Pediococcus, Streptococcus and Pseudoramibacter-related. A qualitative multiplex system and a real-time quantitative PCR could be developed for most isolates, realized by the selection of specific primers. Their role in biogas plants was discussed on the basis of the quantitative results and on physiological data of the isolates.

Antioxidant capacity of phenolic compounds on human cell lines as affected by grape-tyrosinase and Botrytis-laccase oxidation.

Phenolic components (PCs) are well-known for their positive impact on human health. In addition to their action as radical scavengers, they act as activators for the intrinsic cellular antioxidant system. Polyphenol oxidases (PPOs) such as tyrosinase and laccase catalyze the enzymatic oxidation of PCs and thus, can alter their scavenging and antioxidative capacity. In this study, oxidation by tryosinase was shown to increase the antioxidant capacity of many PCs, especially those that lack adjacent aromatic hydroxyl groups. In contrast, oxidation by laccase tended to decrease the antioxidant capacity of red wine and distinct PCs. This was clearly demonstrated for p-coumaric acid and resveratrol, which is associated with many health benefits. While oxidation by tyrosinase increased their antioxidant activity laccase treatment resulted in a decreased activity and also of that for red wines.

Roles and Responsibilities in the Provision of Accredited Continuing Medical Education/Continuing Professional Development.

The Cologne Consensus Conference 2015 has focused on "Providers in accredited CME[continuing medical education]/CPD [continuing professional development]". As an outcome of the CCC 2015, the authors of this paper, who were part of the faculty, propose a contemporary definition of the roles and responsibilities of stakeholders involved in the different stages of planning, delivery and evaluation of CME/CPD.

Genomics and prevalence of bacterial and archaeal isolates from biogas-producing microbiomes.

BACKGROUND: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. RESULTS: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. CONCLUSIONS: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members.

Analysis of propionate-degrading consortia from agricultural biogas plants.

In order to investigate the propionate-degrading community of agricultural biogas plants, four propionate-degrading consortia (Ap1a, N12, G12, and Wp2a) were established from different biogas plants which were fed with renewable resources. The consortia were cultivated in a batch for a period of 2-4 years and then analyzed in an 8-week batch experiment for microbial succession during propionate degradation. Community shifts showed considerable propagation of Syntrophobacter sulfatireducens, Cryptanaerobacter sp./Pelotomaculum sp., and "Candidatus Cloacamonas sp." in the course of decreasing propionate concentration. Methanogenic species belonged mainly to the genera Methanosarcina, Methanosaeta, and Methanoculleus. Due to the prevalent presence of the syntrophic acetate-oxidizing species Tepidanaerobacter acetatoxydans and potentially autotrophic homoacetogenic bacteria (Moorella sp., Thermacetogenium sp.), a theoretical involvement of syntrophic acetate oxidation and autotrophic homoacetogenesis in stable and efficient propionate degradation was indicated. Considering theoretical Gibbs free energy values at different hydrogen partial pressures, it is noticeable that syntrophic acetate oxidation and autotrophic homoacetogenesis have the potential to counterbalance adverse hydrogen partial pressure fluctuations, stabilizing most probably continuous and stable propionate degradation.

Isolation of acetic, propionic and butyric acid-forming bacteria from biogas plants.

In this study, acetic, propionic and butyric acid-forming bacteria were isolated from thermophilic and mesophilic biogas plants (BGP) located in Germany. The fermenters were fed with maize silage and cattle or swine manure. Furthermore, pressurized laboratory fermenters digesting maize silage were sampled. Enrichment cultures for the isolation of acid-forming bacteria were grown in minimal medium supplemented with one of the following carbon sources: Na(+)-dl-lactate, succinate, ethanol, glycerol, glucose or a mixture of amino acids. These substrates could be converted by the isolates to acetic, propionic or butyric acid. In total, 49 isolates were obtained, which belonged to the phyla Firmicutes, Tenericutes or Thermotogae. According to 16S rRNA gene sequences, most isolates were related to Clostridium sporosphaeroides, Defluviitoga tunisiensis and Dendrosporobacter quercicolus. Acetic, propionic or butyric acid were produced in cultures of isolates affiliated to Bacillus thermoamylovorans, Clostridium aminovalericum, Clostridium cochlearium/Clostridium tetani, C. sporosphaeroides, D. quercicolus, Proteiniborus ethanoligenes, Selenomonas bovis and Tepidanaerobacter sp. Isolates related to Thermoanaerobacterium thermosaccharolyticum produced acetic, butyric and lactic acid, and isolates related to D. tunisiensis formed acetic acid. Specific primer sets targeting 16S rRNA gene sequences were designed and used for real-time quantitative PCR (qPCR). The isolates were physiologically characterized and their role in BGP discussed.

