Natalia Belkin

Dinitrogen Fixation in Aphotic Oxygenated Marine Environments

We measured N2 fixation rates from oceanic zones that have traditionally been ignored as sources of biological N2 fixation; the aphotic, fully oxygenated, nitrate (NO−3)-rich, waters of the oligotrophic Levantine Basin (LB) and the Gulf of Aqaba (GA). N2 fixation rates measured from pelagic aphotic waters to depths up to 720 m, during the mixed and stratified periods, ranged from 0.01 nmol N L−1 d−1 to 0.38 nmol N L−1 d−1. N2 fixation rates correlated significantly with bacterial productivity and heterotrophic diazotrophs were identified from aphotic as well as photic depths. Dissolved free amino acid amendments to whole water from the GA enhanced bacterial productivity by 2–3.5 fold and N2 fixation rates by ~2-fold in samples collected from aphotic depths while in amendments to water from photic depths bacterial productivity increased 2–6 fold while N2 fixation rates increased by a factor of 2 to 4 illustrating that both BP and heterotrophic N2 fixation were carbon limited. Experimental manipulations of aphotic waters from the LB demonstrated a significant positive correlation between transparent exopolymeric particle (TEP) concentrations and N2 fixation rates. This suggests that sinking organic material and high carbon (C): nitrogen (N) micro-environments (such as TEP-based aggregates or marine snow) could support high heterotrophic N2 fixation rates in oxygenated surface waters and in the aphotic zones. Indeed, our calculations show that aphotic N2 fixation accounted for 37 to 75% of the total daily integrated N2 fixation rates at both locations in the Mediterranean and Red Seas with rates equal or greater to those measured from the photic layers. Moreover, our results indicate that that while N2 fixation may be limited in the surface waters, aphotic, pelagic N2 fixation may contribute significantly to new N inputs in other oligotrophic basins, yet it is currently not included in regional or global N budgets.

The primary transcriptome of the marine diazotroph Trichodesmium erythraeum IMS101

Blooms of the dinitrogen-fixing marine cyanobacterium Trichodesmium considerably contribute to new nitrogen inputs into tropical oceans. Intriguingly, only 60% of the Trichodesmium erythraeum IMS101 genome sequence codes for protein, compared with ~85% in other sequenced cyanobacterial genomes. The extensive non-coding genome fraction suggests space for an unusually high number of unidentified, potentially regulatory non-protein-coding RNAs (ncRNAs). To identify the transcribed fraction of the genome, here we present a genome-wide map of transcriptional start sites (TSS) at single nucleotide resolution, revealing the activity of 6,080 promoters. We demonstrate that T. erythraeum has the highest number of actively splicing group II introns and the highest percentage of TSS yielding ncRNAs of any bacterium examined to date. We identified a highly transcribed retroelement that serves as template repeat for the targeted mutation of at least 12 different genes by mutagenic homing. Our findings explain the non-coding portion of the T. erythraeum genome by the transcription of an unusually high number of non-coding transcripts in addition to the known high incidence of transposable elements. We conclude that riboregulation and RNA maturation-dependent processes constitute a major part of the Trichodesmium regulatory apparatus.

Enhanced salinities, as a proxy of seawater desalination discharges, impact coastal microbial communities of the eastern Mediterranean Sea.

Seawater desalination plants increase local coastal salinities by discharging concentrated brine back to the sea with ∼ 50% higher than ambient salinities. The impacts of high salinities on microbial coastal populations of the eastern Mediterranean Sea (EMS) were examined in two mesocosm experiments; first, during the mixed-spring and second, during the stratified-summer periods with average salinity of ∼ 39. Ambient salinities were increased by 5% and 15%. Higher salinity (15%) mesocosms induced rapid (within 2 h) declines in both primary productivity (PP) and algal biomass parallel to an increase in bacterial productivity. Subsequently, for the duration of the experiments (11-12 days), both Chlorophyll a and PP rates increased (2 to 5 and 1.5 to 2.5-fold, respectively) relative to unamended controls. The initial assemblages of the ambient microbial populations and intensity of salinity enrichments influenced the community responses. During the mixed-spring experiment, the composition of prokaryotic and eukaryotic populations shifted only slightly, suggesting high functional plasticity of the initial populations. While during the stratified-summer experiment, high salinity changed the composition and reduced the biodiversity of the microbial communities. In an ultra-oligotrophic environment such as the EMS, salinity induced declines in microbial diversity may provide a tipping point destabilizing the local aquatic food web.

