The Baltic Sea receives large nitrogen inputs by diazotrophic (N 2 -fixing) heterocystous cyanobacteria but the significance of heterotrophic N 2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N 2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of 410 and o10 lm size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta-and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 Â 10 7 nifH copies l À 1 . Abundant nifH transcripts (up to 3.2 Â 10 4 transcripts l À 1 ) within nifH cluster III and co-occurring N 2 fixation (0.44 ± 0.26 nmol l À 1 day À 1 ) in deep water suggests that heterotrophic diazotrophs are fixing N 2 in anoxic ammonium-rich waters. Our results reveal that N 2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N 2 fixation could also be of importance in other suboxic regions of the world's oceans. The ISME Journal (2013Journal ( ) 7, 1413Journal ( -1423 doi:10.1038/ismej.2013 published online 28 February 2013 Subject Category: microbial ecology and functional diversity of natural habitats

Nitrogen (N) fixation is fueling planktonic production in a multitude of aquatic environments. In meso-and poly-haline estuaries, however, the contribution of N by pelagic N 2 fixation is believed to be insignificant due to the high input of N from land and the presumed absence of active N 2 -fixing organisms. Here we report N 2 fixation rates, nifH gene composition and nifH gene transcript abundance for key diazotrophic groups over 1 year in two contrasting, temperate, estuarine systems: Roskilde Fjord (RF) and the Great Belt (GB) strait. Annual pelagic N 2 fixation rates averaged 17 and 61 mmol N m À 2 per year at the two sites, respectively. In RF, N 2 fixation was mainly accompanied by transcripts related to heterotrophic (for example, Pseudomonas sp.) and photoheterotrophic bacteria (for example, unicellular diazotrophic cyanobacteria group A). In the GB, the first of two N 2 fixation peaks coincided with a similar nifH-expressing community as in RF, whereas the second peak was synchronous with increased nifH expression by an array of diazotrophs, including heterotrophic organisms as well as the heterocystous cyanobacterium Anabaena. Thus, we show for the first time that significant planktonic N 2 fixation takes place in mesohaline, temperate estuaries and that the importance of heterotrophic, photoheterotrophic and photosynthetic diazotrophs is clearly variable in space and time.

SummaryMicroorganisms are of great importance to aquaculture where they occur naturally, and can be added artificially, fulfilling different roles. They recycle nutrients, degrade organic matter and, occasionally, they infect and kill the fish, their larvae or the live feed. Also, some microorganisms may protect fish and larvae against disease. Hence, monitoring and manipulating the microbial communities in aquaculture environments hold great potential; both in terms of assessing and improving water quality, but also in terms of controlling the development of microbial infections. Using microbial communities to monitor water quality and to efficiently carry out ecosystem services within the aquaculture systems may only be a few years away. Initially, however, we need to thoroughly understand the microbiomes of both healthy and diseased aquaculture systems, and we need to determine how to successfully manipulate and engineer these microbiomes. Similarly, we can reduce the need to apply antibiotics in aquaculture through manipulation of the microbiome, i.e. by the use of probiotic bacteria. Recent studies have demonstrated that fish pathogenic bacteria in live feed can be controlled by probiotics and that mortality of infected fish larvae can be reduced significantly by probiotic bacteria. However, the successful management of the aquaculture microbiota is currently hampered by our lack of knowledge of relevant microbial interactions and the overall ecology of these systems.

The community composition of N2 -fixing microorganisms (diazotrophs) was investigated in copepods (primarily Acartia spp.) in parallel to that of seawater in coastal waters off Denmark (Øresund) and New England, USA. The unicellular cyanobacterial diazotroph UCYN-A was detected from seawater and full-gut copepods, suggesting that the new N contributed by UCYN-A is directly transferred to higher trophic levels in these waters. Deltaproteobacterial and Cluster 3 nifH sequences were detected in > 1 μm seawater particles and full-gut copepods, suggesting that they associate with copepods primarily via feeding. The dominant communities in starved copepods were Vibrio spp. and related Gammaproteobacteria, suggesting they represent the most permanent diazotroph associations in the copepods. N2 fixation rates were up to 3.02 pmol N copepod(-1) day(-1). Although at a typical copepod density in estuarine waters, these volumetric rates are low; considering the small size of a copepod, these mesozooplanktonic crustaceans may serve as hotspots of N2 fixation, at 12.9-71.9 μmol N dm(-3) copepod biomass day(-1). Taken together, diazotroph associations range from more permanent attachments to copepod feeding on some groups. Similar diazotroph groups detected on the eastern and western Atlantic Ocean suggest that these associations are a general phenomenon and play a role in the coastal N cycles.

The ability to reduce atmospheric nitrogen (N2) to ammonia, known as N2 fixation, is a widely distributed trait among prokaryotes that accounts for an essential input of new N to a multitude of environments. Nitrogenase reductase gene (nifH) composition suggests that putative N2-fixing heterotrophic organisms are widespread in marine bacterioplankton, but their autecology and ecological significance are unknown. Here, we report genomic and ecophysiology data in relation to N2 fixation by three environmentally relevant heterotrophic bacteria isolated from Baltic Sea surface water: Pseudomonas stutzeri strain BAL361 and Raoultella ornithinolytica strain BAL286, which are gammaproteobacteria, and Rhodopseudomonas palustris strain BAL398, an alphaproteobacterium. Genome sequencing revealed that all were metabolically versatile and that the gene clusters encoding the N2 fixation complex varied in length and complexity between isolates. All three isolates could sustain growth by N2 fixation in the absence of reactive N, and this fixation was stimulated by low concentrations of oxygen in all three organisms (≈4 to 40 µmol O2 liter−1). P. stutzeri BAL361 did, however, fix N at up to 165 µmol O2 liter−1, presumably accommodated through aggregate formation. Glucose stimulated N2 fixation in general, and reactive N repressed N2 fixation, except that ammonium (NH4+) stimulated N2 fixation in R. palustris BAL398, indicating the use of nitrogenase as an electron sink. The lack of correlations between nitrogenase reductase gene expression and ethylene (C2H4) production indicated tight posttranscriptional-level control. The N2 fixation rates obtained suggested that, given the right conditions, these heterotrophic diazotrophs could contribute significantly to in situ rates.