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Marine sponges represent an excellent example of host-microbe symbiosis. Yet, most sponge-associated microorganisms remain uncultured, hampering efforts in determining interactions with their sponge host. This project aimed at enhancing our understanding of the sponge-bacteria interplay, focusing on both cultured and yet-uncultured sponge-associated lineages. Several novel sponge-associated taxa were elucidated by resolving their phylogeny and reconstructing their primary and secondary metabolism. Genome-based predictions revealed that the sponge microbiome mediates the interactions of the holobiont via heterotrophy, potential metabolite exchange and chemical defense. Sponge symbionts also possessed an extended genomic repertoire of defense systems implying adaptation to host environment. Moreover, genome mining for biosynthetic gene clusters (BGCs) and bioactivity screening of sponge-associated strains detected many candidate BGCs potentially involved in observed anticancer and antibacterial activity. Altogether, this work shed light into novel sponge-associated phylotypes, features that point at a symbiotic lifestyle and a rich secondary metabolite biosynthesis arsenal with biotechnological potential.

In the deep ocean symbioses between microbes and invertebrates are emerging as key drivers of ecosystem health and services. We present a large-scale analysis of microbial diversity in deep-sea sponges (Porifera) from scales of sponge individuals to ocean basins, covering 52 locations, 1077 host individuals translating into 169 sponge species (including understudied glass sponges), and 469 reference samples, collected anew during 21 ship-based expeditions. We demonstrate the impacts of the sponge microbial abundance status, geographic distance, sponge phylogeny, and the physical- biogeochemical environment as drivers of microbiome composition, in descending order of relevance. Our study further discloses that fundamental concepts of sponge microbiology apply robustly to sponges from the deep-sea across distances of >10,000 km. Deep-sea sponge microbiomes are less complex, yet more heterogeneous, than their shallow-water counterparts. Our analysis underscores the uniqueness of each deep-sea sponge ground based on which we provide critical knowledge for conservation of these vulnerable ecosystems.

The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.

Chloroform (CF) is an environmental contaminant that can be naturally formed in various environments ranging from forest soils to salt lakes. Here we investigated CF removal potential in sediments obtained from hypersaline lakes in Western Australia. Reductive dechlorination of CF to dichloromethane (DCM) was observed in enrichment cultures derived from sediments of Lake Strawbridge, which has been reported as a natural source of CF. No CF removal was observed in abiotic control cultures without artificial electron donors, indicating biotic CF dechlorination in the enrichment cultures. Increasing vitamin B12 concentration from 0.04 to 4 µM in enrichment cultures enhanced CF removal and reduced DCM formation. In cultures amended with 4 µM vitamin B12 and 13C labelled CF, formation of 13CO2 was detected. Known organohalide-respiring bacteria and reductive dehalogenase genes were neither detected using quantitative PCR nor metagenomic analysis of the enrichment cultures. Rather, members of the order Clostridiales, known to co-metabolically transform CF to DCM and CO2, were detected. Accordingly, metagenome-assembled genomes of Clostridiales encoded enzymatic repertoires for the Wood-Ljungdahl pathway and cobalamin biosynthesis, which are known to be involved in fortuitous and nonspecific CF transformation. This study indicates that hypersaline lake microbiomes may act as a filter to reduce CF emission to the atmosphere

Research described in this thesis aimed to investigate the microalgal microbial ecology by integrating both cultivation-dependent and cultivation-independent approaches. In addition, we examined effects of bacteria on growth of microalgae by developing a coculture system. Understanding the bacterial community composition and functional associations between microalgae and bacteria will provide important insights for both aquatic ecology and algal biotechnology.Microalgae are living with diverse microbial communities and interactions between microalgae and bacteria are prevalent and strongly influence carbon and nutrient cycling in aquatic ecosystems. A brief overview of the current knowledge of algae-bacteria interactions and current research methodologies was given in chapter 1. This chapter furthermore introduced the microalgae production facilities at AlgaePARC, and highlighted two research objects (Nannochloropsis sp. and Botryococcus braunii), and current knowledge on their associated bacteria.In chapter 2 we comprehensively reviewed the recent research progress on algae-bacteria interactions and summarized the current knowledge on functional aspects of algae-associated microbiomes. In addition, we discussed the applications of knowledge on algae-bacteria interactions in algal biomass production and various other related biotechnological innovations. Finally, we concluded that principles of algae-bacteria interactions can be integrated into many aspects of the algal production chain, which may help push the limit of the algal industry.In chapter 3, we studied the bacterial community associated with 12 strains of B. braunii. The changes of bacterial community composition were monitored over a period of 12 days. It was clear from this study that B. braunii hosts a wide variety of bacterial species, among which the bacterial families Rhizobiaceae, Bradyrhizobiaceae and Comamonadaceae were found in all 12 strains. These families that all belong to the phylum Proteob

