Prokaryotic community dynamics and nitrogen-cycling genes in an oxygen-deficient upwelling system during La Niña and El Niño conditions.

Dissolved oxygen regulates microbial distribution and nitrogen cycling and, therefore, ocean productivity and Earth's climate. To date, the assembly of microbial communities in relation to oceanographic changes due to El Niño Southern Oscillation (ENSO) remains poorly understood in oxygen minimum zones (OMZ). The Mexican Pacific upwelling system supports high productivity and a permanent OMZ. Here, the spatiotemporal distribution of the prokaryotic community and nitrogen-cycling genes was investigated along a repeated transect subjected to varying oceanographic conditions associated with La Niña in 2018 and El Niño in 2019. The community was more diverse during La Niña and in the aphotic OMZ, dominated by the Subtropical Subsurface water mass, where the highest abundances of nitrogen-cycling genes were found. The largest proportion of the Gulf of California water mass during El Niño provided warmer, more oxygenated, and nutrient-poor waters towards the coast, leading to a significant increase of Synechococcus in the euphotic layer compared with the opposite conditions during La Niña. These findings suggest that prokaryotic assemblages and nitrogen genes are linked to local physicochemical conditions (e.g. light, oxygen, nutrients), but also to oceanographic fluctuations associated with ENSO phases, indicating the crucial role of climate variability in microbial community dynamics in this OMZ.

Enhanced nitrogen and carbon removal in natural seawater by electrochemical enrichment in a bioelectrochemical reactor.

Municipal and industrial wastewater discharges in coastal and marine environments are of major concern due to their high carbon and nitrogen loads and the resulted phenomenon of eutrophication. Bioelectrochemical reactors (BERs) for simultaneous nitrogen and carbon removal have gained attention owing to their cost efficiency and versatility, as well as the possibility of electrochemical enrich specific groups. This study presented a scalable two-chamber BERs using graphite granules as electrode material. BERs were inoculated and operated for 37 days using natural seawater with high concentrations of ammonium and acetate. The BERs demonstrated a maximum current density of 0.9 A m-3 and removal rates of 7.5 mg NH4+-N L-1 d-1 and 99.5 mg L-1 d-1 for total organic carbon (TOC). Removals observed for NH4+-N and TOC were 96.2% and 68.7%, respectively. The results of nutrient removal (i.e., ammonium, nitrate, nitrite and TOC) and microbial characterization (i.e., next-generation sequencing of the 16S rRNA gene and fluorescence in situ hybridization) showed that BERs operated with a poised cathode at -260 mV (vs. Ag/AgCl) significantly enriched nitrifying microorganisms in the anode and denitrifying microorganisms and planctomycetes in the cathode. Interestingly, the electrochemical enrichment did not increase the total number of microorganisms in the formed biofilms but controlled their composition. Thus, this work shows the first successful attempt to electrochemically enrich marine nitrifying and denitrifying microorganisms and presents a technique to accelerate the start-up process of BERs to remove dissolved inorganic nitrogen and total organic carbon from seawater.

Dark Diazotrophy during the Late Summer in Surface Waters of Chile Bay, West Antarctic Peninsula.

