Harder, Jens
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Harder, Jens
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Harder, Jens
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Harder, J.
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jharder@mpi-bremen.de
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Item-typ:Veröffentlichung, Microbiome in shrimp Litopenaeus vannamei aquaculture : dynamic changes and bacterial lifestyles(2019-05-29); ; ; The Pacific white leg shrimp Litopenaeus vannamei is the most frequently cultured shrimp species, comprising more than 70% of the world shrimp commodities since 2010. Shrimp indoor and outdoor aquacultures are affected by water quality deteriorations and bacterial diseases. These cultivation problems lead to a substantial annual loss of shrimp harvest. Traditionally, water quality is quantified by plankton density, total suspended solids/particulate matter, pH, salinity and inorganic nutrients, particularly ammonium, nitrite and phosphate. Especially, the quantitative assessment of the carbon cycle in shrimp aquaculture is required to determine oxygen demand and depletion. However, optimal and acceptable but potentially stressful conditions have not systematically been determined in pond aquaculture. Excess biomass formed by photosynthesis of phytoplankton and by heterotrophic bacteria in the microbial loop poses a particular problem in shrimp aquaculture. While particulate matter in pond water may serve as additional feed source for shrimps, it also provides niches for particle-associated bacteria, such as pathogenic Vibrio species. In this study, water quality parameters as well as bacterial community compositions were observed in Indonesian shrimp aquacultures over the complete rearing process. Semi-intensive and intensive aquacultures differed in suspended particulate matter content, chlorophyll a, pH, and dissolved oxygen, while inorganic nutrient concentrations and the population size of cultivable heterotrophic bacteria were comparable. Halomonas, Salegentibacter, and Sulfitobacter were the most abundant free-living bacteria, whereas particle-associated bacteria were dominated by Halomonas and Psychrobacter. Vibrio were more frequently found in the intensive aquaculture system, particularly in the particle-associated fraction and at low pH conditions. White feces disease (WFD) event, which is a disease repeatedly occurring in shrimp aquaculture, was further documented in this thesis. To better understand pond water conditions as well as bacterial community dynamics during the disease event, the quantification of physico-chemical water parameters was combined with molecular analyses of the microbiome of shrimps, feces, and aquaculture water including the particles. The WFD event coincided with a low water pH and a high proportion of Alteromonas in the feces. Virulence genes of Vibrio, i.e toxin regulator (toxR) and termolabile hemolysin (tlh) were detected in the particle fraction ( 3 AAmicrometre), in the intestine of healthy shrimps and feces of WFD-infected shrimps. An increase of pH above 8 via limestone addition enabled a recovery from WFD. In addition, the bacterial community composition also changed with the rise in pH. This observation led to a recommendation, that aquacultures of L. vannamei should maintain a pH above 8. Lastly, bacterial dynamic on aggregates, which form in large numbers in shrimp aquaculture, was investigated to monitor the growth of potential pathogenic Vibrio species. In the rolling tank experiments, the addition of carbon-rich molasses was shown to rapidly reduce toxic ammonium and nitrite pools. Furthermore, it enabled the fast growth of halophilic heterotrophic bacteria, such as Halomonas, Psychrobacter, and Salegentibacter. Conversely, in the presence of Chlorella vulgaris aggregates V. parahaemolyticus population remained constant without decay. The algal biomass seemed to maintain the Vibrio population and density. I conclude that water parameters such as salinity and pH shape bacterial communities in shrimp pond aquaculture, and that a deterioration of water quality may cause detrimental shifts in bacterial community composition. However, bacterial communities will recover to the initial composition if water parameters are adjusted to former condition. To improve shrimp farming practices, I propose to perform regular pond water assessment, not only for the physicochemical parameters, but also for bacterial community composition. For this purpose, I recommend to analyze the bacterial communities in the particle fraction, including