Molecular identification of deep-sea blind lobster Willemoesia forceps (Crustacea: Decapoda: Polychelidae) from the Central Indian Ridge.

The deep-sea blind lobster Willemoesia forceps A. Milne-Edwards, 1880 was collected at a water depth of 3433 m and is reported for the first time from the Central Indian Ridge (CIR). The species was identified based on morphological examination and supported by mitochondrial Cytochrome Oxidase Subunit I (mtCOI) gene. The phylogenetic analysis shows that W. leptodactyla is sister species of W. forceps. Our findings expand the distributional range of the specimen in the southern hemisphere.

Changes in morphology and metabolism enable Mn-oxidizing bacteria from mid-oceanic ridge environment to counter metal-induced stress.

Mn-oxidizing potential of two metal-tolerant bacterial strains - Halomonas meridiana and Marinobacter algicola isolated from the South West Indian Ridge waters were compared at varying concentrations of Mn (II), i.e., 1, 10, and 100 µmol and mmol L-1 . Accompanying changes in their morphology and metabolism were also determined. At concentrations >1 mmol L-1 Mn (II), Mn-oxidizing potential of M. algicola was 2-7 times greater than that of H. meridiana. Scanning electron microscopy revealed that exposure to elevated metal content prompted bacterial cells especially those of M. algicola to been enveloped in exopolymeric material and form aggregates. Energy dispersive spectrometric analysis showed that exopolymeric material acts as a nucleation site for Mn deposition and oxide formation which occurs in the form of microspherical aggregates. These features show striking resemblance to biogenically produced Fe-Mn oxide deposits from Lau Basin. Surprisingly, diffractograms of auto-oxidized and bacterially formed Mn-oxide showed similarities to the hydrothermal vein mineral Rhodochrosite indicating that it can also be produced biotically. Elongation of cells by up to 4× the original size and distortion in cell shape were evident at Mn (II) concentrations >100 µmol L-1 . Marked differences in C-substrate utilization by the test strains were also observed in presence of Mn (II). A shift in use of substrates that are readily available in oceanic waters like N-acetyl-d-glucosamine to those that can be used under changing redox conditions (d-cellobiose) or in the presence of metal ions (d-arabinose, l-asparagine) were observed. These findings highlight the significant role of autochthonous bacteria in transforming reduced metal ions and aiding in the formation of metal oxides. Under natural or laboratory conditions, the mode of bacterially generated Mn-oxide tends to remain the same.

Grazing of particle-associated bacteria-an elimination of the non-viable fraction

Abstract Quantification of bacteria being grazed by microzooplankton is gaining importance since they serve as energy subsidies for higher trophic levels which consequently influence fish production. Hence, grazing pressure on viable and non-viable fraction of free and particle-associated bacteria in a tropical estuary controlled mainly by protist grazers was estimated using the seawater dilution technique. In vitro incubations over a period of 42 h showed that at the end of 24 h, growth coefficient (k) of particle-associated bacteria was 9 times higher at 0.546 than that of free forms. Further, ‘k’ value of viable cells on particles was double that of free forms at 0.016 and 0.007, respectively. While bacteria associated with particles were grazed (coefficient of removal (g) = 0.564), the free forms were relatively less grazed indicating that particle-associated bacteria were exposed to grazers in these waters. Among the viable and non-viable forms, ‘g’ of non-viable fraction (particle-associated bacteria = 0.615, Free = 0.0086) was much greater than the viable fraction (particle-associated bacteria = 0.056, Free = 0.068). Thus, grazing on viable cells was relatively low in both the free and attached states. These observations suggest that non-viable forms of particle-associated bacteria were more prone to grazing and were weeded out leaving the viable cells to replenish the bacterial standing stock. Particle colonization could thus be a temporary refuge for the “persistent variants” where the viable fraction multiply and release their progeny.

Grazing of particle-associated bacteria-an elimination of the non-viable fraction.

Quantification of bacteria being grazed by microzooplankton is gaining importance since they serve as energy subsidies for higher trophic levels which consequently influence fish production. Hence, grazing pressure on viable and non-viable fraction of free and particle-associated bacteria in a tropical estuary controlled mainly by protist grazers was estimated using the seawater dilution technique. In vitro incubations over a period of 42h showed that at the end of 24h, growth coefficient (k) of particle-associated bacteria was 9 times higher at 0.546 than that of free forms. Further, 'k' value of viable cells on particles was double that of free forms at 0.016 and 0.007, respectively. While bacteria associated with particles were grazed (coefficient of removal (g)=0.564), the free forms were relatively less grazed indicating that particle-associated bacteria were exposed to grazers in these waters. Among the viable and non-viable forms, 'g' of non-viable fraction (particle-associated bacteria=0.615, Free=0.0086) was much greater than the viable fraction (particle-associated bacteria=0.056, Free=0.068). Thus, grazing on viable cells was relatively low in both the free and attached states. These observations suggest that non-viable forms of particle-associated bacteria were more prone to grazing and were weeded out leaving the viable cells to replenish the bacterial standing stock. Particle colonization could thus be a temporary refuge for the "persistent variants" where the viable fraction multiply and release their progeny.