Although crucial for the addition of new nitrogen in marine ecosystems, dinitrogen (N2) fixation remains an understudied process, especially under dark conditions and in polar coastal areas, such as the West Antarctic Peninsula (WAP). New measurements of light and dark N2 fixation rates in parallel with carbon (C) fixation rates, as well as analysis of the genetic marker nifH for diazotrophic organisms, were conducted during the late summer in the coastal waters of Chile Bay, South Shetland Islands, WAP. During six late summers (February 2013 to 2019), Chile Bay was characterized by high NO3− concentrations (~20 µM) and an NH4+ content that remained stable near 0.5 µM. The N:P ratio was approximately 14.1, thus close to that of the Redfield ratio (16:1). The presence of Cluster I and Cluster III nifH gene sequences closely related to Alpha-, Delta- and, to a lesser extent, Gammaproteobacteria, suggests that chemosynthetic and heterotrophic bacteria are primarily responsible for N2 fixation in the bay. Photosynthetic carbon assimilation ranged from 51.18 to 1471 nmol C L−1 d−1, while dark chemosynthesis ranged from 9.24 to 805 nmol C L−1 d−1. N2 fixation rates were higher under dark conditions (up to 45.40 nmol N L−1 d−1) than under light conditions (up to 7.70 nmol N L−1 d−1), possibly contributing more than 37% to new nitrogen-based production (≥2.5 g N m−2 y−1). Of all the environmental factors measured, only PO43- exhibited a significant correlation with C and N2 rates, being negatively correlated (p < 0.05) with dark chemosynthesis and N2 fixation under the light condition, revealing the importance of the N:P ratio for these processes in Chile Bay. This significant contribution of N2 fixation expands the ubiquity and biological potential of these marine chemosynthetic diazotrophs. As such, this process should be considered along with the entire N cycle when further reviewing highly productive Antarctic coastal waters and the diazotrophic potential of the global marine ecosystem.

Surface Ammonia-Oxidizer Abundance During the Late Summer in the West Antarctic Coastal System.

Marine ammonia oxidizers that oxidize ammonium to nitrite are abundant in polar waters, especially during the winter in the deeper mixed-layer of West Antarctic Peninsula (WAP) waters. However, the activity and abundance of ammonia-oxidizers during the summer in surface coastal Antarctic waters remain unclear. In this study, the ammonia-oxidation rates, abundance and identity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were evaluated in the marine surface layer (to 30 m depth) in Chile Bay (Greenwich Island, WAP) over three consecutive late-summer periods (2017, 2018, and 2019). Ammonia-oxidation rates of 68.31 nmol N L-1 day-1 (2018) and 37.28 nmol N L-1 day-1 (2019) were detected from illuminated 2 m seawater incubations. However, high ammonia-oxidation rates between 267.75 and 109.38 nmol N L-1 day-1 were obtained under the dark condition at 30 m in 2018 and 2019, respectively. During the late-summer sampling periods both stratifying and mixing events occurring in the water column over short timescales (February-March). Metagenomic analysis of seven nitrogen cycle modules revealed the presence of ammonia-oxidizers, such as the Archaea Nitrosopumilus and the Bacteria Nitrosomonas and Nitrosospira, with AOA often being more abundant than AOB. However, quantification of specific amoA gene transcripts showed number of AOB being two orders of magnitude higher than AOA, with Nitrosomonas representing the most transcriptionally active AOB in the surface waters. Additionally, Candidatus Nitrosopelagicus and Nitrosopumilus, phylogenetically related to surface members of the NP-ε and NP-γ clades respectively, were the predominant AOA. Our findings expand the known distribution of ammonium-oxidizers to the marine surface layer, exposing their potential ecological role in supporting the marine Antarctic system during the productive summer periods.

Proteorhodopsin Phototrophy in Antarctic Coastal Waters.

