Uncovering the role of oxygen on organic carbon cycling: insights from a continuous culture study with a facultative anaerobic bacterioplankton species (Shewanella baltica)

Deoxygenation is tied to organic carbon (Corg) supply and utilization in marine systems. Under oxygen-depletion, bacteria maintain Corg respiration using alternative electron acceptors such as nitrate. Since anaerobic respiration’s energy yield is lower, Corg remineralization may be reduced and its residence time increased. We investigated the influence of oxygen and alternative electron acceptors’ availability on Corg cycling by heterotrophic bacteria during a continuous culture experiment with Shewanella baltica, a facultative anaerobic γ-Proteobacteria in the Baltic Sea. We tested six different oxygen levels, from suboxic (<5 µmol L-1) to fully oxic conditions, using a brackish (salinity=14 g L-1) media supplied with high (HighN) or low (LowN) inorganic nitrogen concentrations relative to glucose as labile Corg source. Our results show that suboxia limited DOC (glucose) uptake and cell growth only under LowN, while higher availability of alternative electron acceptors seemingly compensated oxygen limitation under HighN. N-loss was observed under suboxia in both nitrogen treatments. Under HighN, N-loss was highest and a C:N loss ratio of ~2.0 indicated that Corg was remineralized via denitrification. Under LowN, the C:N loss ratio under suboxia was higher (~5.5), suggesting the dominance of other anaerobic respiration pathways, such as dissimilatory nitrate reduction to ammonium (DNRA). Bacterial growth efficiency was independent of oxygen concentration but higher under LowN (34 ± 3.0%) than HighN (26 ± 1.6%). Oxygen concentration also affected dissolved organic matter (DOM) cycling. Under oxic conditions, the release of dissolved combined carbohydrates was enhanced, and the amino acid-based degradation index (DI) pointed to more diagenetically altered DOM. Our results suggest bacterial Corg uptake in low-oxygen systems dominated by S. baltica can be limited by oxygen but compensated by high nitrate availability. Hence, suboxia diminishes Corg remineralisation only when alternative electron acceptors are lacking. Under high nitrate:Corg supply, denitrification leads to a higher N:C loss ratio, potentially counteracting eutrophication in the long run. Low nitrate:Corg supply may favour other anaerobic respiration pathways like DNRA, which sustains labile nitrogen in the system, potentially intensifying the cycle of eutrophication. Going forward, it will be crucial to establish the validity of our findings for S. baltica in natural systems with diverse organic substrates and microbial consortia

A semi-quantitative spectrophotometric, dye-binding assay for determination of Coomassie Blue stainable particles

Coomassie stainable particles (CSP) are protein-containing transparent particles that can be stained with Coomassie brilliant blue (CBB) and are found abundantly in aquatic systems; however, their distribution and role remain poorly known, in part due to the lack of an efficient method to study them. We developed a new, simple, and low cost semi-quantitative spectrophotometric method for determination of CSP in aquatic systems. The method is analogous to that used to quantify polysaccharide-rich gel particles called transparent exopolymeric particles (TEP). CSP concentration is determined relative to bovine serum albumin (BSA) standard aggregates (in a manner similar to how TEP is quantified relative to xanthan gum). The method is based on the linear relationship between CSP concentration and the absorbance of the eluted dye from a CBB-protein complex, which has an absorbance maximum (λmax) at 615 nm. The limit of detection and the precision (%RSD) for the proposed method are 6 μg BSA equivalent and 11%, respectively. The new spectrophotometric method was validated with the existing microscopic method. This new method to quantify CSP is simple, enables rapid measurements, and allows a more efficient comparison with TEP concentrations than the present microscopic method. The spectrophotometric analyses will further the investigation of the abundance, distribution, and role of CSP in the biogeochemistry of the ocean

Effects of Higher CO2 and Temperature on Exopolymer Particle Content and Physical Properties of Marine Aggregates

