Estuarine Bacteria Research Articles

Uncovering the biotechnological capacity of marine and brackish water Planctomycetota

An appealing strategy for finding novel bioactive molecules in Nature consists in exploring underrepresented and -studied microorganisms. Here, we investigated the antimicrobial and tumoral anti-proliferative bioactivities of twenty-three marine and estuarine bacteria of the fascinating phylum Planctomycetota. This was achieved through extraction of compounds produced by the Planctomycetota cultured in oligotrophic medium followed by an antimicrobial screening against ten relevant human pathogens including Gram-positive and Gram-negative bacteria, and fungi. Cytotoxic effects of the extracts were also evaluated against five tumoral cell lines. Moderate to potent activities were obtained against Enterococcus faecalis, methicillin-sensitive and methicillin-resistant Staphylococcus aureus and vancomycin-sensitive and vancomycin-resistant Enterococcus faecium. Anti-fungal effects were observed against Trichophyton rubrum, Candida albicans and Aspergillus fumigatus. The highest cytotoxic effects were observed against human breast, pancreas and melanoma tumoral cell lines. Novipirellula caenicola and Rhodopirellula spp. strains displayed the widest spectrum of bioactivities while Rubinisphaera margarita ICM_H10T affected all Gram-positive bacteria tested. LC-HRMS analysis of the extracts did not reveal the presence of any known bioactive natural product, suggesting that the observed activities are most likely caused by novel molecules, that need identification. In summary, we expanded the scope of planctomycetal species investigated for bioactivities and demonstrated that various strains are promising sources of novel bioactive compounds, which reenforces the potential biotechnological prospects offered by Planctomycetota.

Aeromonas Species in Meat Samples Obtained From Some Markets in Parts of Plateau State, Nigeria

Aeromonas species are emerging food borne pathogens with serious potentials for causing food borne diseases. They are estuarine bacteria and are ubiquitous in fresh water, ground water, bottled water, meats and fresh vegetables. A total of 800 meat samples, comprising of 4 different meat types; Chicken (200), beef (200), Fish (200) and chevon (goat meat) (200) were collected in the period of one year (from June 2017—June 2018) in Jos, Miango, Bukuru, Vom and Lamingo areas of Plateau state. These meat samples were analyzed for the presence of Aeromonas species using Aeromonas Dextrin agar and MacConkey agar. Overall 114 meat samples out of the 800 samples analyzed were positive for Aeromonas species with the beef having the highest percentage prevalence of 9.25% while the chevon had the lowest percentage prevalence of 5.75%. The result of this study revealed the presence of potentially pathogenic Aeromonas species in the meat samples obtained from these locations.

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria.

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell-cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as β-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound β-glucosidase and leucine-aminopeptidase activities significantly increased in 4%-68% of strains but decreased in 0%-21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.

Extracellular polymeric substances mediate the coaggregation of aquatic biofilm-forming bacteria

Coaggregation, a phenomenon contributing to biofilm formation, occurs among biofilm bacteria from different aquatic environments. However, not much is known about molecules involved in aggregation. In this study, freshwater, estuarine and marine biofilm bacteria were evaluated for aggregation capabilities, and their cell-bound extracellular polymeric substances (CB-EPS), known to play an important role in biofilm formation, were characterized for functional groups, and sugar composition via Fourier-transform infrared spectroscopy and high-pressure liquid chromatography. Biofilm-forming potential of estuarine and freshwater biofilm bacteria was higher as indicated by their coaggregation scores, attributed to CB-EPS with distinct sugar types, compared to marine. Most of the biofilm bacteria lost their ability to coaggregate after removal of CB-EPS, indicating its importance in coaggregation. Estuarine (Bacillus indicus, Bacillus cereus), and freshwater (Exiguobacterium spp., B. cereus) bacterial pairs, retained their aggregation capability probably via expression of lipids and proteins, suggesting their ability to rebuild themselves by expressing specific biomolecules under stressed conditions. A similar expression pattern was observed when these strains were exposed to abrupt salinity change (environmental stressor), indicating modulation of cell surface chemistry as a strategy to protect biofilm bacteria in harsh conditions. Unravelling role of these biomolecules as cues for settlement of macrofoulers is a step ahead.