Microbial proton-pumping rhodopsins are considered the simplest strategy among phototrophs to conserve energy from light. Proteorhodopsins are the most studied rhodopsins thus far because of their ubiquitous presence in the ocean, except in Antarctica, where they remain understudied. We analyzed proteorhodopsin abundance and transcriptional activity in the Western Antarctic coastal seawaters. Combining quantitative PCR (qPCR) and metagenomics, the relative abundance of proteorhodopsin-bearing bacteria accounted on average for 17, 3.5, and 29.7% of the bacterial community in Chile Bay (South Shetland Islands) during 2014, 2016, and 2017 summer-autumn, respectively. The abundance of proteorhodopsin-bearing bacteria changed in relation to environmental conditions such as chlorophyll a and temperature. Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were the main bacteria that transcribed the proteorhodopsin gene during day and night. Although green light-absorbing proteorhodopsin genes were more abundant than blue-absorbing ones, the latter were transcribed more intensely, resulting in >50% of the proteorhodopsin transcripts during the day and night. Flavobacteriia were the most abundant proteorhodopsin-bearing bacteria in the metagenomes; however, Alphaproteobacteria and Gammaproteobacteria were more represented in the metatranscriptomes, with qPCR quantification suggesting the dominance of the active SAR11 clade. Our results show that proteorhodopsin-bearing bacteria are prevalent in Antarctic coastal waters in late austral summer and early autumn, and their ecological relevance needs to be elucidated to better understand how sunlight energy is used in this marine ecosystem. IMPORTANCE Proteorhodopsin-bearing microorganisms in the Southern Ocean have been overlooked since their discovery in 2000. The present study identify taxonomy and quantify the relative abundance of proteorhodopsin-bearing bacteria and proteorhodopsin gene transcription in the West Antarctic Peninsula's coastal waters. This information is crucial to understand better how sunlight enters this marine environment through alternative ways unrelated to chlorophyll-based strategies. The relative abundance of proteorhodopsin-bearing bacteria seems to be related to environmental parameters (e.g., chlorophyll a, temperature) that change yearly at the coastal water of the West Antarctic Peninsula during the austral late summers and early autumns. Proteorhodopsin-bearing bacteria from Antarctic coastal waters are potentially able to exploit both the green and blue spectrum of sunlight and are a prevalent group during the summer in this polar environment.

A source of isotopically light organic carbon in a low-pH anoxic marine zone.

Geochemical and stable isotope measurements in the anoxic marine zone (AMZ) off northern Chile during periods of contrasting oceanographic conditions indicate that microbial processes mediating sulfur and nitrogen cycling exert a significant control on the carbonate chemistry (pH, AT, DIC and pCO2) of this region. Here we show that in 2015, a large isotopic fractionation between DIC and POC, a DIC and N deficit in AMZ waters indicate the predominance of in situ dark carbon fixation by sulfur-driven autotrophic denitrification in addition to anammox. In 2018, however, the fractionation between DIC and POC was significantly lower, while the total alkalinity increased in the low-pH AMZ core, suggesting a predominance of heterotrophic processes. An isotope mass-balance model demonstrates that variations in the rates of sulfur- and nitrogen-mediated carbon fixation in AMZ waters contribute ~7-35% of the POC exported to deeper waters. Thus, dark carbon fixation should be included in assessments of future changes in carbon cycling and carbonate chemistry due to AMZ expansion.

Coastal Bacterial Community Response to Glacier Melting in the Western Antarctic Peninsula.

Current warming in the Western Antarctic Peninsula (WAP) has multiple effects on the marine ecosystem, modifying the trophic web and the nutrient regime. In this study, the effect of decreased surface salinity on the marine microbial community as a consequence of freshening from nearby glaciers was investigated in Chile Bay, Greenwich Island, WAP. In the summer of 2016, samples were collected from glacier ice and transects along the bay for 16S rRNA gene sequencing, while in situ dilution experiments were conducted and analyzed using 16S rRNA gene sequencing and metatranscriptomic analysis. The results reveal that certain common seawater genera, such as Polaribacter, Pseudoalteromonas and HTCC2207, responded positively to decreased salinity in both the bay transect and experiments. The relative abundance of these bacteria slightly decreased, but their functional activity was maintained and increased the over time in the dilution experiments. However, while ice bacteria, such as Flavobacterium and Polaromonas, tolerated the increased salinity after mixing with seawater, their gene expression decreased considerably. We suggest that these bacterial taxa could be defined as sentinels of freshening events in the Antarctic coastal system. Furthermore, these results suggest that a significant portion of the microbial community is resilient and can adapt to disturbances, such as freshening due to the warming effect of climate change in Antarctica.

Greenhouse gases, nutrients and the carbonate system in the Reloncaví Fjord (Northern Chilean Patagonia): Implications on aquaculture of the mussel, Mytilus chilensis, during an episodic volcanic eruption.