virulence genes of pathogenic bacteria. In addition, sludge discharge and regular addition of lime stones are necessary to improve and maintain shrimp production. As consequence, sustainable shrimp pond farming systems should include sludge as well as clean water reservoirs.Dissertation552 498 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, The anaerobic linalool metabolism in the betaproteobacteria Castellaniella defragrans 65Phen and Thauera linaloolentis 47Lol(2015-12-11); ; ; The betaproteobacteria Castellaniella defragrans 65Phen and Thauera linaloolentis 47Lol were recently isolated on monoterpenes as sole carbon and energy source under denitrifying conditions. C. defragrans 65Phen metabolizes the hydrocarbon monoterpene beta-myrcene. Its activation is catalyzed by the bifunctional enzyme linalool dehydratase/isomerase. In the presented work, an improved purification protocol was developed to yield high amounts of protein for structural analysis by X-ray crystallography. The structure of the enzyme and a proposed mechanism are described. T. linaloolentis 47Lol uses the tertiary monoterpene alcohol linalool as sole carbon source. It is isomerized into the primary alcohol geraniol by the enzyme linalool isomerase. The presented work describes an enrichment of the enzyme from protein extracts and its characterization. Further degradation of geraniol via the acyclic terpene utilization pathway was shown by cultivation based and enzymatic experiments.Dissertation324 149 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Isolierung und genetische Charakterisierung von Stämmen des Planctomycetengenus Rhodopirellula und die Biogeographie in europäischen Gewässern(2009-05-25); ; ; The isolation of new strains of the planctomycete genus Rhodopirellula and their genetic characterisation should provide new insights into the diversity and biogeography of an attached- living bacterium. Within this work 130 different samples of marine water, sediment, algae and plankton have been collected and examined by cultivation experiments. Out of 19 different samples 70 new strains close related to Rhodopirellula baltica were isolated. The majority of strains was gained from European Seas with Iceland as northernmost and Greece as southernmost site. Also strains from Tansania and the Philippines were obtained. All strains showed a high similarity to R. baltica by 16S rDNA sequences and further experiments approved these results. It was also possible to distinguish different groups of isolates by techniques like DNA-DNA-hybridization and MLSA. An electron microscopic study of selected strains showed the special intracytoplasmatic compartimentation, typical for Planctomycetes.Dissertation366 382 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Biogeography of Rhodopirellula in European coastal sediments(2015-09-02); ; ; Rhodopirellula is a marine representative of the globally distributed and environmentally important bacterial phylum Planctomycetes. In cultivation studies, 70 strains had been isolated from European coastal seas and sediments and several of those strains shared a sequence identity over 99.5% of the 16S rRNA gene. A multilocus sequence analysis (MLSA) in combination with DNA-DNA hybridization experiments had arranged those strains into 13 operational taxonomic units (OTUs), suggesting a high diversity and a limited habitat size for Rhodopirellula species in European seas and sediments. The classification of microorganisms using only differences in 16S rRNA gene sequences is limited due to the high degree of conservation of this gene. Closely related species with nearly identical 16S rRNA gene sequences often show ecologically meaningful genetic and physiological differences. Thus, other genes have to be investigated to characterize biogeographic structures in environmental studies at the species level. To investigate the diversity and biogeography of Rhodopirellula in the environment, without cultivation and beyond the resolution of the commonly used 16S rRNA gene, we performed a comparison of the genomes of Rhodopirellula strains and related planctomycetes and identified the carB gene, coding for the large subunit of carbamoylphosphate synthetase, and the acsA gene, coding for acetyl CoA synthetase, as suitable molecular markers. The carB primers were designed to detect all known and potentially new Rhodopirellula species. The detection of 12 species of Rhodopirellula in a single sample from Sylt island in the North Sea demonstrated that the carB gene is a powerful molecular marker for detecting Rhodopirellula species in the