Institut für Nutztierwissenschaften

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The genome reduction excludes the ribosomal rescue system in acholeplasmataceae
(2022) Zübert, Christina; Ilic, Anna-Marie; Duduk, Bojan; Kube, Michael
The trans-translation process is a ribosomal rescue system for stalled ribosomes processing truncated mRNA. The genes ssrA and smpB fulfil the key functions in most bacteria, but some species have either lost these genes or the function of the ribosomal rescue system is taken over by other genes. To date, the ribosomal rescue system has not been analysed in detail for the Acholeplasmataceae. This family, in the Mollicutes class, comprises the genus Acholeplasma and the provisional taxon “Candidatus Phytoplasma”. Despite their monophyletic origin, the two clades can be separated by traits such as not representing primary pathogens for acholeplasmas versus being phytopathogenic for the majority of phytoplasmas. Both taxa share reduced genomes, but only phytoplasma genomes are characterised by a remarkable level of instability and reduction. Despite the general relevance of the ribosomal rescue system, information is lacking on coding, the genomic context and pseudogenisation of smpB and ssrA and their possible application as a phylogenetic marker. Herein, we provide a comprehensive analysis of the ribosomal rescue system in members of Acholeplasmataceae. The examined Acholeplasmataceae genomes encode a ribosomal rescue system, which depends on tmRNA encoded by ssrA acting in combination with its binding protein SmpB. Conserved gene synteny is evident for smpB, while ssrA shows a less conserved genomic context. Analysis of the tmRNA sequences highlights the variability of proteolysis tag sequences and short conserved sites at the 5′- and 3′-ends. Analyses of smpB provided no hints regarding the coding of pseudogenes, but they did suggest its application as a phylogenetic marker of Acholeplasmataceae – in accordance with 16S rDNA topology. Sequence variability of smpB provides sufficient information for species assignment and phylogenetic analysis.
Treatment of benzene and ammonium contaminated groundwater using microbial electrochemical technology and constructed wetlands
(2018) Wei, Manman; Seifert, Jana
With the rapid development of modern industry, energy shortage and environmental pollution are getting more and more serious. Groundwater pollution is one of the most important problems. A multitude of remediation techniques in situ or ex situ have been used to treat contaminated groundwater. This thesis was to investigate whether groundwater contaminated mainly by benzene and ammonium can be remediated by constructed wetlands in combination with microbial electrochemical technology. The objectives of this thesis are (i) to develop and test systems for removing pollutants and simultaneously recovering energy from contaminated groundwater, (ii) to maximize the benefits of both constructed wetland and microbial electrochemical technology while treating contaminated groundwater, (iii) to elucidate the underlying electrochemical reactions and pollutant degradation pathways, and (iv) to investigate microbial active species and functional proteins involved in benzene degradation and ammonium removal. A microbial fuel cell (MFC) equipped with an aerated cathode and a control without aeration at the cathode were designed to remove benzene and ammonium from contaminated groundwater collected in the Leuna site (Saxony-Anhalt, Germany). The performance of pollutant removal and electricity generation was investigated and compared in the two reactors. Electrochemical processes occurring in the MFC were determined by benzene and ammonium spiking experiments as well as oxygen interruption experiments. Additionally, the biodegradation pathways and dominant organisms were elucidated by compound specific stable isotope analysis (CSIA) and Illumina sequencing. The results indicated the principal feasibility of treating benzene and ammonium contaminated groundwater by a MFC equipped with an aerated cathode. Benzene (~15 mg/L) was completely removed in the MFC, of which 80% disappeared already at the anoxic anode. Ammonium (~20 mg/L) was oxidized to nitrate at the cathode; this reaction was not directly linked to electricity generation. The maximum power density was 316 mW/m3 net anoxic compartment (NAC) at a current density of 0.99 A/m3 NAC. Coulombic and energy efficiencies of 14% and 4% were obtained based on the anodic benzene degradation. Benzene was initially activated by enzymatic monohydroxylation at the oxygen-limited anode; the further anaerobic oxidation of the intermediate metabolites released electrons accompanied by electrons transfer to the anode. Dominant phylotypes at the MFC anode revealed by 16S rRNA Illumina sequencing were affiliated to the Chlorobiaceae, Rhodocyclaceae and Comamonadaceae, presumably associated with benzene degradation. Nitrification took place at the aerated cathode of the MFC and was catalyzed by phylotypes belonging to the Nitrosomonadales and Nitrospirales. The control reactor failed to generate electricity, although phylotypes affiliated to the Chlorobiaceae, Rhodocyclaceae and Comamonadaceae were dominant as well; the control reactor can be thus regarded as a mesocosm in which granular graphite was colonized by benzene degraders, but showed a lower benzene removal efficiency compared to the MFC. In order to enhance benzene and ammonium removal while simultaneously harvesting energy, a constructed wetland integrated with microbial electrochemical technology (MET-CW) was established by embedding four anode modules into the