This study investigates the immediate and mid-term effects of the biogeochemical variables input into the Reloncaví fjord (41°40'S; 72°23'O) as a result of the eruption of Calbuco volcano. Reloncaví is an estuarine system supporting one of the largest mussels farming production within Northern Chilean-Patagonia. Field-surveys were conducted immediately after the volcanic eruption (23-30 April 2015), one month (May 2015), and five months posterior to the event (September 2015). Water samples were collected from three stations along the fjord to determine greenhouse gases [GHG: methane (CH4), nitrous oxide (N2O)], nutrients [NO3-, NO2-, PO43-, Si(OH)4, sulphate (SO42-)], and carbonate systems parameters [total pH (pHT), temperature, salinity, dissolved oxygen (O2), and total alkalinity (AT)]. Additionally, the impact of physicochemical changes in the water column on juveniles of the produced Chilean blue mussel, Mytilus chilensis, was also studied. Following the eruption, a large phytoplankton bloom led to an increase in pHT, due to the uptake of dissolved-inorganic carbon in photic waters, potentially associated with the runoff of continental soil covered in volcanic ash. Indeed, high surface SO42- and GHG were observed to be associated with river discharges. No direct evidence of the eruption was observed within the carbonate system. Notwithstanding, a vertical pattern was observed, with an undersaturation of aragonite (ΩAr < 1) both in brackish surface (<3 m) and deep waters (>10 m), and saturated values in subsurface waters (3 to 7 m). Simultaneously, juvenile mussel shells showed maximized length and weight at 4 m depth. Results suggest a localized impact of the volcanic eruption on surface GHG, nutrients and short-term effects on the carbonate system. Optimal conditions for mussel calcification were identified within a subsurface refuge in the fjord. These specific attributes can be integrated into adaptation strategies by the mussel aquaculture industry to confront ocean acidification and changing runoff conditions.

Bacterial community structure in a sympagic habitat expanding with global warming: brackish ice brine at 85-90 °N.

Larger volumes of sea ice have been thawing in the Central Arctic Ocean (CAO) during the last decades than during the past 800,000 years. Brackish brine (fed by meltwater inside the ice) is an expanding sympagic habitat in summer all over the CAO. We report for the first time the structure of bacterial communities in this brine. They are composed of psychrophilic extremophiles, many of them related to phylotypes known from Arctic and Antarctic regions. Community structure displayed strong habitat segregation between brackish ice brine (IB; salinity 2.4-9.6) and immediate sub-ice seawater (SW; salinity 33.3-34.9), expressed at all taxonomic levels (class to genus), by dominant phylotypes as well as by the rare biosphere, and with specialists dominating IB and generalists SW. The dominant phylotypes in IB were related to Candidatus Aquiluna and Flavobacterium, those in SW to Balneatrix and ZD0405, and those shared between the habitats to Halomonas, Polaribacter and Shewanella. A meta-analysis for the oligotrophic CAO showed a pattern with Flavobacteriia dominating in melt ponds, Flavobacteriia and Gammaproteobacteria in solid ice cores, Flavobacteriia, Gamma- and Betaproteobacteria, and Actinobacteria in brine, and Alphaproteobacteria in SW. Based on our results, we expect that the roles of Actinobacteria and Betaproteobacteria in the CAO will increase with global warming owing to the increased production of meltwater in summer. IB contained three times more phylotypes than SW and may act as an insurance reservoir for bacterial diversity that can act as a recruitment base when environmental conditions change.

A new method to evaluate the vulnerability of watersheds facing several stressors: A case study in mediterranean Chile.