Bioflocculation: Chemical free, pre-treatment technology for the desalination industry

Rapid sand filtration (RSF), proceeded by chemical coagulation and flocculation, is a commonly used, effective pretreatment in the desalination industry. We designed and tested a novel, large pilot-scale, two-stage granular Rapid Bioflocculation Filter (RBF) based on a first-stage Bioflocculator (BF) unit followed by a mixed-media bed filter (MBF). The BF filter bed consisted of an extremely porous volcanic Tuff granular medium which provided an enlarged surface area for microbial development and biofilm proliferation. We compared the efficiency of the pilot RBF to that of a full-scale RSF, operating with upstream chemical coagulation, by measuring the removal from the same untreated seawater feed of key factors related to membrane clogging: SDI, turbidity, chlorophyll a (Chl a) and transparent exopolymer particles (TEP). After 2 weeks of operation, the Tuff grains were colonized extensively by coccoid bacteria that formed biofilm along the entire BF. With bacterial colonization and biofilm development, numerous aggregates of bacteria and some algal cells embedded in an amorphous organic matrix were formed on and within the Tuff grains. By 1-3 months, the biotic diversity within the Tuff filter bed had increased to include filamentous bacteria, cyanobacteria, fungi, protista and even crustaceans and marine worms. During and for ≈ 24 h after each cleaning cycle (carried out every 5 to 7 days by upward flushing with air and water), large numbers of floc-like particles, from ≈ 15 μm to ≈ 2 mm in size were observed in the filtrate of the BF unit. Microscopic examination of these flocs (stained with Alcian Blue and SYTO(R) 9) showed that they were aggregates of many smaller particles with associated bacteria and algae within a polysaccharide gel-like matrix. These biogenic flocs (bioflocs) were observed to form during normal operation of the RBF, accumulating as aggregates of inorganic and organic material on the Tuff surfaces. With each flush cleaning cycle, these bioflocs were released into the BF effluent but were retained by the second phase MBF unit. No flocs were seen in the MBF filtrate. Over a year-long study, both the pilot RBF and the full-scale RSF showed similar filtration efficiencies, measured as the percentage removal of Chl a, TEP, turbidity and SDI from the same seawater feed. These results indicate the potential of the bioflocculation approach with no chemical additives as an alternative to conventional RSF pretreatment for large SWRO facilities.

Benefit from decline: the primary transcriptome of Alteromonas macleodii str. Te101 during Trichodesmium demise

Interactions between co-existing microorganisms deeply affect the physiology of the involved organisms and, ultimately, the function of the ecosystem as a whole. Copiotrophic Alteromonas are marine gammaproteobacteria that thrive during the late stages of phytoplankton blooms in the marine environment and in laboratory co-cultures with cyanobacteria such as Trichodesmium. The response of this heterotroph to the sometimes rapid and transient changes in nutrient supply when the phototroph crashes is not well understood. Here, we isolated and sequenced the strain Alteromonas macleodii str. Te101 from a laboratory culture of Trichodesmium erythraeum IMS101, yielding a chromosome of 4.63 Mb and a single plasmid of 237 kb. Increasing salinities to ≥43 ppt inhibited the growth of Trichodesmium but stimulated growth of the associated Alteromonas. We characterized the transcriptomic responses of both microorganisms and identified the complement of active transcriptional start sites in Alteromonas at single-nucleotide resolution. In replicate cultures, a similar set of genes became activated in Alteromonas when growth rates of Trichodesmium declined and mortality was high. The parallel activation of fliA, rpoS and of flagellar assembly and growth-related genes indicated that Alteromonas might have increased cell motility, growth, and multiple biosynthetic activities. Genes with the highest expression in the data set were three small RNAs (Aln1a-c) that were identified as analogs of the small RNAs CsrB-C in E. coli or RsmX-Z in pathogenic bacteria. Together with the carbon storage protein A (CsrA) homolog Te101_05290, these RNAs likely control the expression of numerous genes in responding to changes in the environment.

Trimethylated homoserine functions as the major compatible solute in the globally significant oceanic cyanobacterium Trichodesmium

Significance Trichodesmium spp. are globally significant contributors of new nitrogen to the surface ocean. Marine organisms must accumulate compatible solutes in their cells, counteracting the high exterior osmotic pressure. However, Trichodesmium does not possess any known genes for the synthesis of compatible solutes, making its proliferation in the high-salinity environment enigmatic. We demonstrate that Trichodesmium cultures in the laboratory as well as natural populations in the ocean synthesize homoserine betaine, previously unknown as a compatible solute, and elucidated the biosynthetic pathway. The high intracellular concentrations will lead to a major injection of this organic compound into the oligotrophic ocean, when natural Trichodesmium blooms lyse. Such sudden releases of homoserine betaine could impact the biogeochemical cycling of carbon and nitrogen. The oceanic N2-fixing cyanobacterium Trichodesmium spp. form extensive surface blooms and contribute significantly to marine carbon and nitrogen cycles in the oligotrophic subtropical and tropical oceans. Trichodesmium grows in salinities from 27 to 43 parts per thousand (ppt), yet its salt acclimation strategy remains enigmatic because the genome of Trichodesmium erythraeum strain IMS101 lacks all genes for the biosynthesis of any known compatible solute. Using NMR and liquid chromatography coupled to mass spectroscopy, we identified the main compatible solute in T. erythraeum strain IMS101 as the quaternary ammonium compound N,N,N-trimethyl homoserine (or homoserine betaine) and elucidated its biosynthetic pathway. The identification of this compatible solute explains how Trichodesmium spp. can thrive in the marine system at varying salinities and provides further insight into the diversity of microbial salt acclimation.