sand bed and connecting it to a cathode placed in the open pond inside a bench-scale horizontal subsurface flow constructed wetland (HSSF-CW). Compared with the control CW, enhanced benzene and ammonium removal efficiencies were found in the MET-CW. The electrochemical performances of anode modules located at the four different depths were compared; the results showed that anode modules located in the deep layer (Module 3 and 4) had the relatively high power densities whereas the power densities located in the upper layer (modules 1 and 2) were extremely low. The initial activity mechanism of benzene degradation was analyzed by CSIA. Ammonium removal processes were assessed using nitrogen isotope fractionation of ammonium. Functional proteins and active microbial species involved in nitrogen transformation processes were detected using protein-based stable isotope probing (protein-SIP) with in situ feeding of 15N-NH4+. Additionally, potential denitrification and anammox rates were measured using Nitrogen isotope tracing. The results demonstrated that benzene and ammonium removal in a CW can be improved by combination with microbial electrochemical technology. The enhanced benzene removal was linked to the use of the anode modules as electron acceptor, whereas efficient ammonium removal was probably attributed to the elimination of inhibition effects by the co-contaminant benzene. Benzene was initially activated by monohydroxylation, forming intermediates which were subsequently oxidized accompanied by extracellular electron transfer, leading to current production. Partial nitrification accompanied by either heterotrophic denitrification or nitrifier-denitrification was mainly responsible for NH4+-N removal in the MET-CW, whereas anammox played a minor role. However, the contribution of anammox was markedly increased at the location near to the anode modules. In summary, this research indicated that microbial electrochemical technology can be used to improve the performance of pollutant removal while simultaneously harvesting energy from contaminated groundwater. Especially, the combination of MET with other traditional treatment approaches (e.g. constructed wetland) is a promising alternative to treat contaminated water.
Untersuchung der Energie- und Nährstoffflüsse mikrobieller Gemeinschaften
(2017) Starke, Robert; Seifert, Jana
The activity of microorganisms was heavily investigated using the incorporation of stabile isotopes in the last decade. Here, all biomolecules but predominantly DNA, RNA, proteins and phospholipid derived fatty acids are used to trace the label in the biomass of active microbes. Thereby, the phylogenetic information decreases from DNA and RNA to proteins whereas the latter allow to describe the actual phenotype. In this work, protein stable isotope probing (protein-SIP) was applied to two different microbial systems: (a) the anaerobic mineralization of benzene and (b) the assimilation of plant-derived organic matter in soil. Labeling of the secondary metabolism of the benzene-mineralizing and sulfate-reducing community using 13C2-acetate: The well-described microbial community enriched from the Zeitz aquifer was fed with the postulated and fully 13C-labeled intermediate of syntrophic benzene fermentation, acetate, to unveil detailed secondary utilization processes. Additional acetate amended to the ongoing benzene mineralization showed no influence on sulfide produced by sulfate reduction. Instead, labeled acetate was incorporated by Campylobacterales, Syntrophobacterales, Archaeoglobales, Clostridiales and Desulfobacterales in descending order. The epsilonproteobacterial Campylobacterales featured the fastest and the highest 13C-incorporation to confirm previous metagenome-based studies and to assign a physiological role to this phylotype of the community for the first time. Metagenome based labeling of the secondary metabolism of the benzene-mineralizing and sulfate-reducing community: In this study, the population genome of the primary acetate utilizer was reconstructed from the metagenome of the benzene mineralizing community obtained by whole-genome shotgun sequencing. Genomic DNA originated from a starvation enrichment culture previously metabolizing m-xylen and enriched in the identical epsilonproteobacterial phylotype of this community. The presence of the sulfide quinone oxidoreductase (sqr) and the polysulfide reductase (psr) suggested a key role in sulfur cycling. Hence, the epsilonproteobacterial phylotype is able to oxidize otherwise toxic sulfid produced by sulfate reduction to polysulfide via SQR and its subsequent reduction to sulfide via PSR. Further, the detection of an acetate transporter (actP) and the acetyl-CoA synthetase (acsA) for acetate activation approved direct assimilation as shown in the previous study. Short-term assimilation of plant-derived organic matter in soil: In this protein-SIP study, the short-term assimilation of plant-derived organic matter in soil was demonstrated using 15N-labeled tobacco for the first time. In contrast to the postulated model in which fungi degrade plant-derived complex compounds and secrete low molecular weight compounds which are then degraded by bacteria, our study demonstrated the dominance of bacteria over fungi during the short-term assimilation of plant-derived organic matter. Bacteria outcompete fungi for the easy available plant-derived compounds until complex compounds such as cellulose and lignin are enriched and degraded by slow growing fungi. The use of multiOMIC techniques resulted in a multidimensional scheme to easily group and categorize different behaviours of microorganisms.