Freshwater systems are subjected to multiple anthropogenic stressors and natural disturbances that act as debilitating agents and modifiers of river systems, causing cumulative and synergistic effects that deteriorate their health and result in watershed vulnerability. This study proposes an easy-to-apply spatial method of watershed vulnerability evaluation using Geographic Information Systems (GIS) in the Andalién River watershed, located in the Chilean mediterranean. A watershed vulnerability index (WVI) based on three sub-indices - anthropogenic stressors, environmental fragility and natural disturbances - was developed. To determine the index grouping weights, expert surveys were carried out using the Delphi method. We subsequently normalized and integrated the factors of each sub-index with relative weights. The ranges of each thematic layer were re-classified to establish vulnerability scores. The watershed was divided into three sections: headwaters zone, transfer zone and depositional zone. The watershed vulnerability index showed that 41% of the watershed had very low vulnerability and 42% had medium vulnerability, while only 1% - in the depositional zone - had high vulnerability. A one-way ANOVA was carried out to analyze the vulnerability differences among the three sections of the watershed; it showed significant differences (F (2, 16) = 8.15: p < 0.05). The a posteriori test showed differences between the headwaters and depositional zones (Tukey test, p = 0.005) and between the transfer and depositional zones (Tukey test, p = 0.014). To validate the WVI, water quality was measured at 16 stations in the watershed; there was a significant correlation between vulnerability level and NO2- levels (r = 0.8; p = 0.87; α = 0.05) and pH (r = 0.8; p = 0.80; α = 0.05). The WVI showed the cumulative effects of multiple stressors in the depositional zone of the watershed. This is the first study to evaluate and validate non-regulated watershed vulnerability with GIS using multiple anthropogenic and natural stressors.

Microbial activity during a coastal phytoplankton bloom on the Western Antarctic Peninsula in late summer.

Phytoplankton biomass during the austral summer is influenced by freezing and melting cycles as well as oceanographic processes that enable nutrient redistribution in the West Antarctic Peninsula (WAP). Microbial functional capabilities, metagenomic and metatranscriptomic activities as well as inorganic 13C- and 15N-assimilation rates were studied in the surface waters of Chile Bay during two contrasting summer periods in 2014. Concentrations of Chlorophyll a (Chla) varied from 0.3 mg m-3 in February to a maximum of 2.5 mg m-3 in March, together with a decrease in nutrients; however, nutrients were never depleted. The microbial community composition remained similar throughout both sampling periods; however, microbial abundance and activity changed with Chla levels. An increased biomass of Bacillariophyta, Haptophyceae and Cryptophyceae was observed along with night-grazing activity of Dinophyceae and ciliates (Alveolates). During high Chla conditions, HCO3- uptake rates during daytime incubations increased 5-fold (>2516 nmol C L-1 d-1), and increased photosynthetic transcript numbers that were mainly associated with cryptophytes; meanwhile night time NO3- (>706 nmol N L-1 d-1) and NH4+ (41.7 nmol N L-1 d-1) uptake rates were 2- and 3-fold higher, respectively, due to activity from Alpha-/Gammaproteobacteria and Bacteroidetes (Flavobacteriia). Due to a projected acceleration in climate change in the WAP, this information is valuable for predicting the composition and functional changes in Antarctic microbial communities.

Unravel biophysical factors on river water quality response in Chilean Central-Southern watersheds.

Identifying the key anthropogenic (land uses) and natural (topography and climate) biophysical drivers affecting river water quality is essential for efficient management of water resources. We tested the hypothesis that water quality can be predicted by different biophysical factors. Multivariate statistics based on a geographical information system (GIS) were used to explore the influence of factors (i.e., precipitation, topography, and land uses) on water quality (i.e., nitrate (NO 3 (-)), phosphate (PO 4 (3-)), silicate (Si(OH)4), dissolved oxygen (DO), suspended solids (TSS), biological oxygen demand (DO), temperature (T), conductivity (EC), and pH) for two consecutive years in the Itata and Biobío river watersheds, Central Chile (36° 00' and 38° 30'). The results showed that (NO 3 (-)), (PO 4 (3-)), Si(OH)4, TSS, EC, and DO were higher during rainy season (austral fall, winter, and spring), whereas BOD and temperature were higher during dry season. The spatial variation of these parameters in both watersheds was related to land use, topography (e.g., soil moisture, soil hydrological group, and erodability), and precipitation. Soil hydrological group and soil moisture were the strongest explanatory predictors for PO 4 (3-) , Si(OH)4 and EC in the river, followed by land use such as agriculture for NO 3 (-) and DO and silviculture for TSS and Si(OH)4. High-resolution water leaching and runoff maps allowed us to identify agriculture areas with major probability of water leaching and higher probability of runoff in silviculture areas. Moreover, redundancy analysis (RDA) revealed that land uses (agriculture and silviculture) explained in 60 % the river water quality variation. Our finding highlights the vulnerability of Chilean river waters to different biophysical drivers, rather than climate conditions alone, which is amplified by human-induced degradation.