Seasonal variations in antibiotic resistance genes in estuarine sediments and the driving mechanisms

Estuary sediments are chemically contaminated by adjacent coastal industrial cities, but the impact of organic pollutants on antibiotic resistance genes (ARGs) in estuarine sediments is unknown. We comprehensively analyzed the complex interactions between chemical pollutants (heavy metals and organic pollutants), mobile genetic elements (MGEs), and ARGs in estuarine sediments during various seasons. The results indicate that under the effects of the chemically polluted river water, the number of different estuarine sediment ARGs increased by 76.9%–92.3% in summer and 5.9%–35.3% in winter, and the abundance of these ARGs increased by 29–5195 times in summer and 48–239 times in winter. The abundance of sediment ARGs in distinct estuaries showed different seasonal trends. Seasonal changes had a greater impact on the abundance of estuarine sediment ARGs than on their diversity. The diversity of estuarine sediment ARGs was positively correlated with the chemical pollution levels. Furthermore, chemical pollution was positively correlated with MGEs, and MGEs were correlated with ARG abundance. These results indicate that ARGs are enriched in bacteria via horizontal gene transfer triggered by chemical pollution, promoting multi-antibiotic resistance in estuarine sediment bacteria. These findings have implications for our understanding of the distribution and propagation of ARGs in chemically polluted estuarine sediments.

Chromate detoxification potential of Staphylococcus sp. isolates from an estuary.

Chromium (Cr) pollution is an emerging environmental problem. The present study was carried out to isolate Cr-resistant bacteria and characterize their Cr detoxification and resistance ability. Bacteria screened by exposure to chromate (Cr6+) were isolated from Mandovi estuary Goa, India. Two isolates expressed high resistance to Cr6+ (MIC ≥ 300 µg mL-1), Cr3+ (MIC ≥ 900 µg mL-1), other toxic heavy metals and displayed a pattern of resistance to cephalosporins and ß-lactams. Biochemical and 16 S rRNA gene sequence analysis indicated that both isolates tested belonged to the Staphylococcus genus and were closely related to S. saprophyticus and S. arlettae. Designated as strains NIOER176 and NIOER324, batch experiments demonstrated that both removed 100% of 20 and 50 µg mL-1 Cr6+ within 4 and 10 days, respectively. The rate of reduction in both peaked at 0.260 µg mL-1 h-1. ATP-binding cassette (ABC) transporter gene involved in transport of a variety of substrates including efflux of toxicants was present in strain NIOER176. Through SDS-PAGE analysis, whole-cell proteins extracted from both strains indicated chromium-induced specific induction and up-regulation of 24 and 40 kDa proteins. Since bacterial ability to ameliorate Cr6+ is of practical significance, these findings demonstrate strong potential of some estuarine bacteria to detoxify Cr6+ even when its concentrations far exceed the concentrations reported from many hazardous effluents and chromium contaminated natural habitats. Such potential of salt tolerant bacteria would help in Cr6+ bioremediation efforts.

Distribution of thermophilic endospores in a temperate estuary indicate that dispersal history structures sediment microbial communities.

SummaryEndospores of thermophilic bacteria are found in cold and temperate sediments where they persist in a dormant state. As inactive endospores that cannot grow at the low ambient temperatures, they are akin to tracer particles in cold sediments, unaffected by factors normally governing microbial biogeography (e.g., selection, drift, mutation). This makes thermophilic endospores ideal model organisms for studying microbial biogeography since their spatial distribution can be directly related to their dispersal history. To assess dispersal histories of estuarine bacteria, thermophilic endospores were enriched from sediments along a freshwater‐to‐marine transect of the River Tyne in high temperature incubations (50°C). Dispersal histories for 75 different taxa indicated that the majority of estuarine endospores were of terrestrial origin; most closely related to bacteria from warm habitats associated with industrial activity. A subset of the taxa detected were marine derived, with close relatives from hot deep marine biosphere habitats. These patterns are consistent with the sources of sediment in the River Tyne being predominantly terrestrial in origin. The results point to microbial communities in estuarine and marine sediments being structured by bi‐directional currents, terrestrial run‐off and industrial effluent as vectors of passive dispersal and immigration.

