Isotopic signatures induced by upwelling reveal regional fish stocks in Lake Tanganyika.

Lake Tanganyika's pelagic fish sustain the second largest inland fishery in Africa and are under pressure from heavy fishing and global warming related increases in stratification. The strength of water column stratification varies regionally, with a more stratified north and an upwelling-driven, biologically more productive south. Only little is known about whether such regional hydrodynamic regimes induce ecological or genetic differences among populations of highly mobile, pelagic fish inhabiting these different areas. Here, we examine whether the regional contrasts leave distinct isotopic imprints in the pelagic fish of Lake Tanganyika, which may reveal differences in diet or lipid content. We conducted two lake-wide campaigns during different seasons and collected physical, nutrient, chlorophyll, phytoplankton and zooplankton data. Additionally, we analyzed the pelagic fish-the clupeids Stolothrissa tanganicae, Limnothrissa miodon and four Lates species-for their isotopic and elemental carbon (C) and nitrogen (N) compositions. The δ13C values were significantly higher in the productive south after the upwelling/mixing period across all trophic levels, implying that the fish have regional foraging grounds, and thus record these latitudinal isotope gradients. By combining our isotope data with previous genetic results showing little geographic structure, we demonstrate that the fish reside in a region for a season or longer. Between specimens from the north and south we found no strong evidence for varying trophic levels or lipid contents, based on their bulk δ15N and C:N ratios. We suggest that the development of regional trophic or physiological differences may be inhibited by the lake-wide gene flow on the long term. Overall, our findings show that the pelagic fish species, despite not showing evidence for genetic structure at the basin scale, form regional stocks at the seasonal timescales. This implies that sustainable management strategies may consider adopting regional fishing quotas.

Hydrodynamic regimes modulate nitrogen fixation and the mode of diazotrophy in Lake Tanganyika.

The factors that govern the geographical distribution of nitrogen fixation are fundamental to providing accurate nitrogen budgets in aquatic environments. Model-based insights have demonstrated that regional hydrodynamics strongly impact nitrogen fixation. However, the mechanisms establishing this physical-biological coupling have yet to be constrained in field surveys. Here, we examine the distribution of nitrogen fixation in Lake Tanganyika - a model system with well-defined hydrodynamic regimes. We report that nitrogen fixation is five times higher under stratified than under upwelling conditions. Under stratified conditions, the limited resupply of inorganic nitrogen to surface waters, combined with greater light penetration, promotes the activity of bloom-forming photoautotrophic diazotrophs. In contrast, upwelling conditions support predominantly heterotrophic diazotrophs, which are uniquely suited to chemotactic foraging in a more dynamic nutrient landscape. We suggest that these hydrodynamic regimes (stratification versus mixing) play an important role in governing both the rates and the mode of nitrogen fixation.

Attenuation of wind intensities exacerbates anoxic conditions leading to sulfur plume development off the coast of Peru.

The release of vast quantities of sulfide from the sediment into the water column, known as a sulfidic event, has detrimental consequences on fish catches, including downstream effects on other linked element cycles. Despite being frequent occurrences in marine upwelling regions, our understanding of the factors that moderate sulfidic event formation and termination are still rudimentary. Here, we examined the biogeochemical and hydrodynamic conditions that underpinned the formation/termination of one of the largest sulfur plumes to be reported in the Peruvian upwelling zone. Consistent with previous research, we find that the sulfur-rich plume arose during the austral summer when anoxic conditions (i.e., oxygen and nitrate depletion) prevailed in waters overlying the upper shelf. Furthermore, the shelf sediments were organically charged and characterized by low iron-bound sulfur concentrations, further enabling the diffusion of benthic-generated sulfide into the water column. While these biogeochemical conditions provided a predicate to sulfidic event formation, we highlight that attenuations in local wind intensity served as an event trigger. Namely, interruptions in local wind speed constrained upwelling intensity, causing increased stratification over the upper shelf. Moreover, disturbances in local wind patterns likely placed additional constraints on wind-driven mesoscale eddy propagation, with feedback effects on coastal elemental sulfur plume (ESP) formation. We suggest ESP development occurs as a result of a complex interaction of biogeochemistry with regional hydrodynamics.