Biological N2O fixation in the Eastern South Pacific Ocean and marine cyanobacterial cultures.

Despite the importance of nitrous oxide (N2O) in the global radiative balance and atmospheric ozone chemistry, its sources and sinks within the Earth's system are still poorly understood. In the ocean, N2O is produced by microbiological processes such as nitrification and partial denitrification, which account for about a third of global emissions. Conversely, complete denitrification (the dissimilative reduction of N2O to N2) under suboxic/anoxic conditions is the only known pathway accountable for N2O consumption in the ocean. In this work, it is demonstrated that the biological assimilation of N2O could be a significant pathway capable of directly transforming this gas into particulate organic nitrogen (PON). N2O is shown to be biologically fixed within the subtropical and tropical waters of the eastern South Pacific Ocean, under a wide range of oceanographic conditions and at rates ranging from 2 pmol N L(-1) d(-) to 14.8 nmol N L(-1) d(-1) (mean ± SE of 0.522 ± 1.06 nmol N L(-1) d(-1), n = 93). Additional assays revealed that cultured cyanobacterial strains of Trichodesmium (H-9 and IMS 101), and Crocosphaera (W-8501) have the capacity to directly fix N2O under laboratory conditions; suggesting that marine photoautotrophic diazotrophs could be using N2O as a substrate. This metabolic capacity however was absent in Synechococcus (RCC 1029). The findings presented here indicate that assimilative N2O fixation takes place under extreme environmental conditions (i.e., light, nutrient, oxygen) where both autotrophic (including cyanobacteria) and heterotrophic microbes appear to be involved. This process could provide a globally significant sink for atmospheric N2O which in turn affects the oceanic N2O inventory and may also represent a yet unexplored global oceanic source of fixed N.

Nitrogen fixation in denitrified marine waters.

Nitrogen fixation is an essential process that biologically transforms atmospheric dinitrogen gas to ammonia, therefore compensating for nitrogen losses occurring via denitrification and anammox. Currently, inputs and losses of nitrogen to the ocean resulting from these processes are thought to be spatially separated: nitrogen fixation takes place primarily in open ocean environments (mainly through diazotrophic cyanobacteria), whereas nitrogen losses occur in oxygen-depleted intermediate waters and sediments (mostly via denitrifying and anammox bacteria). Here we report on rates of nitrogen fixation obtained during two oceanographic cruises in 2005 and 2007 in the eastern tropical South Pacific (ETSP), a region characterized by the presence of coastal upwelling and a major permanent oxygen minimum zone (OMZ). Our results show significant rates of nitrogen fixation in the water column; however, integrated rates from the surface down to 120 m varied by ∼30 fold between cruises (7.5±4.6 versus 190±82.3 µmol m(-2) d(-1)). Moreover, rates were measured down to 400 m depth in 2007, indicating that the contribution to the integrated rates of the subsurface oxygen-deficient layer was ∼5 times higher (574±294 µmol m(-2) d(-1)) than the oxic euphotic layer (48±68 µmol m(-2) d(-1)). Concurrent molecular measurements detected the dinitrogenase reductase gene nifH in surface and subsurface waters. Phylogenetic analysis of the nifH sequences showed the presence of a diverse diazotrophic community at the time of the highest measured nitrogen fixation rates. Our results thus demonstrate the occurrence of nitrogen fixation in nutrient-rich coastal upwelling systems and, importantly, within the underlying OMZ. They also suggest that nitrogen fixation is a widespread process that can sporadically provide a supplementary source of fixed nitrogen in these regions.