Microbial Antibiotic Resistance and Heavy Metal Tolerance and their Implications and prospects with Environment

Most natural waterways include microorganisms, which are extensively dispersed across the natural world. Their diversity and abundance can serve as indicators of whether a body of water is suitable for fish, wildlife, or leisure and recreational uses. Hundreds of antimicrobial medicines have been produced for use in humans and animals since the discovery of penicillin in the late 1920s, significantly lowering the morbidity and mortality linked to a variety of infectious illnesses. Approximately half of the over one million tons of antibiotics that have been discharged into the biosphere in the past fifty years are thought to have entered through veterinary and agricultural systems the ongoing exposure of microbial populations to substances chosen for resistance strains, such as antibiotics, chemical compounds, heavy metals, and other agents. Bacteria that have adapted to the presence of heavy metals and evolved effective metal resistance mechanisms live in ecosystems that have been contaminated by dangerous quantities of heavy metals. Based on the degree of pollution, the frequency of plasmids differed from site to site, according to ecological research on the incidence of plasmids in natural populations of freshwater, marine, estuarine, and terrestrial bacteria.

Vibrio parahaemolyticus and Vibrio vulnificus Recovered from Oysters during an Oyster Relay Study.

Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring estuarine bacteria and are the leading causes of seafood-associated infections and mortality in the United States. Though multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus strains have been reported, resistance patterns in vibrios are not as well documented as those of other foodborne bacterial pathogens. Salinity relaying (SR) is a postharvest processing (PHP) treatment to reduce the abundances of these pathogens in shellfish harvested during the warmer months. The purpose of this study was to evaluate the antimicrobial susceptibility (AMS), pathogenicity, and genetic profiles of V. parahaemolyticus and V. vulnificus recovered from oysters during an oyster relay study. Isolates (V. parahaemolyticus [n = 296] and V. vulnificus [n = 94]) were recovered from oysters before and during the 21-day relaying study to detect virulence genes (tdh and trh) and genes correlated with virulence (vcgC) using multiplex quantitative PCR (qPCR). AMS to 20 different antibiotics was investigated using microbroth dilution, and pulsed-field gel electrophoresis (PFGE) was used to study the genetic profiles of the isolates. Twenty percent of V. vulnificus isolates were vcgC+, while 1 and 2% of V. parahaemolyticus were tdh+ and trh+, respectively. More than 77% of the V. vulnificus isolates and 30% of the V. parahaemolyticus isolates were resistant to at least one antimicrobial. Forty-eight percent of V. vulnificus and 8% of V. parahaemolyticus isolates were resistant to two or more antimicrobials. All isolates demonstrated a high genetic diversity, even among those isolated from the same site and having a similar AMS profile. No significant effects of the relaying process on AMS, virulence genes, or PFGE profiles of V. vulnificus and V. parahaemolyticus were observed.IMPORTANCE Analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from oysters during this study indicated that more than 48% of V. vulnificus isolates were resistant to two or more antimicrobials, including those recommended by the CDC for treating Vibrio infections. Also, the V. parahaemolyticus isolates showed high MICs for some of the Vibrio infection treatment antibiotics. Monitoring of AMS profiles of this bacterium is important to ensure optimal treatment of infections and improve food safety. Our study showed no significant differences in the AMS profiles of V. vulnificus (P = 0.26) and V. parahaemolyticus (P = 0.23) isolated from the oysters collected before versus after relaying. This suggests that the salinity of the relaying sites did not affect the AMS profiles of the Vibrio isolates, although it did reduce the numbers of these bacteria in oysters (S. Parveen et al., J Food Sci 82:484-491, 2017, https://doi.org/10.1111/1750-3841.13584).

