Effects of heat shocks on biofilm formation and the influence on corrosion and scaling in a geothermal plant in the North German Basin

At geothermal plants, process failures often occur due to corrosion and scaling processes. Especially after heat extraction, sulfate reducing bacteria contribute to corrosion processes by producing reduced sulfur compounds. In biofilms containing scales such as iron sulfides, corrosion processes are enhanced. In a mobile bypass system located at the geothermal plant in Neubrandenburg (North German Basin), the influence of biofilm formation on corrosion and scaling was investigated. Short-term heat shocks were successfully tested in the bypass system in order to reduce biofilm formation and thus to diminish corrosion and scaling processes

Relationship between process failures and the microbial biocenosis in biogas plants of the waste industry

In dieser Doktorarbeit wurden die Zusammenhänge zwischen Prozessstörungen und der mikrobiellen Biozönose in großtechnischen Biogasanlagen aus der Abfallwirtschaft und in Laborversuchen mikrobiologisch und chemisch analysiert und verschiedene Gegenmaßnahmen untersucht. Unter Einsatz von Calciumoxid als Additiv wurde die Raumbelastung in einem Laborreaktor stufenweise bis auf 9,5 kg oTS m-3 d-1 erhöht und ein Frühwarnindikator gegen Übersäuerung zur Steuerung der Zugabe eingesetzt. Die Zusammensetzung der methanogenen Gemeinschaft blieb unter dem Einfluss höherer Raumbelastungen konstant und gewährleistete eine stabile Biogasproduktion. Genetische Fingerprintanalysen zeigten, dass syntrophe Mikroorganismen aus Wasserstoff-produzierenden Bakterien und Wasserstoff-oxidierenden Archaeen die Gemeinschaft dominierten. Infolge der Calciumoxid-Zugabe bildeten sich Aggregate, die zur Stabilität des Prozesses beitrugen und wahrscheinlich als Mikrohabitat für die Mikroorganismen fungierten. Ein Ausfall der Rührwerke und die daraus resultierende ungünstige Durchmischung verursachten eine Schwimmschichtbildung sowie eine drohende Übersäuerung in einer großtechnischen Biogasanlage. Infolge des Rührwerksausfalls und der ungleichmäßigen Verteilung des organischen Materials, verstärkten sich lokale Heterogenitäten, Säuren reicherten sich an und die Gasproduktion fiel ab. Die mikrobielle Biozönose veränderte sich durch die ungünstige Durchmischung und passte sich nach der Wiederherstellung der Durchmischung wieder an. In Verbindung mit der Zugabe von hydrophoben Substanzen wurde das filamentöse Bakterium Microthrix parvicella als Verursacher einer Schaumbildung in einer Mischschlamm und Fett Co-vergärenden großtechnischen Biogasanlage identifiziert. Die Schaumbildung ereignete sich, wenn Fett zugeführt und ein Schwellenwert von 2x108 M. parvicella Genkopien überschritten wurde. Die Erhöhung der Genkopienzahlen im nachgeschalteten Reaktor der Kaskade wies auf anaerobes Wachstum ohne Nitrat hin. Der Einfluss einer Temperaturerhöhung von 37 °C auf 56 °C auf die Abundanz von Microthrix parvicella und die damit verbundene Bildung einer Schwimmschicht wurde in zwei parallel betriebenen Laborreaktoren untersucht. Die mikrobielle Biozönose passte sich an die Temperaturerhöhung von 1 °C pro Woche an, so dass der Biogasproduktionsprozess stabil ablief. Die Abundanz von M. parvicella verringerte sich bei einer Erhöhung von 37 °C auf 39-41 °C um den Faktor 10. Die Schwimmschicht löste sich bei der Erhöhung von 37 °C auf 56 °C auf.In this PhD thesis, the interaction of the microbial biocenosis and process disturbances in full-scale biogas plants and laboratory-scale reactors were analysed microbiologically and chemically and several countermeasures were investigated. In a sewage sludge and raps oil fed laboratory-scale reactor, addition of calcium oxide was used to increase the organic loading rate stepwise up to 9.5 kg VS m-3 d-1. The process was controlled by applying an early warning indicator regarding overacidification. Genetic fingerprint analysis showed that the methanogenic community composition stayed constant during the increase in organic loading rate warranting a stable biogas production process. Syntrophic microorganisms of hydrogen producing bacteria and hydrogen consuming archaea dominated the biocenosis. Due to the addition of calcium oxide, granules were formed, which contributed to the stabilization of the process and probably served as microhabitat for the microorganisms. A formation of floating sludge as well as a threatening overacidification in a full-scale biogas plant were caused by a disturbance of the agitators. Due to the mixing failure and the uneven distribution of the organic material, local heterogeneities appeared, organic acids accumulated and the gas production decreased. The microbial biocenosis changed due to the agitator breakdown and adapted again after the mixing was re-established. The introduction of hydrophobic substances and the filamentous bacterium Microthrix parvicella were identified as the cause of a foam formation in a full-scale biogas plant fed with sewage sludge and fat, oil and grease (FOG). Foaming was caused if FOG was fed and a threshold amount of 2x108 M. parvicella gene copies was exceeded. As the biogas plant is operated as a cascade, increased gene copy numbers of M. parvicella in the downstream reactor indicated anaerobic growth without reduction of nitrate as the energy source. The influence of a temperature increase from 37 °C to 56 °C on the presence of Microthrix parvicella and the associated formation of floating sludge was investigated in two parallel operated laboratory-scale reactors. The microbial biocenosis was shown to adapt to the increase in temperature by 1 °C per week, so that the biogas production process worked properly. The occurrence of M. parvicella already decreased with an increase from 37 °C to 39-41 °C by a factor of 10. The floating sludge dissolved completely after increasing the temperature from 37 °C to 56 °C.BMU, 03KB018A-F, Vergleichende Untersuchungen an großtechnischen Biogasreaktoren - Verfahrenstechnische, mikrobiologische, ökologische und ökonomische Bewertung und Optimierung/Optga