We investigated how future ocean conditions, and specifically the interaction between temperature and CO2, might affect marine aggregate formation and physical properties. Initially, mesocosms filled with coastal seawater were subjected to three different treatments of CO2 concentration and temperature: (1) 750 ppm CO2, 16°C, (2) 750 ppm CO2, 20°C, and (3) 390 ppm CO2, 16°C. Diatom-dominated phytoplankton blooms were induced in the mesocosms by addition of nutrients. In aggregates produced in roller tanks using seawater taken from the mesocosms during different stages of the bloom, we measured sinking velocity, size, chlorophyll a, particulate organic carbon and nitrogen, and exopolymer particle content; excess density and mass were calculated from the sinking velocity and size of the aggregates. As has been seen in previous experiments, no discernable differences in overall nutrient uptake, chlorophyll-a concentration, or exopolymer particle concentrations could be related to the acidification treatment in the mesocosms. In addition, in the aggregates formed during the roller tank experiments (RTEs), we observed no statistically significant differences in chemical composition among the treatments during Pre-Bloom, Bloom, and Post-Bloom periods. However, physical characteristics were different and showed a synergistic effect of warmer temperature and higher CO2 during the Pre-Bloom period; at this time, temperature had a larger effect than CO2 on aggregate sinking velocity. In RTEs with warmer and acidified treatment (future conditions), aggregates were larger, heavier, and settled faster than aggregates formed at present-day or only acidified conditions. During the Post-Bloom, however, aggregates formed under present and future conditions had similar physical properties. In acidified tanks at ambient temperature, aggregates were slower, smaller and less dense than those formed at the same temperature but under present CO2 or under warmer and acidified conditions. Thus, the sinking velocity of aggregates formed in acidified tanks at ambient temperature was slower than the other two cases. Our findings point out the potential of ocean acidification and warming to modify physical properties of sinking aggregates but also emphasize the need of future experiments investigating multiple environmental stressors to clarify the importance of each factor

On the effect of low oxygen concentrations on bacterial degradation of sinking particles

In marine oxygen (O2) minimum zones (OMZs), the transfer of particulate organic carbon (POC) to depth via the biological carbon pump might be enhanced as a result of slower remineralisation under lower dissolved O2 concentrations (DO). In parallel, nitrogen (N) loss to the atmosphere through microbial processes, such as denitrification and anammox, is directly linked to particulate nitrogen (PN) export. However it is unclear (1) whether DO is the only factor that potentially enhances POC transfer in OMZs, and (2) if particle fluxes are sufficient to support observed N loss rates. We performed a degradation experiment on sinking particles collected from the Baltic Sea, where anoxic zones are observed. Sinking material was harvested using surface-tethered sediment traps and subsequently incubated in darkness at different DO levels, including severe suboxia (<0.5 mg l−1 DO). Our results show that DO plays a role in regulating POC and PN degradation rates. POC(PN) degradation was reduced by approximately 100% from the high to low DO to the lowest DO. The amount of NH4+ produced from the pool of remineralising organic N matched estimations of NH4+ anammox requirements during our experiment. This anammox was likely fueled by DON degradation rather than PON degradation

Composition and Vertical Flux of Particulate Organic Matter to the Oxygen Minimum Zone of the Central Baltic Sea: Impact of a sporadic North Sea inflow