Reprint of: Contribution of enrichments and resampling for sulfate reducing bacteria diversity assessment by high-throughput cultivation

The development of new high-throughput cultivation methods aims to increase the isolation efficiency as compared to standard techniques that often require enrichment procedures to compensate the low microbial recovery. In the current study, estuarine sulfate-reducing bacteria were isolated using an anaerobic isolation procedure in 384-well microplates. Ninety-nine strains were recovered from initial sediments. Isolates were identified according to their partial 16S rRNA sequences and clustered into 13 phylotypes. Besides, the increase in species richness obtained through enrichments or resampling was investigated. Forty-four enrichment procedures were conducted and shifts in sulfate-reducing bacterial communities were investigated through dsrAB gene fingerprinting. Despite efforts in conducting numerous enrichment conditions only few of them were statistically different from initial sample. The cultural diversity obtained from 3 of the most divergent enrichments, as well as from resampled sediments equally contributed to raise the sulfate-reducing diversity up to 22 phylotypes. Enrichments (selection of metabolism) or resampling (transient populations and micro-heterogeneity) may still be helpful to assess new microbial phylotypes. Nevertheless, all the newly cultivated strains were all representatives of minor Operational Taxonomic Units and could eventually be recovered by maintaining high-throughput isolation effort from the initial sediments.

Bacterial production, abundance and cell properties in boreal estuaries: relation to dissolved organic matter quantity and quality

Estuarine bacteria are key modifiers of allochthonous matter entering the sea, and thereby control major biogeochemical processes such as nutrient cycling and organic matter transformation. In a highly dynamic estuarine environment, bacterial growth and activity are regulated by multiple factors including the availability of organic carbon, inorganic nutrient concentrations, salinity and temperature. The variability of estuarine bacterial communities in relation to environmental factors and dissolved organic matter (DOM) quantity and quality was studied in three Baltic Sea estuaries with different catchment characteristics, flow and mixing regimes. The work was conducted during six consecutive seasons over 2 years. Bacterial activity, biomass and cytometric cell population characteristics were studied against an suite of environmental parameters and DOM quality and quantity (Salinity, DOC, DON and DIN concentration, SUVA254, UV Slope275–295, Fluoresecence peaks A and T, and Apparent mean molecular weight). Environmental variables and DOM quality varied significantly between rivers, indicating influence of catchment characteristics. Bacterial biomass varied significantly between the sampling seasons, and bacterial production also varied between the rivers, indicating an influence of DOM quality on bacterial activity. Based on cytometric grouping into low- and high- nucleic acid bacterial populations (LNA and HNA, respectively) salinity-dependent and salinity-independent clusters of bacterial variables were identified. The results suggest that the LNA clusters were not directly dependent on salinity, but possibly linked to humic substance degradation, whereas the HNA clusters were linked to salinity-correlated environmental variables reflecting DOM quality. Mixed-effects regression modeling of bacterial biomass turnover time suggests DOM fluorescence properties to be useful proxies of bioavailability. Our study shows that growth and community dynamics of estuarine bacteria may be partially regulated by quality of riverine DOM, and cytometric sub-populations of estuarine bacteria have differing regulative factors.

Methanesulfonate (MSA) Catabolic Genes from Marine and Estuarine Bacteria.