Microbial Nitrogen Transformation Potential in Sediments of Two Contrasting Lakes Is Spatially Structured but Seasonally Stable.

The nitrogen (N) cycle is of global importance, as N is an essential element and a limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N fertilizer usage threatens sensitive downstream aquatic ecosystems. Although freshwater lake sediments remove N through various microbially mediated processes, few studies have investigated the microbial communities involved. In an integrated biogeochemical and microbiological study on a eutrophic and oligotrophic lake, we estimated N removal rates from pore water concentration gradients in sediments. Simultaneously, the abundance of different microbial N transformation genes was investigated using metagenomics on a seasonal and spatial scale. We observed that contrasting nutrient concentrations in sediments were associated with distinct microbial community compositions and significant differences in abundances of various N transformation genes. For both characteristics, we observed a more pronounced spatial than seasonal variability within each lake. The eutrophic Lake Baldegg showed a higher denitrification potential with higher nosZ gene (N2O reductase) abundances and higher nirS:nirK (nitrite reductase) ratios, indicating a greater capacity for complete denitrification. Correspondingly, this lake had a higher N removal efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for nitrification. Specifically, it harbored a high abundance of Nitrospira, including some with the potential for comammox. Our results demonstrate that knowledge of the genomic N transformation potential is important for interpreting N process rates and understanding how the lacustrine sedimentary N cycle responds to variations in trophic conditions. IMPORTANCE Anthropogenic nitrogen (N) inputs can lead to eutrophication in surface waters, especially in N-limited coastal ecosystems. Lakes effectively remove reactive N by transforming it to N2 through microbial denitrification or anammox. The rates and distributions of these microbial processes are affected by factors such as the amount and quality of settling organic material and nitrate concentrations. However, the microbial communities mediating these N transformation processes in freshwater lake sediments remain largely unknown. We provide the first seasonally and spatially resolved metagenomic analysis of the N cycle in sediments of two lakes with different trophic states. We show that lakes with different trophic states select for distinct communities of N-cycling microorganisms with contrasting functional potentials for N transformation.

Nitrogen removal processes in lakes of different trophic states from on-site measurements and historic data.

Freshwater lakes are essential hotspots for the removal of excessive anthropogenic nitrogen (N) loads transported from the land to coastal oceans. The biogeochemical processes responsible for N removal, the corresponding transformation rates and overall removal efficiencies differ between lakes, however, it is unclear what the main controlling factors are. Here, we investigated the factors that moderate the rates of N removal under contrasting trophic states in two lakes located in central Switzerland. In the eutrophic Lake Baldegg and the oligotrophic Lake Sarnen, we specifically examined seasonal sediment porewater chemistry, organic matter sedimentation rates, as well as 33-year of historic water column data. We find that the eutrophic Lake Baldegg, which contributed to the removal of 20 ± 6.6 gN m-2 year-1, effectively removed two-thirds of the total areal N load. In stark contrast, the more oligotrophic Lake Sarnen contributed to 3.2 ± 4.2 gN m-2 year-1, and had removed only one-third of the areal N load. The historic dataset of the eutrophic lake revealed a close linkage between annual loads of dissolved N (DN) and removal rates (NRR = 0.63 × DN load) and a significant correlation of the concentration of bottom water nitrate and removal rates. We further show that the seasonal increase in N removal rates of the eutrophic lake correlated significantly with seasonal oxygen fluxes measured across the water-sediment interface (R2 = 0.75). We suggest that increasing oxygen enhances sediment mineralization and stimulates nitrification, indirectly enhancing denitrification activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00027-021-00795-7.

Anoxic chlorophyll maximum enhances local organic matter remineralization and nitrogen loss in Lake Tanganyika.

In marine and freshwater oxygen-deficient zones, the remineralization of sinking organic matter from the photic zone is central to driving nitrogen loss. Deep blooms of photosynthetic bacteria, which form the suboxic/anoxic chlorophyll maximum (ACM), widespread in aquatic ecosystems, may also contribute to the local input of organic matter. Yet, the influence of the ACM on nitrogen and carbon cycling remains poorly understood. Using a suite of stable isotope tracer experiments, we examined the transformation of nitrogen and carbon under an ACM (comprising of Chlorobiaceae and Synechococcales) and a non-ACM scenario in the anoxic zone of Lake Tanganyika. We find that the ACM hosts a tight coupling of photo/litho-autotrophic and heterotrophic processes. In particular, the ACM was a hotspot of organic matter remineralization that controlled an important supply of ammonium driving a nitrification-anammox coupling, and thereby played a key role in regulating nitrogen loss in the oxygen-deficient zone.

