Bioinformatic progress and applications in metaproteogenomics for bridging the gap between genomic sequences and metabolic functions in microbial communities.

Metaproteomics of microbial communities promises to add functional information to the blueprint of genes derived from metagenomics. Right from its beginning, the achievements and developments in metaproteomics were closely interlinked with metagenomics. In addition, the evaluation, visualization, and interpretation of metaproteome data demanded for the developments in bioinformatics. This review will give an overview about recent strategies to use genomic data either from public databases or organismal specific genomes/metagenomes to increase the number of identified proteins obtained by mass spectrometric measurements. We will review different published metaproteogenomic approaches in respect to the used MS pipeline and to the used protein identification workflow. Furthermore, different approaches of data visualization and strategies for phylogenetic interpretation of metaproteome data are discussed as well as approaches for functional mapping of the results to the investigated biological systems. This information will in the end allow a comprehensive analysis of interactions and interdependencies within microbial communities.

Hydrodynamic disintegration of thickened excess sludge and maize silage to intensify methane production: Energy effect and impact on microbial communities.

The aim of this project was to study the combination of two methods to increase methane production: feedstock pretreatment by hydrodynamic disintegration and co-digestion of maize silage (MS) with thickened excess sludge (TES). Disintegration of TES alone resulted in a 15% increase in specific methane production from 0.192 Nml/gVS (TES + MS) to 0.220 Nml/gVS (pretreated TES + MS). The energy balance revealed additional energy (0.14 Wh) would cover only the energy expenditure for the mechanical pretreatment and would not allow for net energy profit. Identification of the methanogenic consortia by 16S rRNA gene amplicon sequencing revealed that Chloroflexi, Bacteroidota, Firmicutes, Proteobacteria and Actinobacteriota were five most abundant bacteria phyla, with Methanothrix and Methanolinea as the dominant methanogens. Principal component analysis did not show any effect of feedstock pretreatment on methanogenic consortia. Instead, the composition of inoculum was the decisive factor in shaping the microbial community structure.

Identification and ecophysiological characterization of epiphytic protein-hydrolyzing saprospiraceae ("Candidatus Epiflobacter" spp.) in activated sludge.

The identity and ecophysiology of a group of uncultured protein-hydrolyzing epiphytic rods attached to filamentous bacteria in activated sludge from nutrient removal plants were investigated by using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. The epiphytic group consists of three closely related clusters, each containing 11 to 16 clones. The closest related cultured isolate is the type strain Haliscomenobacter hydrossis (ATCC 27775) (<87% similarity) in the family Saprospiraceae of the phylum Bacteroidetes. Oligonucleotide probes at different hierarchical levels were designed for each cluster and used for ecophysiological studies. All three clusters behaved similarly in their physiology and were specialized in protein hydrolysis and used amino acids as energy and carbon sources. They were not involved in denitrification. No storage of polyphosphate and polyhydroxyalkanoates was found. They all colonized probe-defined filamentous bacteria belonging to the phyla Chloroflexi, Proteobacteria, and candidate phylum TM7, with the exception of cluster 1, which did not colonize TM7 filaments. The three epiphytic clusters were all widespread in domestic and industrial wastewater treatment plants with or without biological phosphorus removal, constituting, in total, up to 9% of the bacterial biovolume. A new genus, "Candidatus Epiflobacter," is proposed for this epiphytic group in activated-sludge treatment plants, where it presumably plays an important role in protein degradation.

Quenching effects in the application of multi-channel fluorescence in activated sludge suspended solids.

The potential of multi-channel fluorescence as an in-line monitor of the activated sludge process has been investigated. Humic substances are known to be present as an important constituent in most activated sludges, and it was tested whether humic substances influenced the fluorescence signals from protein and NADH/NADPH. A negative correlation between the humic acid (HA) signal and the signals from both protein and NADH/NADPH was found. This phenomenon was investigated in detail by studying the interaction between a standard HA (Janssen) and a standard protein (bovine serum albumin, BSA). When mixing HA and BSA, the fluorescence signal from BSA declined or even disappeared. The quenching of the fluorescence from BSA was shown to relate to an adsorption of HA onto the protein as demonstrated by capillary electrophoresis. This quenching phenomenon was also observed in activated sludge, which was spiked with HA. As 250 mg HA/L was added to activated sludge, the fluorescence signal from both protein and NADH/NADPH was reduced by 20-30%. The results indicate that it is difficult to carry out quantitative analyses of the components in activated sludge by multi-channel fluorescence, and that variations in HA can affect the signals representing the biomass and the biological activity.