Quantitatively, methanesulfonate (MSA) is a very relevant compound in the global biogeochemical sulfur cycle. Its utilization by bacteria as a source of carbon and energy has been described and a specific enzyme, methanesulfonate monooxygenase (MSAMO), has been found to perform the first catabolic step of its oxidation. Other proteins seemingly involved in the import of MSA into bacterial cells have been reported. In this study, we obtained novel sequences of genes msmA and msmE from marine, estuary and soil MSA-degraders (encoding the large subunit of the MSAMO enzyme and the periplasmic component of the import system, respectively). We also obtained whole-genome sequences of two novel marine Filomicrobium strains, Y and W, and annotated two full msm operons in these genomes. Furthermore, msmA and msmE sequences were amplified from North Atlantic seawater and analyzed. Good conservation of the MsmA deduced protein sequence was observed in both cultured strains and metagenomic clones. A long spacer sequence in the Rieske-type [2Fe-2S] cluster-binding motif within MsmA was found to be conserved in all instances, supporting the hypothesis that this feature is specific to the large (α) subunit of the MSAMO enzyme. The msmE gene was more difficult to amplify, from both cultivated isolates and marine metagenomic DNA. However, 3 novel msmE sequences were obtained from isolated strains and one directly from seawater. With both genes, our results combined with previous metagenomic analyses seem to imply that moderate to high-GC strains are somehow favored during enrichment and isolation of MSA-utilizing bacteria, while the majority of msm genes obtained by cultivation-independent methods have low levels of GC%, which is a clear example of the misrepresentation of natural populations that culturing, more often than not, entails. Nevertheless, the data obtained in this work show that MSA-degrading bacteria are abundant in surface seawater, which suggests ecological relevance for this metabolic group of bacteria.

Contribution of enrichments and resampling for sulfate reducing bacteria diversity assessment by high-throughput cultivation

The development of new high-throughput cultivation methods aims to increase the isolation efficiency as compared to standard techniques that often require enrichment procedures to compensate the low microbial recovery. In the current study, estuarine sulfate-reducing bacteria were isolated using an anaerobic isolation procedure in 384-well microplates. Ninety-nine strains were recovered from initial sediments. Isolates were identified according to their partial 16S rRNA sequences and clustered into 13 phylotypes. Besides, the increase in species richness obtained through enrichments or resampling was investigated. Forty-four enrichment procedures were conducted and shifts in sulfate-reducing bacterial communities were investigated through dsrAB gene fingerprinting. Despite efforts in conducting numerous enrichment conditions only few of them were statistically different from initial sample. The cultural diversity obtained from 3 of the most divergent enrichments, as well as from resampled sediments equally contributed to raise the sulfate-reducing diversity up to 22 phylotypes. Enrichments (selection of metabolism) or resampling (transient populations and micro-heterogeneity) may still be helpful to assess new microbial phylotypes. Nevertheless, all the newly cultivated strains were all representatives of minor Operational Taxonomic Units and could eventually be recovered by maintaining high-throughput isolation effort from the initial sediments.

Initial community and environment determine the response of bacterial communities to dispersant and oil contamination

Bioremediation of seawater by natural bacterial communities is one potential response to coastal oil spills, but the success of the approach may vary, depending on geographical location, oil composition and the timing of spill. The short term response of coastal bacteria to dispersant, oil and dispersed oil was characterized using 16S rRNA gene tags in two mesocosm experiments conducted two months apart. Despite differences in the amount of oil-derived alkanes across the treatments and experiments, increases in the contributions of hydrocarbon degrading taxa and decreases in common estuarine bacteria were observed in response to dispersant and/or oil. Between the two experiments, the direction and rates of changes in particulate alkane concentrations differed, as did the magnitude of the bacterial response to oil and/or dispersant. Together, our data underscore large variability in bacterial responses to hydrocarbon pollutants, implying that bioremediation success varies with starting biological and environmental conditions.

A direct pre-screen for marine bacteria producing compounds inhibiting quorum sensing reveals diverse planktonic bacteria that are bioactive.