This thesis aims to investigate the impact of depth on prokaryotic community composition and metabolite profiles, including antimicrobial activities, in marine sponges. Understanding both the prokaryotic community and metabolite profiles of sponges in relation to depth will provide important insights for marine ecology and future bioprospecting efforts of bioactive compounds from the sponge holobiont. A brief overview of prokaryotic community composition in sponges from shallow water to the deep sea and motivation of this research is described in chapter 1. Chapter 2 is a comprehensive literature review on antimicrobial activities (antiviral, antibacterial, antifungal and antiprotozoal) of microbial isolates from marine sponges. The most potent antimicrobial compounds against microbial targets documented to date based on in vitro tests are: 2-undecyl-4-quinolone (human immunodeficiency virus 1 (HIV-1), truncateol M (influenza A virus), thiopeptide YM-266183 (nosocomial Gram positive bacteria), sydonic acid (Escherichia coli), naphthacene glycoside SF2446A2 (Chlamydia trachomatis), manzamine A (Plasmodium spp. and Leishmania donovani), valinomycin and staurosporine (Trypanosoma brucei), and saadamycin (Candida albicans and dermatophytic fungi). Furthermore, we identified Streptomyces, Pseudovibrio, Bacillus, Aspergillus and Penicillium as the leading producers of currently known antimicrobial compounds.In chapter 3, the impact of depth on prokaryotic community composition and antimicrobial activities of two demosponges, Xestospongia muta and Agelas sventres, was investigated. Sponges were sampled from three depth categories: shallow (< 30 m), medium (30 – 60m) and deep (> 60 m) from the Curaçao Sea. Our findings showed that specific OTUs assigned to Cyanobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Proteobacteria and Thaumarchaeota contributed to the significant variance in prokaryotic community composition observed along the depth gradient. Shallow sponge s

The research described in this thesis had the goal to increase our understanding of marine sponge microbial ecology by integrating both cultivation-dependent and –independent approaches. Simultaneously, steps towards accessing the biotechnological potential of sponge-associated microbes are presented in this work. Thus, the insights presented here will deepen our understanding of sponge microbial ecology as well as provide directions for further bioprospecting efforts targeting marine sponges, especially Aplysina species. Sponges harbor dense and diverse microbial communities, and are key members of marine ecosystems. Chapter 1 introduced the sponge, provided an overview of the importance of these animals in their environment and summarized the current knowledge on functional aspects of their associated microbiomes. This chapter furthermore outlined the biotechnological potential inherent to sponge-associated microorganisms, such as the production of secondary metabolites with antibiotic, antiviral and anticancer properties. Furthermore, a brief introduction of microbial cultivation was given, and previous efforts on obtaining sponge-associated microbes in culture were highlighted. In many cases, the microbes inhabiting sponges have been demonstrated to be the actual producers of often halogenated bioactive secondary metabolites. Microorganisms attach halogen atoms such as chlorine or bromine to organic scaffolds using specialized enzymes, including halogenases. Such enzymes are of major biotechnological interest for the production of pharmaceutical or agrochemical compounds, since they halogenate regioselectively and under mild reaction conditions. In Chapter 2, six sponge species from the genus Aplysina were screened for flavin-dependent tryptophan halogenase sequence variants as well as the composition and structure of their bacterial communities using a PCR-based approach. In these sponge species from the Mediterranean and Caribbean seas we detected four phyloge

