Temporal variation in the prokaryotic community of a nearshore marine environment.

Prokaryotic communities inhabiting surface waters of temperate areas exhibit patterns of seasonal succession. Generally, studies describing these temporal changes are not performed in the proximity to the coast. In the present study, temporal variation of these communities was determined in surface waters at two stations located in the close proximity to the eastern shore of the northern Adriatic Sea. Sequencing of the V4 region of the 16S rRNA gene identified the highest community richness in December with distinct shifts in community structure between periods from April to May, June to October, and November to March. Temperature was shown to be the main environmental force explaining community temporal variation. The NS5 marine group, uncultured Cryomorphaceae, SAR86 clade, and Synechococcus were present throughout the year. Members without know relatives within Rhodobacteraceae and the NS4 marine group were more pronounced in the period from April to May, the AEGEAN-169 marine group, SAR11 subclade III, and HIMB11 in the period from June to October, and SAR11 subclade Ia and Archaea in the period from November to March. Litoricola and OM60 (NOR5) clade were characteristic for both the community sampled from April to May and November to March. Taken together, prokaryotic communities inhabiting nearshore surface waters exhibit a general pattern in community structure similar to other surface associated assemblages of temperate areas. However, the identified specific community composition and temporal patterns differ from other coastal areas.

Seasonal Dynamics of Epiphytic Microbial Communities on Marine Macrophyte Surfaces.

Surfaces of marine macrophytes are inhabited by diverse microbial communities. Most studies focusing on epiphytic communities of macrophytes did not take into account temporal changes or applied low sampling frequency approaches. The seasonal dynamics of epiphytic microbial communities was determined in a meadow of Cymodocea nodosa invaded by Caulerpa cylindracea and in a monospecific settlement of C. cylindracea at monthly intervals. For comparison the ambient prokaryotic picoplankton community was also characterized. At the OTU level, the microbial community composition differed between the ambient water and the epiphytic communities exhibiting host-specificity. Also, successional changes were observed connected to the macrophyte growth cycle. Taxonomic analysis, however, showed similar high rank taxa (phyla and classes) in the ambient water and the epiphytic communities, with the exception of Desulfobacterota, which were only found on C. cylindracea. Cyanobacteria showed seasonal changes while other high rank taxa were present throughout the year. In months of high Cyanobacteria presence the majority of cyanobacterial sequences were classified as Pleurocapsa. Phylogenetic groups present throughout the year (e.g., Saprospiraceae, Rhodobacteraceae, members without known relatives within Gammaproteobacteria, Desulfatitalea, and members without known relatives within Desulfocapsaceae) constituted most of the sequences, while less abundant taxa showed seasonal patterns connected to the macrophyte growth cycle. Taken together, epiphytic microbial communities of the seagrass C. nodosa and the macroalga C. cylindracea appear to be host-specific and contain taxa that undergo successional changes.

Selective DNA and Protein Isolation From Marine Macrophyte Surfaces.

Studies of unculturable microbes often combine methods, such as 16S rRNA sequencing, metagenomics, and metaproteomics. To apply these techniques to the microbial community inhabiting the surfaces of marine macrophytes, it is advisable to perform a selective DNA and protein isolation prior to the analysis to avoid biases due to the host material being present in high quantities. Two protocols for DNA and protein isolation were adapted for selective extractions of DNA and proteins from epiphytic communities inhabiting the surfaces of two marine macrophytes, the seagrass Cymodocea nodosa and the macroalga Caulerpa cylindracea. Protocols showed an almost complete removal of the epiphytic community regardless of the sampling season, station, settlement, or host species. The obtained DNA was suitable for metagenomic and 16S rRNA sequencing, while isolated proteins could be identified by mass spectrometry. Low presence of host DNA and proteins in the samples indicated a high specificity of the protocols. The procedures are based on universally available laboratory chemicals making the protocols widely applicable. Taken together, the adapted protocols ensure an almost complete removal of the macrophyte epiphytic community. The procedures are selective for microbes inhabiting macrophyte surfaces and provide DNA and proteins applicable in 16S rRNA sequencing, metagenomics, and metaproteomics.

Seasonal variations in extracellular enzymatic activity in marine snow-associated microbial communities and their impact on the surrounding water.

Seasonal changes of microbial abundance and associated extracellular enzymatic activity in marine snow and in seawater were studied in the northern Adriatic during a three-year period. Marine snow was present during the entire period of investigation, although in higher concentrations during summer than during winter. Microorganisms densely colonized marine snow and aggregate-associated enzymatic activity was substantially higher (up to 105 times) than in seawater. Alkaline phosphatase activity (APA) and aminopeptidase activity in marine snow showed seasonal variations with higher values in late spring-summer than in autumn-winter, probably in response to changes in the quantity and quality of organic matter. The highest cell-specific bacterial activity was found for phosphatase, followed by peptidase, and the lowest was for glucosidases. Differential hydrolysis of marine snow-derived organic matter points to the well-known phosphorus limitation of the northern Adriatic and indicates preferential utilization of phosphorus- and nitrogen-rich organic compounds by microbes, while hydrolysis of polysaccharides seemed to be less important. In oligotrophic conditions during summer, organic matter released from marine snow might represent a significant source of substrate for free-living bacteria in seawater. For the first time microorganisms producing APA in marine snow were identified, revealing that dense populations of bacteria expressed APA, while cyanobacteria did not. Cyanobacteria proliferating in marine snow could benefit from phosphorus release by bacteria and nanoflagellates.