A promising new strategy in antibacterial research is inhibition of the bacterial communication system termed quorum sensing. In this study, a novel and rapid pre-screening method was developed to detect the production of chemical inhibitors of this system (quorum-quenching compounds) by bacteria isolated from marine and estuarine waters. This method involves direct screening of mixed populations on an agar plate, facilitating specific isolation of bioactive colonies. The assay showed that between 4 and 46 % of culturable bacteria from various samples were bioactive, and of the 95 selectively isolated bacteria, 93.7 % inhibited Vibrio harveyi bioluminescence without inhibiting growth, indicating potential production of quorum-quenching compounds. Of the active isolates, 21 % showed further activity against quorum-sensing-regulated pigment production by Serratia marcescens. The majority of bioactive isolates were identified by 16S ribosomal DNA (rDNA) amplification and sequencing as belonging to the genera Vibrio and Pseudoalteromonas. Extracts of two strongly bioactive Pseudoalteromonas isolates (K1 and B2) were quantitatively assessed for inhibition of growth and quorum-sensing-regulated processes in V. harveyi, S. marcescens and Chromobacterium violaceum. Extracts of the isolates reduced V. harveyi bioluminescence by as much as 98 % and C. violaceum pigment production by 36 % at concentrations which had no adverse effect on growth. The activity found in the extracts indicated that the isolates may produce quorum-quenching compounds. This study further supports the suggestion that quorum quenching may be a common attribute among culturable planktonic marine and estuarine bacteria.

Processing of humic-rich riverine dissolved organic matter by estuarine bacteria: effects of predegradation and inorganic nutrients

The bioavailability of predegraded dissolved organic matter (DOM) from a humic-rich, boreal river to estuarine bacteria from the Baltic Sea was studied in 39-day bioassays. The river waters had been exposed to various degrees of bacterial degradation by storing them between 0 and 465 days in dark prior to the bioassay. The resulting predegraded DOM was inoculated with estuarine bacteria and the subsequent changes in DOM quantity and quality measured. During the incubations, dissolved organic carbon (DOC) and oxygen concentrations decreased, indicating heterotrophic activity. Coloured DOM was degraded less than DOC, indicating a selective utilization of DOM, and humic-like fluorescence components increased during the incubations. The amount of DOC degraded was not affected by the length of DOM predegradation. The percentage of bioavailable DOC (%BDOC) was higher in experiment units with added inorganic nitrogen and phosphorus than without addition (on average 13.5 % and 9.0, respectively), but had no effect on the degradation of fresh, non-predegraded, DOC (%BDOC 12.0 %). Bacterial growth efficiency (BGE) was highest (65 ± 2 %) in the units with fresh DOM, and lowest in units with predegraded DOM and no added inorganic nutrients (11 ± 4 %). The addition of inorganic nutrients increased the BGE of predegraded DOM units by an average of 28 ± 4 %. There was no significant effect on BGE by length of predegradation after the initial drop (<3 months). This study suggests that both the length of predegradation and the inorganic nutrient status in the receiving estuary has consequences to carbon cycling and will determine the amount of terrestrial-derived DOC being ultimately assimilated into marine food webs.

Impact of freshwater inflow on bacterial abundance and activity in the estuarine system Ria de Aveiro

The influence of freshwater flow on bacterial communities in the estuarine system Ria de Aveiro (Portugal) was investigated at two sites differently impacted by river inputs, representative of the marine and brackish water zones of the estuary. Sampling events were clustered based on hydrological features. The hydrodynamic was simulated with a Lagrangian model and related to microbiological parameters. Estuarine bacteria responded to different freshwater regimes developing distinct patterns of abundance and activity at the marine and brackish water zones. A circulation pattern induced by high river inflow produced vertical stratification in the marine zone, promoting a seaward flux of bacterioplankton, and stimulating the import of riverine phytoplankton and particle-attached bacteria to the brackish water zone. Advective transport and resuspension processes contributed to a 3-times increase in abundance of particle-attached bacteria during intense freshwater inputs. Additionally, bacterial activity in the estuary was controlled by inorganic nitrogen, responding to different freshwater inputs, which, in association with different prevailing sources of organic substrates induced significant changes in bacterial production. The dynamic and main controlling factors of bacterial communities are clearly impacted by freshwater inputs. Therefore, significant changes in the recycling of nutrients by microbial activities can be expected from alterations in freshwater inputs either related to global climate change or regional hydrological regimes.