environment and may be used for the taxonomic evaluation of new strains. R. baltica, 'R. europaea' and 'R. islandica' were present in Sylt sediments. The biogeography of these three species in intertidal sediments around Europe was investigated targeting the acsA partial gene sequence with specific primers. Growth experiments in different temperature, salinity and light conditions showed that the three Rhodopirellula species have different optimal growth conditions, however, they all grew in all the conditions tested. The acsA study, in agreement with the growth experiments, revealed the presence of all three species in all regions sampled and provided evidence that the Rhodopirellula species are ubiquitously present as a seed bank in European and Philippines samples.Dissertation259 146 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Cultivation of Flavobacteria and other in situ abundant bacteria from the North Sea(2013-06-17); ; ; The isolation and cultivation of heterotrophic marine bacteria opens possibilities to study their physiology and genomes with respect to their function in the marine environment. In the pelagic marine realm bacteria remineralize more than half of the photosynthetically produced biomass, and thus play an important role in the biogeochemical cycling of elements. Flavobacteria are abundant of up to 30% in the North Sea. In previous studies marine Gammaproteobacteria, Alphaproteobacteria and Actinobacteria were predominantly cultivated, but cultures of Flavobacteria were infrequently obtained. This thesis addresses the isolation of phylogenetically diverse marine Flavobacteria using three new approaches. First, samples were retrieved from various pelagic and benthic habitats of the North Sea. Second, a new marine artificial seawater HaHa medium was developed to facilitate the growth of Flavobacteria. This medium was supplemented with saccharides and proteins as carbon sources at a concentration of 2 g/L. Third, a specific 16S rRNA gene PCR assay was applied to identify Flavobacteria-Cytophagia among the colonies. The molecular screen was preferred over the identification by cell and colony morphology, since the latter has predominantly resulted in the isolation of strains of the genera Arenibacter, Cellulophaga and Maribacter. The 375 Flavobacteriaceae strains isolated on agar plates comprised (i) seven presumably novel genera, (ii) 42 presumably novel species in 22 validly described Flavobacteriaceae genera and (iii) many isolates that were so far not distinguishable from 37 type strains in 16 genera. Thus, in contrast to previous studies, we could show that phylogenetically diverse Flavobacteria from the North Sea can be cultivated on solid medium. The isolation of representative strains of the genera Formosa, Polaribacter, and Reinekea from the North Sea was attempted. In previous studies these bacterial populations were proposed to be of importance during coastal diatom-dominated phytoplankton blooms, based on their high abundance of 15% to 25% of the bacterioplankton and their potential capability to decompose algae derived polysaccharides. A new medium was devised which had the same composition as the marine HaHa medium, but with environmental-like micromolar carbon, nitrogen, and phosphate concentrations. Aerobic dilution cultivation in the HaHa medium led to a high culturability of 35% of the bacterioplankton in spring 2010 and 27% of the bacterioplankton in summer 2010. 23 strains of Flavobacteria, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were obtained directly by dilution cultivation of single cell inocula. One strain that belonged to the genus Reinekea was isolated by generating co-cultivatures of randomly mixed bacterial populations which potentially had a positive effect on the growth of Reinekea. Strains that affiliated with Polaribacter , Formosa, Gillisia (Flavobacteria), the Roseobacter clade associated (RCA) lineage (Alphaproteobacteria), Reinekea, and the OM182 clade (Gammaproteobacteria) had 16S rRNA gene sequence identities of >99.9% with 16S rRNA clones of the bacterioplankton from the North Sea in spring 2009. In addition, draft genomes of Formosa, Polaribacter , and Reinekea strains were used to recruit reads of metagenomes of the bacterioplankton in spring 2009. Thereby, reads of >95% nucleotide identity covered the draft genomes of the Formosa clade B strain to 94%, of Reinekea sp. to 90% and of Polaribacter sp. to 50%. Based on these results we argue that the novel species of Formosa, Polaribacter , and Reinekea are representatives of ecologically relevant clades catalyzing the