Microbial Communities in the Process and Effluents of Seawater Desalination Plants

Abstract Seawater desalination is now being widely regarded as the solution to the ever increasing need for potable water. However, in contrast to the rapid technological developments throughout the desalination process and expanding desalination facilities, not much is known on the environmental and ecological impacts on the marine ecosystem. Specifically, the effects of discharging brine and chemicals, used routinely in the desalination process, have been poorly documented. In this chapter, we summarize the available data examining the impacts of desalination brine and chemicals, such as iron-based coagulants and phosphonate antiscalants, on coastal seawater microbial communities. The data include results from controlled large-scale laboratory experiments and from in situ measurements around desalination plants outfalls. The observed effects could be immediate, on a time scale of several hours, affecting metabolic functions such as primary and bacterial productivity, and on longer temporal scales of days to weeks, influencing community composition and structure. The specific changes depend predominantly on plant location, capacity, chemical utilization, and discharge technologies (open versus submerged outfalls). We conclude with gaps in knowledge and recommendations for SWRO plant operations.

Programmed cell death in diazotrophs and the fate of organic matter in the Western Tropical South Pacific Ocean during the OUTPACE cruise

The fate of diazotroph (N2 fixers) derived carbon (C) and nitrogen (N) and their contribution to vertical export of C and N in the western tropical South Pacific Ocean was studied during OUTPACE (Oligotrophy to UlTraoligotrophy PACific Experiment). Our specific objective during OUTPACE was to determine whether autocatalytic programmed cell death (PCD), occurring in some diazotrophs, is an important mechanism affecting diazotroph mortality and a factor regulating the vertical flux of organic matter and, thus, the fate of the blooms. We sampled at three long duration (LD) stations of 5 days each (LDA, LDB and LDC) where drifting sediment traps were deployed at 150, 325 and 500 m depth. LDA and LDB were characterized by high chlorophyll a (Chl a) concentrations (0.2–0.6 μg L−1) and dominated by dense biomass of the filamentous cyanobacterium Trichodesmium as well as UCYN-B and diatom– diazotroph associations (Rhizosolenia with Richelia-detected by microscopy and het-1 nifH copies). Station LDC was located at an ultra-oligotrophic area of the South Pacific gyre with extremely low Chl a concentration (∼ 0.02 μg L−1) with limited biomass of diazotrophs predominantly the unicellular UCYN-B. Our measurements of biomass from LDA and LDB yielded high activities of caspase-like and metacaspase proteases that are indicative of PCD in Trichodesmium and other phytoplankton. Metacaspase activity, reported here for the first time from oceanic populations, was highest at the surface of both LDA and LDB, where we also obtained high concentrations of transparent exopolymeric particles (TEP). TEP were negatively correlated with dissolved inorganic phosphorus and positively coupled to both the dissolved and particulate organic carbon pools. Our results reflect the increase in TEP production under nutrient stress and its role as a source of sticky carbon facilitating aggregation and rapid vertical sinking. Evidence for bloom decline was observed at both LDA and LDB. However, the physiological status and rates of decline of the blooms differed between the stations, influencing the amount of accumulated diazotrophic organic matter and mass flux observed in the traps during our experimental time frame. At LDA sediment traps contained the greatest export of particulate matter and significant numbers of both intact and decaying Trichodesmium, UCYN-B and het-1 compared to LDB where the bloom decline began only 2 days prior to leaving the station and to LDC where no evidence for bloom or bloom decline was seen. Substantiating previous findings from laboratory cultures linking PCD to carbon export in Trichodesmium, our results from OUTPublished by Copernicus Publications on behalf of the European Geosciences Union. 3894 D. Spungin et al.: Programmed cell death in diazotrophs and the fate of organic matter PACE indicate that nutrient limitation may induce PCD in high biomass blooms such as displayed by Trichodesmium or diatom–diazotroph associations. Furthermore, PCD combined with high TEP production will tend to facilitate cellular aggregation and bloom termination and will expedite vertical flux to depth.