Microalgae are photosynthetic organisms that are found worldwide in many different aquatic environments and therefore display an immense biological diversity. They are a promising source of many useful polymers that have industrial applications such as food, fuel, material and pharmaceutical. One microalga that has gathered quite a research community is Botryococcus braunii. The reason for its scientific club is the fact it can synthetize long chain hydrocarbons molecules from C20 to C40. These hydrocarbons have been found in oil-shales and tests show that it can be used as aviation fuel. Besides producing hydrocarbons, some strains of B. braunii can produce exopolysaccharides (EPS) composed mainly of galactose and a small fraction of fucose. The EPS has interesting rheological properties for the food industry and potential active compounds that could be used in the pharmaceutical industry . Like many other microorganisms, microalgae in the natural environment are usually in the presence of bacteria. The presence of bacteria with microalgae can either have a beneficial or an antagonistic effect. For B. braunii little is known about the bacteria community present especially for the EPS producing strain. For that reason, the aim of this thesis was to investigate B. braunii’s associated bacteria with the aim of improving B. braunii’s biomass growth and hydrocarbon and EPS content. In chapter one, we introduced the topic of microalgae as a potential source of sustainable polymers and we introduced the species B. braunii, describing its characteristics and scientific interest. It is also introduced the topic of microalgae and bacteria associations by looking at other studies from literature. In chapter two, 16 publically available strains of B. braunii were ordered in culture banks and screened for biomass productivity, hydrocarbon and total carbohydrate content. The aim of the study was to identify one or more good strains that displayed high biomass productivity as wel

Pseudovibrio is a marine bacterial genus, members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesized that they form a mutualistic relationship with their hosts. Here, we studied their phylogeny highlighting genetic adaptations that may play a role in host colonization, by comparative genomics of 31 Pseudovibrio strains of which 25 were isolated from sponges., Pseudovibrio is a marine bacterial genus, members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesized that they form a mutualistic relationship with their hosts. Here, we studied their phylogeny highlighting genetic adaptations that may play a role in host colonization, by comparative genomics of 31 Pseudovibrio strains of which 25 were isolated from sponges.

We use a novel approach (an analysis of metatranscriptomics data) to study antibiotic resistance, a topic that poses major health care concerns. We investigate resistance gene expression in microbial communities in a diverse range of environments., We use a novel approach (an analysis of metatranscriptomics data) to study antibiotic resistance, a topic that poses major health care concerns. We investigate resistance gene expression in microbial communities in a diverse range of environments.

Larval zebrafish was subjected to a methodological exploration of the gastrointestinal microbiota and transcriptome. Assessed was the impact of two dietary inclusion levels of a novel protein meal (NPM) of animal origin (ragworm Nereis virens) on the gastrointestinal tract (GIT). Microbial development was assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by pyrosequencing. Differentially expressed genes in the GIT were demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq). Larval zebrafish showed rapid temporal changes in microbial colonization but domination occurred by one to three bacterial species generally belonging to Proteobacteria and Firmicutes. The high iron content of NPM may have led to an increased relative abundance of bacteria that were related to potential pathogens and bacteria with an increased iron metabolism. Functional classification of the 328 differentially expressed genes indicated that the GIT of larvae fed at higher NPM level was more active in transmembrane ion transport and protein synthesis. mRNAseq analysis did not reveal a major activation of genes involved in the immune response or indicating differences in iron uptake and homeostasis in zebrafish fed at the high inclusion level of NPM

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included ß-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