Comparison of sulfide-oxidizing Sulfurimonas strains reveals a new mode of thiosulfate formation in subsurface environments.

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S0 ) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY1 Z1 AB and soxCDY2 Z2 H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY2 Z2 H cluster and not soxXY1 Z1 AB. Despite the absence of the soxXY1 Z1 AB cluster, strain CVO oxidized S0 to thiosulfate and sulfate, demonstrating that soxCDY2 Z2 H genes alone are sufficient for S0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY2 Z2 H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant.

Arcobacter peruensis sp. nov., a Chemolithoheterotroph Isolated from Sulfide- and Organic-Rich Coastal Waters off Peru.

Members of the epsilonproteobacterial genus Arcobacter have been identified to be potentially important sulfide oxidizers in marine coastal, seep, and stratified basin environments. In the highly productive upwelling waters off the coast of Peru, Arcobacter cells comprised 3 to 25% of the total microbial community at a near-shore station where sulfide concentrations exceeded 20 µM in bottom waters. From the chemocline where the Arcobacter population exceeded 106 cells ml-1 and where high rates of denitrification (up to 6.5 ± 0.4 µM N day-1) and dark carbon fixation (2.8 ± 0.2 µM C day-1) were measured, we isolated a previously uncultivated Arcobacter species, Arcobacter peruensis sp. nov. (BCCM LMG-31510). Genomic analysis showed that A. peruensis possesses genes encoding sulfide oxidation and denitrification pathways but lacks the ability to fix CO2 via autotrophic carbon fixation pathways. Genes encoding transporters for organic carbon compounds, however, were present in the A. peruensis genome. Physiological experiments demonstrated that A. peruensis grew best on a mix of sulfide, nitrate, and acetate. Isotope labeling experiments further verified that A. peruensis completely reduced nitrate to N2 and assimilated acetate but did not fix CO2, thus coupling heterotrophic growth to sulfide oxidation and denitrification. Single-cell nanoscale secondary ion mass spectrometry analysis of samples taken from shipboard isotope labeling experiments also confirmed that the Arcobacter population in situ did not substantially fix CO2 The efficient growth yield associated with the chemolithoheterotrophic metabolism of A. peruensis may allow this Arcobacter species to rapidly bloom in eutrophic and sulfide-rich waters off the coast of Peru.IMPORTANCE Our multidisciplinary approach provides new insights into the ecophysiology of a newly isolated environmental Arcobacter species, as well as the physiological flexibility within the Arcobacter genus and sulfide-oxidizing, denitrifying microbial communities within oceanic oxygen minimum zones (OMZs). The chemolithoheterotrophic species Arcobacter peruensis may play a substantial role in the diverse consortium of bacteria that is capable of coupling denitrification and fixed nitrogen loss to sulfide oxidation in eutrophic, sulfidic coastal waters. With increasing anthropogenic pressures on coastal regions, e.g., eutrophication and deoxygenation (D. Breitburg, L. A. Levin, A. Oschlies, M. Grégoire, et al., Science 359:eaam7240, 2018, https://doi.org/10.1126/science.aam7240), niches where sulfide-oxidizing, denitrifying heterotrophs such as A. peruensis thrive are likely to expand.

Metabolites of an Oil Field Sulfide-Oxidizing, Nitrate-Reducing Sulfurimonas sp. Cause Severe Corrosion.