remineralization of coastal diatom-dominated phytoplankton biomass. The physiological characteristics of the strains were investigated focusing on the growth on different mono- and polysaccharides, to provide further evidence that Formosa, Polaribacter and Reinekea species could prevail in different ecological niches during algae decay. Interestingly, Polaribacter strains grew heterotrophically on all tested sulfated (e.g. agar, carrageenan) and non-sulfated polysaccharides (e.g. cellulose, laminarin), whereas Formosa strains grew only on non-sulfated polysaccharides. In contrast, Reinekea sp. did not grow on polysaccharides but on all tested mono-, di-, and trisaccharides including N-acetylneuraminic acid. Finally, I proposed for these novel species the names Formosa flavarachnoidea , Formosa forsetii , Polaribacter forsetii , Polaribacter frigidimaris , Polaribacter adhaesivus , and Reinekea forsetii .Dissertation411 344 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Characterization of a limonene-degrading methanogenic enrichment culture(2024-12-06); ; ; In this study, a limonene-degrading methanogenic enrichment culture was analyzed, revealing new insights into the predatory microbial interactions and potential limonene degradation pathways. The enrichment culture, established in 1999 using activated sludge obtained from a wastewater treatment plant, sustains a complex microbial community despite being supplied only with limonene as a carbon source. Using long-read PacBio sequencing, a metagenome-assembled genome assigned to the Syntrophobacteraceae family was identified as responsible for the initial step of limonene degradation under methanogenic conditions. Within the genome of the Syntrophobacteraceae MAG, two operons were identified, with metatranscriptomic and metaproteomic data verifying the transcription of the genes and the presence of the proteins in the metaproteome. The initial activation of limonene is catalyzed by an enzyme annotated as benzylsuccinate synthase, which forms a new phylogenetic branch within the known and characterized fumarate-adding enzymes and was therefore named limonenylsuccinate synthase. Limonenylsuccinate synthase is possibly the first enzyme detected for monoterpene degradation in a methanogenic environment. With 32 MAGs assembled from the metagenome, the microbial community in the enrichment culture is a complex syntrophic community. Most members of the community can be assigned to syntrophic bacteria, methanogenic archaea, or fermenters, who work together to degrade limonene. However, there is a predator thriving in the culture. Candidatus Velamenicoccus archaeovorus, a member of the candidate phylum Omnitrophota, that was visualized using CARD-FISH in combination with high-resolution microscopy and was found attached to the methanogen Methanothrix soehngenii and other microorganisms within the enrichment culture. Candidatus Velamenicoccus archaeovorus transcribes genes that allow it to attach to its prey, lyse the cells, and consume their biomass, showing similarities to the mechanisms of other predatory bacteria. Furthermore, a group I intron was detected in the predator’s 23S rRNA gene and could be visualized using CARD-FISH in its prey cells, showing the transfer of mobile genetic elements between prey and host. The predation process introduces a new trophic level into the microbial community as it results in the release of necromass. The necromass, consisting of carbohydrates, lipids, and proteins from dead cells, is expected to be fermented by other members of the community, such as Anaerolineaceae and Lentimicrobium, closing the microbial loop and facilitating further syntrophic interactions. The complex interplay between syntrophic bacteria, methanogens, and the predatory bacterium illustrates the intricate dynamics within this anaerobic system. The discovery of the novel limonenylsuccinate synthase enzyme and the predatory role of Candidatus Velamenicoccus archaeovorus highlights the metabolic complexity of microbial biomass turnover in energy-limited ecosystems.Dissertation153 156 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Anaerobic degradation of limonene andp-xylene in freshwater enrichmentcultures(2009-06-09); ; ; The anaerobic degradation of hydrocarbons has been intensively explored in the last decade yielding insights into new physiological capabilities and biochemical pathways. However, for a few hydrocarbons, e.g. p-xylene, it proved to be more difficult to enrich microorganisms and to isolate pure strains. Thermodynamically, the mineralization of hydrocarbons is