Background Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene is transforming the methodological landscape describing microbial diversity within and across complex biomes. However, there is a strong need for standardisation as each new combination of experimental choices affects the results in different ways, restricting true meta-analyses. Results Here we present NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities, specifically designed to challenge issues regarding optimization of routinely used filtering parameters. By sequencing two tandem variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform, the microbial composition of 49 independently amplified mock samples was characterized. This setup allowed for the evaluation of important factors of technical bias in taxonomic classification: 1) run-to-run sequencing variation 2) PCR – error 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 96%. On average 99.94% of the reads could be assigned to at least family level, while assignment to ‘spurious genera’ represented on average only 0.02% of the reads per sample. Pearson correlations between obtained and expected compositions at genus level were as high as 0.94, and Unifrac distance based PCoA plots confirmed biology guided clustering rather than the aforementioned technical aspects. Conclusions NG-Tax demonstrated improved qualitative and quantitative representation of the true sample composition. The high robustness of the pipeline against technical biases associated with 16S rRNA gene amplicon sequencing studies will additionally improve comparability between studies and facilitate efforts towards standardization., Background Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene is transforming the methodological landscape describing microbial diversity within and across complex biomes. However, there is a strong need for standardisation as each new combination of experimental choices affects the results in different ways, restricting true meta-analyses. Results Here we present NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities, specifically designed to challenge issues regarding optimization of routinely used filtering parameters. By sequencing two tandem variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform, the microbial composition of 49 independently amplified mock samples was characterized. This setup allowed for the evaluation of important factors of technical bias in taxonomic classification: 1) run-to-run sequencing variation 2) PCR – error 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 96%. On average 99.94% of the reads could be assigned to at least family level, while assignment to ‘spurious genera’ represented on average only 0.02% of the reads per sample. Pearson correlations between obtained and expected compositions at genus level were as high as 0.94, and Unifrac distance based PCoA plots confirmed biology guided clustering rather than the aforementioned technical aspects. Conclusions NG-Tax demonstrated improved qualitative and quantitative representation of the true sample composition. The high robustness of the pipeline against technical biases associated with 16S rRNA gene amplicon sequencing studies will additionally improve comparability between studies and facilitate efforts towards standardization.

The impact of the installation of a technologically advanced wastewater treatment plant (WWTP) on the benthic microbial community of a vinyl chloride (VC) impacted eutrophic river was examined two years before, and three and four years after installation of the WWTP. Reduced dissolved organic carbon and increased dissolved oxygen concentrations in surface water and reduced total organic carbon and total nitrogen content in the sediment were recorded in the post-WWTP samples. Pyrosequencing of bacterial 16S rRNA gene fragments in sediment cores showed reduced relative abundance of heterotrophs and fermenters such as Chloroflexi and Firmicutes in more oxic and nutrient poor post-WWTP sediments. Similarly, quantitative PCR analysis showed 1-3 orders of magnitude reduction in phylogenetic and functional genes of sulphate reducers, denitrifiers, ammonium oxidizers, methanogens and VC-respiring Dehalococcoides mccartyi. In contrast, members of Proteobacteria adapted to nutrient-poor conditions were enriched in post-WWTP samples. This transition in the trophic state of the hyporheic sediments reduced but did not abolish the VC respiration potential in the post-WWTP sediments as an important hyporheic sediment function. Our results highlight effective nutrient load reduction and parallel microbial ecological state restoration of a human-stressed urban river as a result of installation of a WWTP., The impact of the installation of a technologically advanced wastewater treatment plant (WWTP) on the benthic microbial community of a vinyl chloride (VC) impacted eutrophic river was examined two years before, and three and four years after installation of the WWTP. Reduced dissolved organic carbon and increased dissolved oxygen concentrations in surface water and reduced total organic carbon and total nitrogen content in the sediment were recorded in the post-WWTP samples. Pyrosequencing of bacterial 16S rRNA gene fragments in sediment cores showed reduced relative abundance of heterotrophs and fermenters such as Chloroflexi and Firmicutes in more oxic and nutrient poor post-WWTP sediments. Similarly, quantitative PCR analysis showed 1-3 orders of magnitude reduction in phylogenetic and functional genes of sulphate reducers, denitrifiers, ammonium oxidizers, methanogens and VC-respiring Dehalococcoides mccartyi. In contrast, members of Proteobacteria adapted to nutrient-poor conditions were enriched in post-WWTP samples. This transition in the trophic state of the hyporheic sediments reduced but did not abolish the VC respiration potential in the post-WWTP sediments as an important hyporheic sediment function. Our results highlight effective nutrient load reduction and parallel microbial ecological state restoration of a human-stressed urban river as a result of installation of a WWTP.

Crambe crambe was sampled from the Western Mediterranean and immediately presserved in RNA later. Total RNA was extracted en rRNA and eukaryotic mRNA were removed and the remaining bacterial mRNA (and unremoved rRNAs and eukaryotic mRNAs) were sequenced with the Illumina HiSeq using paired end sequencing., Crambe crambe was sampled from the Western Mediterranean and immediately presserved in RNA later. Total RNA was extracted en rRNA and eukaryotic mRNA were removed and the remaining bacterial mRNA (and unremoved rRNAs and eukaryotic mRNAs) were sequenced with the Illumina HiSeq using paired end sequencing.