Oil reservoir souring and associated material integrity challenges are of great concern to the petroleum industry. The bioengineering strategy of nitrate injection has proven successful for controlling souring in some cases, but recent reports indicate increased corrosion in nitrate-treated produced water reinjection facilities. Sulfide-oxidizing, nitrate-reducing bacteria (soNRB) have been suggested to be the cause of such corrosion. Using the model soNRB Sulfurimonas sp. strain CVO obtained from an oil field, we conducted a detailed analysis of soNRB-induced corrosion at initial nitrate-to-sulfide (N/S) ratios relevant to oil field operations. The activity of strain CVO caused severe corrosion rates of up to 0.27 millimeters per year (mm y-1) and up to 60-µm-deep pitting within only 9 days. The highest corrosion during the growth of strain CVO was associated with the production of zero-valent sulfur during sulfide oxidation and the accumulation of nitrite, when initial N/S ratios were high. Abiotic corrosion tests with individual metabolites confirmed biogenic zero-valent sulfur and nitrite as the main causes of corrosion under the experimental conditions. Mackinawite (FeS) deposited on carbon steel surfaces accelerated abiotic reduction of both sulfur and nitrite, exacerbating corrosion. Based on these results, a conceptual model for nitrate-mediated corrosion by soNRB is proposed.IMPORTANCE Ambiguous reports of corrosion problems associated with the injection of nitrate for souring control necessitate a deeper understanding of this frequently applied bioengineering strategy. Sulfide-oxidizing, nitrate-reducing bacteria have been proposed as key culprits, despite the underlying microbial corrosion mechanisms remaining insufficiently understood. This study provides a comprehensive characterization of how individual metabolic intermediates of the microbial nitrogen and sulfur cycles can impact the integrity of carbon steel infrastructure. The results help explain the dramatic increases seen at times in corrosion rates observed during nitrate injection in field and laboratory trials and point to strategies for reducing adverse integrity-related side effects of nitrate-based souring mitigation.

Oxygen minimum zone cryptic sulfur cycling sustained by offshore transport of key sulfur oxidizing bacteria.

Members of the gammaproteobacterial clade SUP05 couple water column sulfide oxidation to nitrate reduction in sulfidic oxygen minimum zones (OMZs). Their abundance in offshore OMZ waters devoid of detectable sulfide has led to the suggestion that local sulfate reduction fuels SUP05-mediated sulfide oxidation in a so-called "cryptic sulfur cycle". We examined the distribution and metabolic capacity of SUP05 in Peru Upwelling waters, using a combination of oceanographic, molecular, biogeochemical and single-cell techniques. A single SUP05 species, U Thioglobus perditus, was found to be abundant and active in both sulfidic shelf and sulfide-free offshore OMZ waters. Our combined data indicated that mesoscale eddy-driven transport led to the dispersal of U T. perditus and elemental sulfur from the sulfidic shelf waters into the offshore OMZ region. This offshore transport of shelf waters provides an alternative explanation for the abundance and activity of sulfide-oxidizing denitrifying bacteria in sulfide-poor offshore OMZ waters.

Metabolic versatility of a novel N2 -fixing Alphaproteobacterium isolated from a marine oxygen minimum zone.

The N2 -fixing (diazotrophic) community in marine ecosystems is dominated by non-cyanobacterial microorganisms. Yet, very little is known about their identity, function and ecological relevance due to a lack of cultured representatives. Here we report a novel heterotrophic diazotroph isolated from the oxygen minimum zone (OMZ) off Peru. The new species belongs to the genus Sagittula (Rhodobacteraceae, Alphaproteobacteria) and its capability to fix N2 was confirmed in laboratory experiments. Genome sequencing revealed that it is a strict heterotroph with a high versatility in substrate utilization and energy acquisition mechanisms. Pathways for sulfide oxidation and nitrite reduction to nitrous oxide are encoded in the genome and might explain the presence throughout the Peruvian OMZ. The genome further indicates that this novel organism could be in direct interaction with other microbes or particles. NanoSIMS analyses were used to compare the metabolic potential of S. castanea with single-cell activity in situ; however, N2 fixation by this diazotroph could not be detected at the isolation site. While the biogeochemical impact of S. castanea is yet to be resolved, its abundance and widespread distribution suggests that its potential to contribute to the marine N input could be significant at a larger geographical scale.

Enhanced Nitrogen Loss by Eddy-Induced Vertical Transport in the Offshore Peruvian Oxygen Minimum Zone.