Bioprospecting for Genes Encoding Hydrocarbon-Degrading Enzymes from Metagenomic Samples Isolated from Northern Adriatic Sea Sediments.

Three metagenomic libraries were constructed using surface sediment samples from the northern Adriatic Sea. Two of the samples were taken from a highly polluted and an unpolluted site respectively. The third sample from a polluted site had been enriched using crude oil. The results of the metagenome analyses were incorporated in the REDPET relational database (http://redpet.bioinfo.pbf.hr/REDPET), which was generated using the previously developed MEGGASENSE platform. The database includes taxonomic data to allow the assessment of the biodiversity of metagenomic libraries and a general functional analysis of genes using hidden Markov model (HMM) profiles based on the KEGG database. A set of 22 specialised HMM profiles was developed to detect putative genes for hydrocarbon-degrading enzymes. Use of these profiles showed that the metagenomic library generated after selection on crude oil had enriched genes for aerobic n-alkane degradation. The use of this system for bioprospecting was exemplified using potential alkB and almA genes from this library.

Limited influence of primary treated sewage waters on bacterial abundance, production and community composition in coastal seawaters.

The response of bacteria in terms of abundance, production and community structure to changes induced by the discharge of primary treated sewage waters was investigated combining microbiological, chemical and molecular tools. The primary treatment did not affect substantially the bacterial community structure in wastewaters and did not reduce the concentrations of fecal indicators. The spatial distribution of the sewage plume was governed by vertical stratification and currents. Bacterial abundance and production in the sea receiving waste waters depended predominantly on environmental conditions. In the waters with the highest concentration of fecal pollution indicators the bacterial community was characterized by allochthonous bacteria belonging to Epsilonproteobacteria, Firmicutes, Gammaproteobacteria and Bacteroidetes. The latter two taxa were also present in unpolluted waters but had a different structure, typical for oligotrophic environments. Although the impact of primary treated sewage waters was limited, a sanitary risk persisted due to the relevant presence of potentially pathogenic bacteria.

Bacterial diversity of polluted surface sediments in the northern Adriatic Sea.

Samples were collected from sea sediments at seven sites in the northern Adriatic Sea that included six sites next to industrial complexes and one from a tourist site (recreational beach). The samples were assayed for alkanes and polycyclic aromatic hydrocarbons. The composition of the hydrocarbon samples suggested that industrial pollution was present in most cases. A sample from one site was also grown aerobically under crude oil enrichment in order to evaluate the response of indigenous bacterial populations to crude oil exposure. Analysis of 16S rRNA gene sequences showed varying microbial biodiversity depending on the level of pollution--ranging from low (200 detected genera) to high (1000+ genera) biodiversity, with lowest biodiversity observed in polluted samples. This indicated that there was considerable biodiversity in all sediment samples but it was severely restricted after exposure to crude oil selection pressure. Phylogenetic analysis of putative alkB genes showed high evolutionary diversity of the enzymes in the samples and suggested great potential for bioremediation and bioprospecting. The first systematic analysis of bacterial communities from sediments of the northern Adriatic Sea is presented, and it will provide a baseline assessment that may serve as a reference point for ecosystem changes and hydrocarbon degrading potential--a potential that could soon gain importance due to plans for oil exploitation in the area.

Community shift from phototrophic to chemotrophic sulfide oxidation following anoxic holomixis in a stratified seawater lake.

Most stratified sulfidic holomictic lakes become oxygenated after annual turnover. In contrast, Lake Rogoznica, on the eastern Adriatic coast, has been observed to undergo a period of water column anoxia after water layer mixing and establishment of holomictic conditions. Although Lake Rogoznica's chemistry and hydrography have been studied extensively, it is unclear how the microbial communities typically inhabiting the oxic epilimnion and a sulfidic hypolimnion respond to such a drastic shift in redox conditions. We investigated the impact of anoxic holomixis on microbial diversity and microbially mediated sulfur cycling in Lake Rogoznica with an array of culture-independent microbiological methods. Our data suggest a tight coupling between the lake's chemistry and occurring microorganisms. During stratification, anoxygenic phototrophic sulfur bacteria were dominant at the chemocline and in the hypolimnion. After an anoxic mixing event, the anoxygenic phototrophic sulfur bacteria entirely disappeared, and the homogeneous, anoxic water column was dominated by a bloom of gammaproteobacterial sulfur oxidizers related to the GSO/SUP05 clade. This study is the first report of a community shift from phototrophic to chemotrophic sulfide oxidizers as a response to anoxic holomictic conditions in a seasonally stratified seawater lake.