We applied bar-coded 16S rRNA gene amplicon sequencing for species identification. Bacterial diversity in samples from A.aerophoba, P.ficiformis and C.candelabrum was analysed. The cultivable bacterial fraction from these sponges was studied on five different media in the form of 60 communities scraped from plates without antibiotics, as well in the form of individual isolates that were grown on these media supplemented with either one or two of 14 different antibiotics., We applied bar-coded 16S rRNA gene amplicon sequencing for species identification. Bacterial diversity in samples from A.aerophoba, P.ficiformis and C.candelabrum was analysed. The cultivable bacterial fraction from these sponges was studied on five different media in the form of 60 communities scraped from plates without antibiotics, as well in the form of individual isolates that were grown on these media supplemented with either one or two of 14 different antibiotics.

The gut microbiota of fish larvae evolves fast towards a complex community. Both host and environment affect the development of the gut microbiota; however, the relative importance of both is poorly understood. Determining specific changes in gut microbial populations in response to a change in an environmental factor is very complicated. Interactions between factors are difficult to separate and any response could be masked due to high inter-individual variation even for individuals that share a common environment. In this study we characterized and quantified the spatio-temporal variation in the gut microbiota of tilapia larvae, reared in recirculating aquaculture systems (RAS) or active suspension tanks (AS). Our results showed that variation in gut microbiota between replicate tanks was not significantly higher than within tank variation, suggesting that there is no tank effect on water and gut microbiota. However, when individuals were reared in replicate RAS, gut microbiota differed significantly. The highest variation was observed between individuals reared in different types of system (RAS vs. AS). Our data suggest that under experimental conditions in which the roles of deterministic and stochastic factors have not been precisely determined, compositional replication of the microbial communities of an ecosystem is not predictable.

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC- DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots'' of plasmids potentially carrying catabolic genes.

The influence of salt (NaCl) on bacterial and archaeal communities in a submerged fixed bed bioreactor system treating urban wastewater with different saline concentrations was determined by 454 pyrosequencing. Cluster analysis of DGGE fingerprints showed significant differences of the community structure dependent upon the salt concentration applied to the influent. Proteobacteria was found to be the dominant Bacteria Phylum and Euryarchaeota was the main Archaea Phylum in all the experiments. While bacterial α-diversity decreased as salinity increased, the Archaea α-diversity was higher when the NaCl concentration in the influent rose. The differences found between the microbial communities and biodiversity showed that salinity had effects on the structure of microbial communities.

This study unveils microbial eukaryote and fungal diversity in sponges assesed by molecular approach. Non-sponge metazoan, cercozoan and Ichtyosporea are major microbial eukaryotes present in sponge apart from fungi. The findings are considered a major milestones in understanding the dynamics and ecology of sponge-associated fungi., This study unveils microbial eukaryote and fungal diversity in sponges assesed by molecular approach. Non-sponge metazoan, cercozoan and Ichtyosporea are major microbial eukaryotes present in sponge apart from fungi. The findings are considered a major milestones in understanding the dynamics and ecology of sponge-associated fungi.