least favorable under anoxic conditions and especially under methanogenic conditions. In my thesis, two enrichment cultures were characterized, a methanogenic freshwater enrichment culture grown on limonene, the most abundant monoterpene in nature, and a denitrifying freshwater enrichment culture grown on p-xylene. The methanogenic enrichment culture consumed limonene with proportional formation of methane. The full cycle 16S rRNA gene approach revealed the presence of Archaea related to Methanosaeta and Methanoculleus and Bacteria related to Syntrophobacteraceae, Bacteroidetes, and the Candidate Division OP3. This candidate phylum lies within the Planctomycetes, Chlamydiae, Verrucomicrobia, Lentisphaerae superphylum and has no member in culture. Hence nucleic acid probes were developed to target the OP3-phylotype. The probe detected very small spherical cells, which lived alone or attached to larger cells and represented 18% of the total DAPI-stained population. Thus they may play an important role in limonene degradation. The other Bacteria were mainly Deltaproteobacteria (13%) and only 1% were Bacteroidetes. Together with a new EUB-338 probe specific for the OP3 cells, the bacterial 338 probe mixture detected 40% of the total cells whereas the Archaea probe detected 33%. The presence of several phylotypes suggests that more then one syntrophic bacterium and one methanogenic archaeon are involved in limonene degradation to methane gas and carbon dioxide. Syntrophic bacteria were isolated using fumarate and lactate as organic substrates for fermentation. All seven strains belonged phylogenetically to Deltaproteobacteria, with 11% 16S rRNA gene dissimilarity to Desulfoarculus baarsii. Initial co-culture experiments with Methanosarcina mazei showed growth on lactate or fumarate in the presence of limonene. However, their role in limonene degradation could not demonstrated. Denitrifying enrichment cultures were established with a freshwater sediment mix and p-xylene as sole electron donor and carbon source. Several batch transfers and liquid dilution-to-extinction series enriched a curved rod morphotype, 0.5 by 2 μm in size. This dominant morphotype (96% of all cells) was as well identified as the dominant phylotype (91-95%) using the 16S rRNA full cycle approach. The organism affiliated phylogenetically with Betaproteobacteria and not with other anaerobic hydrocarbon degrading microorganisms from the Azoarcus-Thauera clade. Their closest relative was a group of steroid-degraders: Denitratisoma oestradiolicum, Sterolibacterium denitrificans and strain 72Chol.This enrichment cultures coupled complete mineralization of p-xylene to denitrification of nitrate to dinitrogen gas. By gas chromatography mass spectrometric analysis of metabolites found in cell extracts, (4-methylbenzyl)succinate and (4-methylbenzyl)itaconate were identified, supporting an activation mechanism by addition to fumarate. A gene fragment for a benzylsuccinyl synthase could be sequenced, and revealed amino acid similarities with TutD from the well known toluene degrading denitrifier, Thauera aromatica. This study has revealed that anaerobic hydrocarbon degradation involves a broad diversity of microorganism, even outside the phylum Proteobacteria. My thesis established the participation of novel organisms to anaerobic hydrocarbon degradation and revealed for the first time the morphology of Candidate phylum OP3 cells.Dissertation279 228 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Complex algal polysaccharides as substrates for Maribacter strains(2024-10-07); ; ; Marine bacteria play a crucial role in global nutrient cycles, yet much remains to be understood about their ecophysiological niches, metabolic dependencies, and the functions of essential proteins. The research unit FOR 2406 Proteogenomics of Marine Polysaccharide Utilization investigates the mechanisms behind bacterial polysaccharide utilization during marine phytoplankton blooms. The focus is particularly on the functional analyses of marine bacteria within the phylum Bacteroidota. Flavobacteriia, a prominent class within the phylum Bacteroidota, are a significant component of marine bacterioplankton. Free-living members of this class play a pivotal role in the degradation of polysaccharides from lysed algae. They often harbour carbohydrate-active enzymes within polysaccharide utilization loci, operon- like genetic regions encoding proteins responsible for the hydrolysis and transport of polysaccharides. Fierce competition among free-living bacteria has led to genomic streamlining, typically focusing on