Ammonia-oxidizing beta-proteobacteria from the oxygen minimum zone off northern Chile.

The composition of ammonia-oxidizing bacteria from the beta-Proteobacteria subclass (betaAOB) was studied in the surface and upper-oxycline oxic waters (2- to 50-m depth, approximately 200 to 44 microM O(2)) and within the oxygen minimum zone (OMZ) suboxic waters (50- to 400-m depth, < or =10 microM O(2)) of the eastern South Pacific off northern Chile. This study was carried out through cloning and sequencing of genes coding for 16S rRNA and the ammonia monooxygenase enzyme active subunit (amoA). Sequences affiliated with Nitrosospira-like cluster 1 dominated the 16S rRNA gene clone libraries constructed from both oxic and suboxic waters. Cluster 1 consists exclusively of yet-uncultivated betaAOB from marine environments. However, a single clone, out of 224 obtained from the OMZ, was found to belong to Nitrosospira lineage cluster 0. To our knowledge, cluster 0 sequences have been derived from betaAOB isolated only from sand, soil, and freshwater environments. Sequences in clone libraries of the amoA gene from the surface and upper oxycline could be grouped in a marine subcluster, also containing no cultured representatives. In contrast, all 74 amoA sequences originating from the OMZ were either closely affiliated with cultured Nitrosospira spp. from clusters 0 and 2 or with other yet-uncultured betaAOB from soil and an aerated-anoxic Orbal process waste treatment plant. Our results reveal the presence of Nitrosospira-like betaAOB in both oxic and suboxic waters associated with the OMZ but with a clear community shift at the functional level (amoA) along the strong oxygen gradient.

Communities of nirS-type denitrifiers in the water column of the oxygen minimum zone in the eastern South Pacific.

The major sites of water column denitrification in the ocean are oxygen minimum zones (OMZ), such as one in the eastern South Pacific (ESP). To understand the structure of denitrifying communities in the OMZ off Chile, denitrifier communities at two sites in the Chilean OMZ (Antofagasta and Iquique) and at different water depths were explored by terminal restriction fragment length polymorphism analysis and cloning of polymerase chain reaction (PCR)-amplified nirS genes. NirS is a functional marker gene for denitrification encoding cytochrome cd1-containing nitrite reductase, which catalyses the reduction of nitrite to nitric oxide, the key step in denitrification. Major differences were found between communities from the two geographic locations. Shifts in community structure occurred along a biogeochemical gradient at Antofagasta. Canonical correspondence analysis indicated that O2, NO3-, NO2- and depth were important environmental factors governing these communities along the biogeochemical gradient in the water column. Phylogenetic analysis grouped the majority of clones from the ESP in distinct clusters of genes from presumably novel and yet uncultivated denitrifers. These nirS clusters were distantly related to those found in the water column of the Arabian Sea but the phylogenetic distance was even higher compared with environmental sequences from marine sediments or any other habitat. This finding suggests similar environmental conditions trigger the development of denitrifiers with related nirS genotypes despite large geographic distances.

Virulence factors of A. caviae strains isolated from acute diarrheic disease in Cuba.

Fifty Aeromonas caviae strains from intestinal infection in different Cuban provinces were identified by the Aerokey II method and virulence factors were investigated. The strains did not produce haemolysins but other exoenzymes such as proteases, lipases, and DNases; additionally, all isolates adhered to the HEp-2 cell line by the Carrello method and this did not correlate with other virulence factors presence which demonstrates that the haemolysin phenotypic expression is not necessary for these strains to be pathogenic and that pathogenicity is multifactorial, each strain expressing at least one virulence factor.