Baas Becking stated in 1934 that “Everything is everywhere, but the environment selects”. Following this line of thought, one could attempt, probably unsuccessfully, to replicate a microbial community by copying its environment. For the gut of juvenile animals this would be extremely difficult because its microbiota rapidly evolves during early development. Host and environment both affect the development of the gastrointestinal (GI) microbiota. However, the relative importance of both is too poorly understood to predict accurately microbial composition. Furthermore, even for identically reared individuals, inter-individual variation in gut microbial composition can mask treatment effects. We characterized GI microbiota dynamics of Nile tilapia larvae, reared in two replicate recirculating aquaculture systems (RAS) for six weeks starting with first feeding following yolk sack absorption. To evaluate whether similar temporal and replication effects are also observed in other rearing systems, we also monitored variation in replicated active suspension (AS) systems. Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing of PCR-amplified 16S ribosomal RNA gene fragments showed that variation in GI microbiota between replicate tanks, was not significantly higher than within tank variation. However, when individuals were reared in replicated systems, GI microbiota differed significantly. The highest variation was observed between individuals reared in different system types. Our data suggest that compositional replication of the microbial communities of an ecosystem is practically impossible. We recommend to test the effect of experimental treatments on gut microbiota preferentially in tanks within the same system, rather than between different systems, unless systems within each treatment are replicated., Baas Becking stated in 1934 that “Everything is everywhere, but the environment selects”. Following this line of thought, one could attempt, probably unsuccessfully, to replicate a microbial community by copying its environment. For the gut of juvenile animals this would be extremely difficult because its microbiota rapidly evolves during early development. Host and environment both affect the development of the gastrointestinal (GI) microbiota. However, the relative importance of both is too poorly understood to predict accurately microbial composition. Furthermore, even for identically reared individuals, inter-individual variation in gut microbial composition can mask treatment effects. We characterized GI microbiota dynamics of Nile tilapia larvae, reared in two replicate recirculating aquaculture systems (RAS) for six weeks starting with first feeding following yolk sack absorption. To evaluate whether similar temporal and replication effects are also observed in other rearing systems, we also monitored variation in replicated active suspension (AS) systems. Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing of PCR-amplified 16S ribosomal RNA gene fragments showed that variation in GI microbiota between replicate tanks, was not significantly higher than within tank variation. However, when individuals were reared in replicated systems, GI microbiota differed significantly. The highest variation was observed between individuals reared in different system types. Our data suggest that compositional replication of the microbial communities of an ecosystem is practically impossible. We recommend to test the effect of experimental treatments on gut microbiota preferentially in tanks within the same system, rather than between different systems, unless systems within each treatment are replicated.

Zebrafish has been explored as nutritional fish model with the purpose to assess the impact of two dietary inclusion levels of a novel iron-containing protein meal (IPM) of animal origin on the gastrointestinal tract (GIT) of the developing zebrafish. The development of the microbial community has been assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by quantitative PCR (qPCR) and pyrosequencing using a Genome Sequencer FLX. The molecular regulation of physiological processes by differentially expressed genes in the GIT is demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq) using an Illumina HiSeq2000 focusing on genes that are functionally involved in iron uptake and homeostasis. Differential diet dependent phylogenetic diversity occurred. Larvae fed at high inclusion level of IPM differed from those fed at low level in early presence of Bacteroidetes, then an increase of Firmicutes and other phyla at the expense of the Actinobacteria. Finally with Firmicutes and Actinobacteria still present, Proteobacteria dominated. The abundance of Firmicutes in the larvae fed at high inclusion level of IPM at 14 and 21 dpf was much higher than those fed at low inclusion level which probably relates to their iron oxidizing capacity that may coincide with higher pH in the GIT. mRNAseq revealed that 328 genes were differentially expressed: expression of 214 genes was up-regulated and 114 genes down-regulated in larvae fed at high vs. low inclusion levels of IPM. Dominant gene groups representing ribosome components and activity and transport were up-regulated in the GIT of these larvae. 27 genes were identified as involved in iron homeostasis but were non-differentially expressed at a fold change 0.27 – 1.54. Functional classification of genes revealed that the GIT of larvae fed at higher IPM level are more active in transmembrane ion transport and protein synthesis. The marked differences in mi, Zebrafish has been explored as nutritional fish model with the purpose to assess the impact of two dietary inclusion levels of a novel iron-containing protein meal (IPM) of animal origin on the gastrointestinal tract (GIT) of the developing zebrafish. The development of the microbial community has been assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by quantitative PCR (qPCR) and pyrosequencing using a Genome Sequencer FLX. The molecular regulation of physiological processes by differentially expressed genes in the GIT is demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq) using an Illumina HiSeq2000 focusing on genes that are functionally involved in iron uptake and homeostasis. Differential diet dependent phylogenetic diversity occurred. Larvae fed at high inclusion level of IPM differed from those fed at low level in early presence of Bacteroidetes, then an increase of Firmicutes and other phyla at the expense of the Actinobacteria. Finally with Firmicutes and Actinobacteria still present, Proteobacteria dominated. The abundance of Firmicutes in the larvae fed at high inclusion level of IPM at 14 and 21 dpf was much higher than those fed at low inclusion level which probably relates to their iron oxidizing capacity that may coincide with higher pH in the GIT. mRNAseq revealed that 328 genes were differentially expressed: expression of 214 genes was up-regulated and 114 genes down-regulated in larvae fed at high vs. low inclusion levels of IPM. Dominant gene groups representing ribosome components and activity and transport were up-regulated in the GIT of these larvae. 27 genes were identified as involved in iron homeostasis but were non-differentially expressed at a fold change 0.27 – 1.54. Functional classification of genes revealed that the GIT of larvae fed at higher IPM level are more active in transmembrane ion transport and protein synthesis. The marked differences in mi