Influence of drill mud on the microbial communities of sandstone rocks and well fluids at the Ketzin pilot site for CO_{2} storage

At a pilot site for CO_{2} storage in Ketzin (Germany), a drastic decrease in injectivity occurred in a well intended for injection. This was attributed to an obstruction of the pore throats due to microbial degradation of the organic drill mud and subsequent iron sulfide (FeS) precipitation in the highly saline brine (240 g L^{-1}). To better understand the biogeochemical processes, the response of the autochthonous microbial community to drill mud exposure was investigated. Pristine cores of two aquifers with different salinity were incubated under simulated in situ conditions (50 bar, 40 ^{\circ}C and 45 bar, 25 ^{\circ}C, respectively) and CO_{2} atmosphere. For the first time, rock cores obtained from the CO_{2} plume of the storage formation were investigated. The influence of acetate as a biodegradation product of drill mud polymers and the effectiveness of a biocide were additionally tested. Increased microbial diversities were observed in all long-term (8-20 weeks) incubations, even including biocide. Biofilm-like structures and small round-shaped minerals of probable microbiological origin were found. The results indicate that the microbial community remains viable after long-term CO_{2} exposure. Microorganisms hydrolyzing cellulose polymers (e.g., Burkholderia spp., Variovorax spp.) biodegraded organic components of the drill mud and most likely produced low molecular weight acids. Although the effects of drill mud were less strong as observed in situ, it was demonstrated that acetate supports the growth of sulfate-reducing bacteria (i.e., Desulfotomaculum spp.). The microbial-induced precipitation of amorphous FeS reduced the injectivity in the near-well area. Therefore, when using organic drill mud, the well must be cleaned intensively to minimize the hazards of bacterial stimulation

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of ≥8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer

Phylogenetic Tracking of LA-MRSA ST398 Intra-Farm Transmission among Animals, Humans and the Environment on German Dairy Farms

Methicillin-resistant Staphylococcus aureus (MRSA) are a major threat to human and animal health, causing difficult-to-treat infections. The aim of our study was to evaluate the intra-farm transmission of livestock-associated (LA) MRSA sequence type (ST) 398 isolates on German dairy farms. A total of 115 LA-MRSA ST398 isolates originating from animals, humans and the environment of six dairy farms were analyzed by whole-genome sequencing and core genome multilocus sequence typing. Phylogenetic clusters of high allelic similarity were detected on all dairy farms, suggesting a MRSA transmission across the different niches. On one farm, closely related isolates from quarter milk samples (QMS), suckers of calf feeders and nasal cavities of calves indicate that MRSA may be transferred by feeding contaminated milk to calves. Detection of related MRSA isolates in QMS and teat cups (4/6 farms) or QMS and human samples (3/4 farms) pointed out a transmission of MRSA between cows during the milking process and a potential zoonotic risk. In conclusion, LA-MRSA ST398 isolates may spread between animals, humans and the environment on dairy farms. Milking time hygiene and other internal biosecurity measures on farms and pre-treatment of milk before feeding it to calves may reduce the risk of MRSA transmission

Mammaliicoccus spp. from German Dairy Farms Exhibit a Wide Range of Antimicrobial Resistance Genes and Non-Wildtype Phenotypes to Several Antibiotic Classes

Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae

Heat Inactivation of Methicillin-Resistant <i>Staphylococcus aureus</i> Strains from German Dairy farms in Colostrum and Raw Milk

Methicillin-resistant Staphylococcus aureus (MRSA) may cause difficult-to-treat infections in dairy cattle. One possible route of MRSA transmission into calves is via the feeding of contaminated waste milk. We tested the heat resistance of 17 MRSA strains isolated from German dairy farms in colostrum and raw milk in a laboratory approach. Heating colostrum or raw milk at 60 °C for 30 min eliminated all viable MRSA in the milk, provided the MRSA inoculation rate is low (103 cfu mL−1). In contrast, raw milk highly inoculated with MRSA (106 cfu mL−1) required a holding time of at least 30 min at 70 °C to fully eliminate MRSA from it. However, quantitative analysis showed that a heat treatment for 10 min at 60 °C already significantly reduced the number of viable MRSA in highly inoculated raw milk. Heating colostrum and raw milk above 60 °C may destroy immunoglobulins which are crucial for the calf’s health. Therefore, we suggest that colostrum and raw milk that is to be fed to calves on MRSA-positive dairy farms is heated at 60 °C for at least 10 min to reduce the likelihood of transmitting MRSA. In addition, the 60 °C heat-treated colostrum/raw milk should be fed to the calves as soon as possible to avoid re-growth of viable MRSA