Particle sinking is a major form of transport for photosynthetically fixed carbon to below the euphotic zone via the biological carbon pump (BCP). Oxygen (O2) depletion may improve the efficiency of the BCP. However, the mechanisms by which O2 deficiency can enhance particulate organic matter (POM) vertical fluxes are not well understood. Here, we investigate the composition and vertical fluxes of POM in two deep basins of the Baltic Sea (GB: Gotland Basin and LD: Landsort Deep). The two basins showed different O2 regimes resulting from the intrusion of oxygen-rich water from the North Sea that ventilated the water column below 140 m in GB, but not in LD, during the time of sampling. In June 2015, we deployed surface-tethered drifting sediment traps in oxic surface waters (GB: 40 and 60 m; LD: 40 and 55 m), within the oxygen minimum zone (OMZ; GB: 110 m and LD: 110 and 180 m) and at recently oxygenated waters by the North Sea inflow in GB (180 m). The primary objective of this study was to test the hypothesis that the different O2 conditions in the water column of GB and LD affected the composition and vertical flux of sinking particles and caused differences in export efficiency between those two basins. The composition and vertical flux of sinking particles were different in GB and LD. In GB, particulate organic carbon (POC) flux was 18 % lower in the shallowest trap (40 m) than in the deepest sediment trap (at 180 m). Particulate nitrogen (PN) and Coomassie stainable particle (CSP) fluxes decreased with depth, while particulate organic phosphorus (POP), biogenic silicate (BSi), chlorophyll a (Chl a) and transparent exopolymeric particle (TEP) fluxes peaked within the core of the OMZ (110 m); this coincided with the presence of manganese oxide-like (MnOx-like) particles aggregated with organic matter. In LD, vertical fluxes of POC, PN and CSPs decreased by 28 %, 42 % and 56 %, respectively, from the surface to deep waters. POP, BSi and TEP fluxes did not decrease continuously with depth, but they were higher at 110 m. Although we observe a higher vertical flux of POP, BSi and TEPs coinciding with abundant MnOx-like particles at 110 m in both basins, the peak in the vertical flux of POM and MnOx-like particles was much higher in GB than in LD. Sinking particles were remarkably enriched in BSi, indicating that diatoms were preferentially included in sinking aggregates and/or there was an inclusion of lithogenic Si (scavenged into sinking particles) in our analysis. During this study, the POC transfer efficiency (POC flux at 180 m over 40 m) was higher in GB (115 %) than in LD (69 %), suggesting that under anoxic conditions a smaller portion of the POC exported below the euphotic zone was transferred to 180 m than under reoxygenated conditions present in GB. In addition, the vertical fluxes of MnOx-like particles were 2 orders of magnitude higher in GB than LD. Our results suggest that POM aggregates with MnOx-like particles formed after the inflow of oxygen-rich water into GB, and the formation of those MnOx–OM-rich particles may alter the composition and vertical flux of POM, potentially contributing to a higher transfer efficiency of POC in GB. This idea is consistent with observations of fresher and less degraded organic matter in deep waters of GB than LD

Potential role of oxygen and inorganic nutrients on microbial carbon turnover in the Baltic Sea

Oxygen (O2) deficiency and nutrient concentrations in marine systems are impacting organisms from microbes to higher trophic levels. In coastal and enclosed seas, O2 deficiency is often related to eutrophication and high degradation rates of organic matter. To investigate the impact of O2 concentration on bacterial growth and the turnover of organic matter, we conducted multifactorial batch experiments with natural microbial communities of the central Baltic Sea. Water was collected from suboxic (<5 µmol L -1) depths in the Gotland Basin during June 2015. Samples were kept for four days under fully oxygenated and low O2 conditions (mean: 34 µmol L-1 O2), with or without nutrient (ammonium, phosphate, nitrate) and labile carbon (glucose) amendments. We measured bacterial abundance, bacterial heterotrophic production, extracellular enzyme rates (leucine-aminopeptidase) and changes in dissolved and particulate organic carbon concentrations. Our results show that the bacterial turnover of organic matter was limited by nutrients under both oxic and low O2 conditions. In nutrient and glucose replete treatments, low O2 concentrations significantly reduced the net uptake of dissolved organic carbon and lead to higher accumulation of more labile dissolved organic matter. Our results therewith suggest that the combined effects of eutrophication and deoxygenation on heterotrophic bacterial activity may potentially favor the accumulation of dissolved organic carbon in the Baltic Sea

Uncoupled seasonal variability of transparent exopolymer and Coomassie stainable particles in coastal Mediterranean waters: Insights into sources and driving mechanisms

Transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP) are gel-like particles, ubiquitous in the ocean, that affect important biogeochemical processes including organic carbon cycling by planktonic food webs. Despite much research on both groups of particles (especially TEP) over many years, whether they exist as distinctly stainable fractions of the same particles or as independent particles, each with different driving factors, remains unclear. To address this question, we examined the temporal dynamics of TEP and CSP over 2 complete seasonal cycles at 2 coastal sites in the Northwestern Mediterranean Sea, the Blanes Bay Microbial Observatory (BBMO) and the L’Estartit Oceanographic Station (EOS), as well as their spatial distribution along a coast-to-offshore transect. Biological, chemical, and physical variables were measured in parallel. Surface concentrations (mean + standard deviation [SD]) of TEP were 36.7 + 21.5 µg Xanthan Gum (XG) eq L–1 at BBMO and 36.6 + 28.3 µg XG eq L–1 at EOS; for CSP, they were 11.9 + 6.1 µg BSA eq L–1 at BBMO and 13.0 + 5.9 µg BSA eq L–1 at EOS. Seasonal variability was more evident at EOS, where surface TEP and CSP concentrations peaked in summer and spring, respectively, and less predictable at the shore-most station, BBMO. Vertical distributions between surface and 80 m, monitored at EOS, showed highest TEP concentrations within the surface mixed layer during the stratification period, whereas CSP concentrations were highest before the onset of summer stratification. Phytoplankton were the main drivers of TEP and CSP distributions, although nutrient limitation and saturating irradiance also appeared to play important roles. The dynamics and distribution of TEP and CSP were uncoupled both in the coastal sites and along the transect, suggesting that they are different types of particles produced and consumed differently in response to environmental variability

Dynamics and enzymatic degradation of exopolymer particles under increasing concentrations of silver ions and nanoparticles during a marine mesocosm experiment

Pollution of the marine environment is an emerging threat. Nowadays, engineered nanoparticles (<100 nm) such as zinc, copper and silver are widely used as antimicrobial agents, therefore often present in daily-life products. Consequently, the demand and production of nanoparticles are expected to increase. Here, we specifically focus on silver nanoparticles (AgNP). Once released into the environment, AgNPs pose an obvious ecotoxicological risk, potentially affecting ecosystem structure and functioning. For instance, phytoplankton-derived exudates, rich in acidic polysaccharides and amino acids, can abiotically aggregate into microgels such as transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP). Hence, microgels can bridge dissolved and particulate size fractions and facilitate aggregate formation with organic and mineral particles. Both physical and chemical properties make TEP and CSP attractive nutrient hotspots for heterotrophic bacterioplankton. Bacteria, in turn, utilize extracellular enzymes to access these carbon and nitrogen pools. However, knowledge about the mechanisms by which AgNPs might interact with and affect the biogeochemical cycling of TEP and CSP is still insufficient. Therefore, we conducted a mesocosm experiment in the Eastern Mediterranean Sea and investigated the effects of environmentally relevant concentrations of silver ions (Ag+) and AgNP on the properties of TEP and CSP (i.e., area and abundance) along with enzymatic activity measurements. Our results showed that cyanobacteria were likely the primary source of CSP in the ultra-oligotrophic Mediterranean Sea. Also, CSP contributed more to the microgel pool than TEP, as indicated by a strong relationship between CSP and heterotrophic microbial dynamics. While silver (i.e., Ag+ or AgNP) had overall only marginal effects, both species affected the relationships between cell-specific LAPase activity and CSP and cell-specific APase activity and phosphate levels. Thus, Ag+ and AgNP have the potential to regulate microgel dynamics. However, future studies are needed to derive a robust understanding of the effects of silver pollution on the coupling of microgel formation and degradation and the follow-on effect on biogeochemical cycles

Organic matter fluxes and biogeochemical processes in the OMZ off Peru, Cruise No. M138, 01 June - 03 July 2017, Callao (Peru) - Bahia Las Minas (Panama)

The oxygen minimum zone (OMZ) in the eastern tropical South Pacific Ocean is tightly connected to the coastal upwelling system off Peru. The high biological productivity off Peru is therefore, driven by the complex interplay between the amount of nutrients recycled by remineralisation processes in the OMZ and the upwelling which brings these nutrients to the surface layer. However, surprisingly little is known about organic matter cycling and its effects on biogeochemical processes in the OMZ off Peru. To this end we conducted a first comprehensive study on the role of organic matter for the biogeochemical processes and the maintenance of the OMZ off Peru. M138 combined measurements of marine biogeochemistry, microbiology, physical oceanography and air chemistry with foci on (i) the efficiency of the biological pump, (ii) the nitrogen cycle processes in the OMZ, (iii) the ventilation of the OMZ as well as (iv) the air/sea gas exchange across the ocean/atmosphere interface and (v) aerosol deposition. The METEOR cruise M138 was performed as part of the third phase of the SFB754 'Climate-Biogeochemistry Interactions in the Tropical Ocean' (www.sfb754.de)