Untersuchungen zum Potential biotechnologischer Methoden zur Inaktivierung von tier- und humanmedizinischen Krankheitserregern der Schutzstufe 3
(2017) Hartmann, Nadja; Hölzle, Ludwig
With the increasing use of slurry in biogas plants there remains the question of the extent of the potential hazard for human and animal beings due to infectious pathogens which enter biogas plants through contaminated substrates. The aim of this doctoral thesis was to establish a suitable approach for the inactivation of infectious and zoonotic agents. Therefore, the fermentation process itself as well as potential pre- and post-procedures which can contribute to the inactivation of pathogens were analyzed. First of all, a laboratory process was established concerning an increase of temperature and dwelling time to analyze the consequences for the inactivation of the pathogens. Additionally, the possible impact of pasteurization and diverse substrates on the inactivation of different pathogens were investigated. The influence of storage to the contaminated substrates after the biogas process was also considered. Due to the distinctive tenacity of the bovine tuberculosis pathogens M. bovis and M. caprae as well as the paratuberculosis pathogen M. avium ssp. paratuberculosis (MAP). Those pathogens are used within this study. As another major cause of zoonotic, the obligate intracellular bacterium C. burnetii was used. This bacterium occurs also in high concentrations in slurry from animal populations which are tested positively of coxelliosis. Beside bacterial infection and zoonotic pathogens, viral agents are playing a major role, such as the highly contagious foot-and-mouth disease (FMD) and the virus classical swine fever (CSF). FMD was substituted by the equine rhinitis A virus (ERAV) and CSF by the bovine virus diarrhea (BVD). The results can be transferred to FMD and CSF because of the close phylogenetic relation of the surrogate viruses. The inactivation studies of M. bovis through storage over 21 days showed that there are besides the substrate and temperature specific differences also intraspecific differences. This fact should be included in future selection of the inactivation methods. At temperatures of 4 and 20 °C it was possible to detect mycobacteria throughout the entire experimental duration time. At 37 °C already after seven days no pathogens could be detected. Mycobacteria which were suspended in PBS were detectable during the whole experimental time. The storage studies with ERAV and BVDB were performed over 15 days with the different substrates. At low temperatures of 4 and 20 °C there was no significant virus reduction detectable. At higher temperatures (40 – 42 °C) after three days, a significant virus reduction for ERAV was detectable and a complete inactivation for BVD, respectively. Additionally, it could be proven, that the substrate has no impact on the reduction of ERAV. Experiments using ERAV absorbed membranes showed a substrate dependent inactivation at 40 °C. As a consequence, those adsorbed membranes were used for a biogas plant in a laboratory dimension for 24 hours in the style of the Hohenheim biogas yield test. The reduction of the titer could be seen at least after 120 minutes. Furthermore, it could be shown, that there is a significant reduction of the ERAV titer between 120 minutes and 24 hours. In regard to the success of pasteurization dependent on the substrate, no substrate dependency was found for mycobacteria. In experiments with M. bovis and M. caprae no pathogens could be found after 30 minutes incubation time at 60 °C. Investigations with MAP showed after 360 minutes at 70 °C no culturable pathogens. Pasteurization studies with C. burnetii showed no reliable data. The pasteurization studies of ERAV in dependence of three different substrates showed, that the reduction was only caused by temperature. Therefore, BVD was only combined with one substrate which had the most heterogenic composition. Concerning ERAV, after 15 minutes at 55 °C a significant reduction of the titer was detectable. For BVD at 40 °C no significant virus reduction was achieved, but a dependency of the substrate was proven. A significant reduction of the titer was achieved after 60 minutes at 45 °C in the substrate suspended viruses, 30 minutes at 50 °C, and for in cell culture suspended viruses after 60 – 120 minutes at 50 °C. The results of this thesis show the massive effect of the properties of individual pathogens on the duration of inactivation. This subordinate role should be considered within the choice of the suitable biotechnological process. To reduce the infectious risk the procedure should always orientate at the worst case: the pathogen with the highest tenacity.

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