Sponges harbour microbial communities that contribute to the genetic and metabolic potential of their host. Among metabolites produced by sponge-associated microbial communities, halogenated compounds are of special interest because of their biotechnological potential. In this study, we have examined the diversity of the cultivable fraction of the marine demosponge Crambe crambe microbiota. Application of complementary cultivation methods yielded 107 bacterial isolates, some of which may be sponge-specific based on their phylogenetic analysis. Among these, Psychrobacter sp. was found to contain a putative halogenase gene. In addition to the culture-dependent approach for discovering halogenase genes, a cDNA library was constructed to determine the diversity of halogenase genes expressed in situ by the C. crambe microbiota. To this end, seventeen putative tryptophan halogenase cDNA sequences were identified, most of which were only remotely related to known halogenase genes, indicating the potential for novel bioactive compounds being produced by the C. crambe microbiota

Many marine sponges host diverse communities of microorganisms that are often vertically transmitted from mother to oocyte or embryo. However, not all sponges obtain all consistently associated bacteria via vertical transmission. Horizontal transmission (acquisition from the environment) has often been proposed to occur in marine sponges, but the mechanism is poorly understood. To assess the contribution of the mode of transmission to the bacterial composition in the sponge tissue, we analyzed the bacterial fingerprints of three sympatric sponges that have previously been reported to acquire bacteria either via vertical or horizontal transmission. Detailed studies have shown that Petrosia ficiformis does not transmit its associated bacteria vertically. Thus, this species was taken as a model for horizontal transmission. The bacterial fingerprint of Corticium candelabrum adults closely resembles that of C. candelabrum embryos, and hence this sponge was included as a model for vertical transmission. Crambe crambe is in contrast to the other species a low-bacterial-abundance sponge and was included to detect unspecific environmental acquisition of bacteria. The comparative study was performed by pyrosequencing of barcoded PCR-amplified 16S rRNA gene fragments. We found that P. ficiformis and C. candelabrum harbour bacteria similar to other high-microbial-abundance sponges, whereas C. crambe shows some overlap with local seawater bacteria. In addition, the bacteria associated with P. ficiformis were closely related to bacteria that were associated with other sponges for which vertical transmission was proven. Therefore, our results suggest that the mode of transmission has no major impact on the bacterial profile of marine sponges., Many marine sponges host diverse communities of microorganisms that are often vertically transmitted from mother to oocyte or embryo. However, not all sponges obtain all consistently associated bacteria via vertical transmission. Horizontal transmission (acquisition from the environment) has often been proposed to occur in marine sponges, but the mechanism is poorly understood. To assess the contribution of the mode of transmission to the bacterial composition in the sponge tissue, we analyzed the bacterial fingerprints of three sympatric sponges that have previously been reported to acquire bacteria either via vertical or horizontal transmission. Detailed studies have shown that Petrosia ficiformis does not transmit its associated bacteria vertically. Thus, this species was taken as a model for horizontal transmission. The bacterial fingerprint of Corticium candelabrum adults closely resembles that of C. candelabrum embryos, and hence this sponge was included as a model for vertical transmission. Crambe crambe is in contrast to the other species a low-bacterial-abundance sponge and was included to detect unspecific environmental acquisition of bacteria. The comparative study was performed by pyrosequencing of barcoded PCR-amplified 16S rRNA gene fragments. We found that P. ficiformis and C. candelabrum harbour bacteria similar to other high-microbial-abundance sponges, whereas C. crambe shows some overlap with local seawater bacteria. In addition, the bacteria associated with P. ficiformis were closely related to bacteria that were associated with other sponges for which vertical transmission was proven. Therefore, our results suggest that the mode of transmission has no major impact on the bacterial profile of marine sponges.