Genomic characterization of Defluviitoga tunisiensis L3, a key hydrolytic bacterium in a thermophilic biogas plant and its abundance as determined by metagenome fragment recruitment.

The genome sequence of Defluviitoga tunisiensis L3 originating from a thermophilic biogas-production plant was established and recently published as Genome Announcement by our group. The circular chromosome of D. tunisiensis L3 has a size of 2,053,097bp and a mean GC content of 31.38%. To analyze the D. tunisiensis L3 genome sequence in more detail, a phylogenetic analysis of completely sequenced Thermotogae strains based on shared core genes was performed. It appeared that Petrotoga mobilis DSM 10674(T), originally isolated from a North Sea oil-production well, is the closest relative of D. tunisiensis L3. Comparative genome analyses of P. mobilis DSM 10674(T) and D. tunisiensis L3 showed moderate similarities regarding occurrence of orthologous genes. Both genomes share a common set of 1351 core genes. Reconstruction of metabolic pathways important for the biogas production process revealed that the D. tunisiensis L3 genome encodes a large set of genes predicted to facilitate utilization of a variety of complex polysaccharides including cellulose, chitin and xylan. Ethanol, acetate, hydrogen (H2) and carbon dioxide (CO2) were found as possible end-products of the fermentation process. The latter three metabolites are considered to represent substrates for methanogenic Archaea, the key organisms in the final step of the anaerobic digestion process. To determine the degree of relatedness between D. tunisiensis L3 and dominant biogas community members within the thermophilic biogas-production plant, metagenome sequences obtained from the corresponding microbial community were mapped onto the L3 genome sequence. This fragment recruitment revealed that the D. tunisiensis L3 genome is almost completely covered with metagenome sequences featuring high matching accuracy. This result indicates that strains highly related or even identical to the reference strain D. tunisiensis L3 play a dominant role within the community of the thermophilic biogas-production plant.

Unraveling the microbiome of a thermophilic biogas plant by metagenome and metatranscriptome analysis complemented by characterization of bacterial and archaeal isolates.

BACKGROUND: One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood. RESULTS: The microbial community structure of an exemplary thermophilic biogas plant was analyzed by a comprehensive approach comprising the analysis of the microbial metagenome and metatranscriptome complemented by the cultivation of hydrolytic and acido-/acetogenic Bacteria as well as methanogenic Archaea. Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga (5.5 %), Halocella (3.5 %), Clostridium sensu stricto (1.9 %), Clostridium cluster III (1.5 %), and Tepidimicrobium (0.7 %) were most abundant. Among the Archaea, Methanoculleus (2.8 %) and Methanothermobacter (0.8 %) were predominant. As revealed by a metatranscriptomic 16S rRNA analysis, Defluviitoga (9.2 %), Clostridium cluster III (4.8 %), and Tepidanaerobacter (1.1 %) as well as Methanoculleus (5.7 %) mainly contributed to these sequence tags indicating their metabolic activity, whereas Hallocella (1.8 %), Tepidimicrobium (0.5 %), and Methanothermobacter (<0.1 %) were transcriptionally less active. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia, Bacilli, Thermotogae, Methanomicrobia and Methanobacteria were obtained. Genome analyses of isolates support the finding that, besides Clostridium thermocellum and Clostridium stercorarium, Defluviitoga tunisiensis participated in the hydrolysis of hemicellulose producing ethanol, acetate, and H2/CO2. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis. CONCLUSIONS: Obtained results showed that high abundance of microorganisms as deduced from metagenome analysis does not necessarily indicate high transcriptional or metabolic activity, and vice versa. Additionally, it appeared that the microbiome of the investigated thermophilic biogas plant comprised a huge number of up to now unknown and insufficiently characterized species.