a select few polysaccharides. In contrast, particle-associated bacteria face diverse substrate challenges. Some have the ability to sense and migrate toward nutrient sources, while others reside attached to particulate organic matter (POM), resulting in larger genomes compared to their free-living counterparts. Despite their ecological importance, polysaccharide utilization by particle-associated bacteria has received limited attention. Maribacter, observed in particle-associated fractions within marine systems, represent a significant gap in physiological studies. To address this gap, we employed proteomics to investigate the complexity of polysaccharide utilization mechanisms in Maribacter.Dissertation181 280 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Die genetische und biochemische Charakterisierung von Enzymen des anaeroben Monoterpen-Abbaus in Castellaniella defragrans(2011-12-14); ; ; Castellaniella defragrans is a betaproteobacterium metabolizing monoterpenes anaerobically under denitrifying conditions. Metabolite studies as well as a differential proteomic approach led to the identification of a linalool dehydratase-isomerase (ldi/LDI), a geraniol dehydrogenase (gedh/GeDH) and a candidate gene for a geranial dehydrogenase (gadh/GaDH). The LDI and GeDH were subjected to an initial biochemical characterization. In order to gain insight into the in vivo function of these genes a genetic system for C. defragrans was developed based on a suicide vector. After confirmation of the correct genetic background the physiological characaterization of the obtained deletionmutants verified the postulated degradation pathway for β-myrcene. Besides, the results disclosed another monoterpene degradation pathway as well as novel enzyme activities. In addition to the genetic and physiological characterization the enzymes were heterologously expressed in E. coli and biochemically studied. The bifunctionally LDI catalyzes enantiospecifically the hydration of β-myrcene to (S)-( )-linalool with an enantiomeric excess of > 95 % that is further isomerised to geraniol. Chemical modification of cysteine, histidine, aspartic and glutamic acid residues inhibited the enzyme activity. By the deletion of the ldi gene in C. defragrans a phenotype was caused that lost the ability of degrading the acyclic β-myrcene, but cyclic monoterpene degradation was not effected. A novel enzyme activity acting on (R,S)-(±)-linalool was observed, too. The heterologously expressed GeDH was purified to homogeneity and native PAGE in combination with an activity staining revealed the native conformation as dimer. With regards to their kinetic properties recombinant and wild type GeDH correlated well with a high affinity for geraniol. Due to the deletion of the gedh mutant strains revealed an impaired growth on monoterpenes suggesting the presence of further alcohol dehydrogenases acting on geraniol. The candidate gene for a geranial dehydrogenase, which was found to be induced by growth on monoterpenes, interceded specifically the oxidation of geranial. The cis-isomer was not converted. The formation of NAD was in a stoichiometric ratio. To our knowledge, both GeDH and GaDH are the first monoterpenoid specific dehydrogenases originating from a bacterium.Dissertation313 169 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Polyphasische Taxonomie, Kerngenom und Lebenszyklus von Rhodopirellula-Stämmen(2011-07-04); ; ; This thesis focused on three major projects dealing with the planctomycetes of the species Rhodopirellula baltica. In a polyphasic taxonomic study the morphological, physiological and chemotaxonomic characteristics of 17 newly isolated strains were investigated. Based on the obtained results and phylogenetic data ten new Rhodopirellula species were introduced along with one new genus. The physiology of the strains was studied with regard to the influence of ammonium on their life style. Using flow cytometry an increase of aggregated cells compared to single cells was observed parallel to an increase of the ammonium concentration. In chemostat cultures an ammonium shock induced the formation of a R. baltica biofilm by attachment of cells to the fermenter wall. With ten strains the Rhodopirellula core genome was determined by microarray analysis and genome sequencing. Both methods were compared. The R. baltica core genome contains 3314 shared genes according to microarray analysis and 4232 shared genes according to genome annotation. The investigations of this work are the basis for future comparative studies between Rhodopirellula species.Dissertation406 149