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile

Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge Tethya californiana was analyzed based on 16S rRNA gene sequences. One candidate division and nine bacterial phyla were detected, including members of all five subdivisions of Proteobacteria. Moreover, chloroplast-derived Stramenopiles- and Rhodophyta-affiliated 16S rRNA gene sequences were found and Stramenopiles represented the most abundant clones (30%) in the clone library. On the phylum-level, the microbial fingerprint of T. californiana showed a similar pattern as its Mediterranean relative T. aurantium. An interesting difference was that Cyanobacteria that were abundantly present in T. aurantium were not found in T. californiana, but that the latter sponges harbored phototrophic Stramenopiles instead. Surprisingly, the phototrophic microorganisms were evenly distributed over the inner and outer parts of the sponge tissue, which implies that they also reside in regions without direct light exposure. The other phyla were also present in both the outer cortex and the mesohyl of the sponges. These results were confirmed by analysis on the operational taxonomic unit level. This leads to the conclusion that from a qualitative point of view, spatial distribution of microorganisms in T. californiana tissue is quite homogeneous. Thirty-two percent of the operational taxonomic units shared less than 95% similarity with any other known sequence. This indicates that marine sponges are a rich source of previously undetected microbial life

We have characterised the northern Pacific undescribed sponge Haliclona (?gellius) sp. based on rDNA of the sponge and its associated microorganisms. The sponge is closely related to Amphimedon queenslandica from the Great Barrier Reef as the near-complete 18S rDNA sequences of both sponges were identical. The microbial fingerprint of three specimens harvested at different times and of a transplanted specimen was compared to identify stably associated microorganisms. Most bacterial phyla were detected in each sample, but only a few bacterial species were determined to be stably associated with the sponge. A sponge-specific beta- and gamma-Proteobacterium were abundant clones and both of them were present in three of the four specimens analysed. In addition, a Planctomycete and a Crenarchaea were detected in all sponge individuals. Both were closely related to operational taxonomic units that have been found in other sponges, but not exclusively in sponges. Interestingly, also a number of clones that are closely related to intracellular symbionts from insects and amoeba were detected.

Belastingsverhogingen op een weinig draagkrachtige ondergrond, zoals deze in grote delen van Nederland te vinden zijn, gaan gepaard met maaiveld verzakkingen. Deze weinig draagkrachtige ondergrond, gevormd door klei of veen lagen, zijn tevens slecht waterdoorlatend. Bij de veronderstelling dat de bodem volledig verzadigd is en de vereenvoudiging dat zowel water als het basismateriaal onsamendrukbaar zijn, levert een belastingsverhoging instantaan geen zakking van het maaiveld op. Het kan enige tot soms zelfs vele jaren duren voordat het zogenaamde overspannenwater uit de grondlagen is verdreven. Dit proces waarbij het overspannenwater uit de poriën tussen de korrels wordt geperst gaat gepaard met een volumeverkleining van het pakket, en daarmee met een maaiveldzakking. Dit consolidatieproces is het eerste tijdsafhankelijke proces dat optreedt na het aanbrengen van een belastingsverhoging. Daarnaast speelt zich een tweede tijdsafhankelijk proces af dat plastisch van aard is. Dit wil zeggen dat er doorgaande vervormingen optreden bij een gelijkblijvend belastingsniveau, een proces dat ook wel kruip genoemd wordt. Hoewel deze vervormingen optreden bij een gelijkblijvend belastingsniveau is de snelheid waarmee dit proces plaats vindt wel afhankelijk van het effectieve spanningsniveau. Bovendien neemt de snelheid waarmee deze vervorming optreedt af naarmate het materiaal compacter wordt als gevolg van deze vervormingen. Het kruipproces is daarmee dus het tweede tijdsafhankelijke proces. Er bestaan verschillende modellen om dit zettingsproces te beschrijven, waarbij in Nederland meestal gebruik gemaakt wordt van de methode Koppejan en internationaal veelal van de methode Bjerrum. De beperkingen en inconsistenties van de huidige modellen hebben geleid tot de ontwikkeling van het a,b,c-Isotachenmodel. Op gebruiksniveau is er echter slechts in beperkte mate ervaring opgedaan met dit model. Dit rapport is geschreven om inzicht te geven in de gebruikte relaties van het model e, Geo-engineering, Geotechnology, Civil Engineering and Geosciences