Carbon fluxes through estuarine bacteria reflect coupling with phytoplankton

Microbial plankton carbon fluxes were comprehensively assessed at 4 contrasting sites in Waquoit Bay estuary (Massachusetts, USA) in 2008, focussing on the role of phytoplankton as dissolved organic matter suppliers for heterotrophic bacterioplankton. Strong seasonal patterns in environmental and microbial variables were observed, with concurrent summer maxima and minima in December for bacterial biomass (17.7�164.7 µg C l �1 ) and production (4.8�121.5 µg C l �1 d �1 ). Phytoplankton biomass (0.7�32.6 µg chlorophyll l �1 ) and primary production (PP, 164�3570 µg C l �1 d �1 ) varied similarly to bacterial variables, with dissolved PP (DPP), i.e. released extracel- lularly, ranging from 3 to 36% of the total PP. The flux of DPP represented between 0.4 and 20% of bulk dissolved organic carbon (DOC, 92�346 µmol C l �1 ) daily turnover. In addition to temper- ature, moderate bottom-up control of bacterial assemblages was suggested by biomassproduction regressions. Bacteria apparently depended more on phytoplankton-derived DOC than on its total concentration. The difference became larger when the total flux of carbon or bacterial carbon demand (BCD, 19�468 µg C l �1 d �1 ) was considered, with DPP explaining 53% of its variance compared with 37% from bulk DOC. On average, DPP could meet 50% of BCD, indicating a larger than expected trophic dependence of bacteria on phytoplankton in the whole estuarine eco system.

Different resource limitation by carbon, nitrogen and phosphorus between base flow and high flow conditions for estuarine bacteria and phytoplankton

Freshwater inflows can deliver substantial inputs of allochthonous organic carbon to estuaries. The role that allochthonous DOC has on structuring bacterial and phytoplankton communities is still not well understood. We performed a series of 1.25 L bioassay limitation experiments on the Bega and Clyde River estuaries in NSW, Australia, examining what resources limit bacteria and phytoplankton growth. We hypothesized that during base flow conditions bacteria would be carbon limited, and after high flow conditions they would be nutrient limited. A full factorial design was used with additions of carbon (glucose), nitrogen (KNO3) and phosphorus (KH2PO4). During the experiments that took place during base flow conditions bacteria were always primarily C-limited. After high flow conditions, bacteria were P-limited on the Clyde River, and remained C-limited on the Bega River. Phytoplankton growth was limited at all times in each estuary, tending toward N-limitation on the Bega River and P-limitation on the Clyde river. During high flow conditions on the Clyde River, when bacteria and phytoplankton were both primarily P-limited, it appeared that bacteria was able to outcompete phytoplankton for nutrients. These results suggest that freshwater inflows and allochthonous DOC maybe important in structuring estuarine microbial ecosystems and individual estuaries may behave differently in terms of their limiting resources.

Isolation and identification of Aeromonas caviae strain KS-1 as TBTC- and lead-resistant estuarine bacteria

Tributyltin chloride (TBTC)- and lead-resistant estuarine bacterium from Mandovi estuary, Goa, India was isolated and identified as Aeromonas caviae strain KS-1 based on biochemical characteristics and FAME analysis. It tolerates TBTC and lead up to 1.0 and 1.4 mM, respectively, in the minimal salt medium (MSM) supplemented with 0.4 % glucose. Scanning electron microscopy clearly revealed a unique morphological pattern in the form of long inter-connected chains of bacterial cells on exposure to 1 mM TBTC, whereas cells remained unaltered in presence of 1.4 mM Pb(NO₃)₂ but significant biosorption of lead (8 %) on the cell surface of this isolate was clearly revealed by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. SDS-PAGE analysis of whole-cell proteins of this lead-resistant isolate interestingly demonstrated three lead-induced proteins with molecular mass of 15.7, 16.9 and 32.4 kDa, respectively, when bacterial cells were grown under the stress of 1.4 mM Pb (NO₃)₂. This clearly demonstrated their possible involvement exclusively in lead resistance. A. caviae strain KS-1 also showed tolerance to several other heavy metals, viz. zinc, cadmium, copper and mercury. Therefore, we can employ this TBTC and lead-resistant bacterial isolate for lead bioremediation and also for biomonitoring TBTC from lead and TBTC contaminated environment.