The eastern tropical South Pacific (ETSP) upwelling region is one of the ocean's largest sinks of fixed nitrogen, which is lost as N2 via the anaerobic processes of anammox and denitrification. One-third of nitrogen loss occurs in productive shelf waters stimulated by organic matter export as a result of eastern boundary upwelling. Offshore, nitrogen loss rates are lower, but due to its sheer size this area accounts for ~70% of ETSP nitrogen loss. How nitrogen loss and primary production are regulated in the offshore ETSP region where coastal upwelling is less influential remains unclear. Mesoscale eddies, ubiquitous in the ETSP region, have been suggested to enhance vertical nutrient transport and thereby regulate primary productivity and hence organic matter export. Here, we investigated the impact of mesoscale eddies on anammox and denitrification activity using 15N-labelled in situ incubation experiments. Anammox was shown to be the dominant nitrogen loss process, but varied across the eddy, whereas denitrification was below detection at all stations. Anammox rates at the eddy periphery were greater than at the center. Similarly, depth-integrated chlorophyll paralleled anammox activity, increasing at the periphery relative to the eddy center; suggestive of enhanced organic matter export along the periphery supporting nitrogen loss. This can be attributed to enhanced vertical nutrient transport caused by an eddy-driven submesoscale mechanism operating at the eddy periphery. In the ETSP region, the widespread distribution of eddies and the large heterogeneity observed in anammox rates from a compilation of stations suggests that eddy-driven vertical nutrient transport may regulate offshore primary production and thereby nitrogen loss.

Acetate production from oil under sulfate-reducing conditions in bioreactors injected with sulfate and nitrate.

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266-269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.

Improving PCR efficiency for accurate quantification of 16S rRNA genes.

Quantitative real-time PCR is a valuable tool for microbial ecologists. To obtain accurate absolute quantification it is essential that PCR efficiency for pure standards is close to amplification efficiency for test samples. Counter to normal expectation that PCR efficiency might be lower in environmental DNA, due to the presence of PCR inhibitors, we report the counterintuitive observation that PCR efficiency of pure standards can be lower than for environmental DNA. This can lead to overestimation of gene abundances if not corrected. SYBR green-based qPCR assays of 16S rRNA genes targeting Bacteria, Syntrophus and Smithella spp., Marinobacter spp., Methanomicrobiales, Methanosarcinaceae, and Methanosaetaceae in samples from methanogenic crude oil biodegradation enrichments were tested. In five out of the six assays, PCR efficiency was lower with pure standards than with environmental DNA samples. We developed a solution to this problem based on amending pure clone standards with a background of non-target environmental 16S rRNA genes which significantly improved PCR efficiency of standards in the qPCR assays that exhibited this phenomenon. Overall this method of qPCR standard preparation achieved a more reliable and robust quantification of 16S rRNA genes. We believe this may be a potentially common issue in microbial ecology that often goes unreported, as intuitively one would not expect standards to have poorer PCR efficiency than samples.

Microbial community succession in a bioreactor modeling a souring low-temperature oil reservoir subjected to nitrate injection.

Injection of up-flow packed-bed bioreactors with excess volatile fatty acids and limiting concentrations of nitrate and sulfate gave complete reduction of nitrate from 0 to 5.5 cm and complete or near-complete reduction of sulfate from 3.2 to 11.5 cm along the bioreactor flow path. Most of the biomass (85%) and most of the genes for nitrate reduction (narG, 96%; napA, 99%) and for sulfate reduction (dsrB, 91%) were present near the inlet (0-5.5 cm) of the 37-cm-long bioreactor. Microbial community analysis by a combination of denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA amplicons indicated that nitrate-reducing Arcobacter and Pseudomonas species were located from 0 to 3.2 cm and throughout, respectively. Desulfobulbus species were the main sulfate reducers present and acetotrophic methanogens of the genus Methanosaeta predominated at 20-37 cm. Overall, the results indicated a succession of microbial communities along the bioreactor flow path. In the absence of nitrate, the sulfate reduction zone moved nearer to the bioreactor inlet. The sulfide concentration in the bioreactor effluent was temporarily lowered after nitrate injection was re-started. Hence, the bioreactor sulfide output could be disrupted by pulsed, not by constant nitrate injection, as demonstrated also previously in a low-temperature oil field.