Bacterial diversity in relatively pristine and anthropogenically-influenced mangrove ecosystems (Goa, India)

To appreciate differences in benthic bacterial community composition at the relatively pristine Tuvem and the anthropogenically-influenced Divar mangrove ecosystems in Goa, India, parallel tag sequencing of the V6 region of 16S rDNA was carried out. We hypothesize that availability of extraneously-derived anthropogenic substrates could act as a stimulatant but not a deterrent to promote higher bacterial diversity at Divar. Our observations revealed that the phylum Proteobacteria was dominant at both locations comprising 43-46% of total tags. The Tuvem ecosystem was characterized by an abundance of members belonging to the class Deltaproteobacteria (21%), ~ 2100 phylotypes and 1561 operational taxonomic units (OTUs) sharing > 97% similarity. At Divar, the Gammaproteobacteria were ~ 2x higher (17%) than at Tuvem. A more diverse bacterial community with > 3300 phylotypes and > 2000 OTUs mostly belonging to Gammaproteobacteria and a significantly higher DNT (n = 9, p < 0.001, df = 1) were recorded at Divar. These findings suggest that the quantity and quality of pollutants at Divar are perhaps still at a level to maintain high diversity. Using this technique we could show higher diversity at Divar with the possibility of Gammaproteobacteria contributing to modulating excess nitrate.

Bacterial diversity in relatively pristine and anthropogenically-influenced mangrove ecosystems (Goa, India).

To appreciate differences in benthic bacterial community composition at the relatively pristine Tuvem and the anthropogenically-influenced Divar mangrove ecosystems in Goa, India, parallel tag sequencing of the V6 region of 16S rDNA was carried out. We hypothesize that availability of extraneously-derived anthropogenic substrates could act as a stimulatant but not a deterrent to promote higher bacterial diversity at Divar. Our observations revealed that the phylum Proteobacteria was dominant at both locations comprising 43-46% of total tags. The Tuvem ecosystem was characterized by an abundance of members belonging to the class Deltaproteobacteria (21%), ~ 2100 phylotypes and 1561 operational taxonomic units (OTUs) sharing > 97% similarity. At Divar, the Gammaproteobacteria were ~ 2× higher (17%) than at Tuvem. A more diverse bacterial community with > 3300 phylotypes and > 2000 OTUs mostly belonging to Gammaproteobacteria and a significantly higher DNT (n = 9, p < 0.001, df = 1) were recorded at Divar. These findings suggest that the quantity and quality of pollutants at Divar are perhaps still at a level to maintain high diversity. Using this technique we could show higher diversity at Divar with the possibility of Gammaproteobacteria contributing to modulating excess nitrate.

Nitrogen-limited mangrove ecosystems conserve N through dissimilatory nitrate reduction to ammonium.

Earlier observations in mangrove sediments of Goa, India have shown denitrification to be a major pathway for N loss. However, percentage of total nitrate transformed through complete denitrification accounted for <0-72% of the pore water nitrate reduced. Here, we show that up to 99% of nitrate removal in mangrove sediments is routed through dissimilatory nitrate reduction to ammonium (DNRA). The DNRA process was 2x higher at the relatively pristine site Tuvem compared to the anthropogenically-influenced Divar mangrove ecosystem. In systems receiving low extraneous nutrient inputs, this mechanism effectively conserves and re-circulates N minimizing nutrient loss that would otherwise occur through denitrification. In a global context, the occurrence of DNRA in mangroves has important implications for maintaining N levels and sustaining ecosystem productivity. For the first time, this study also highlights the significance of DNRA in buffering the climate by modulating the production of the greenhouse gas nitrous oxide.

Denitrification prevails over anammox in tropical mangrove sediments (Goa, India).

Denitrification, anammox (Anx) and di-nitrogen fixation were examined in two mangrove ecosystems- the anthropogenically influenced Divar and the relatively pristine Tuvem. Stratified sampling at 2 cm increments from 0 to 10 cm depth revealed denitrification as the main process of N2 production in mangrove sediments. At Divar, denitrification was ∼3 times higher than at Tuvem with maximum activity of 224.51 ± 6.63 nmol N2 g⁻¹ h⁻¹ at 0-2 cm. Denitrifying genes (nosZ) numbered up to 2 × 107 copies g⁻¹ sediment and belonged to uncultured microorganisms clustering within Proteobacteria. Anammox was more prominent at deeper depths (8-10 cm) mainly in Divar with highest activity of 101.15 ± 87.73 nmol N2 g⁻¹ h⁻¹ which was 5 times higher than at Tuvem. Di-nitrogen fixation was detected only at Tuvem with a maximum of 12.47 ± 8.36 nmol N2 g⁻¹ h⁻¹. Thus, in these estuarine habitats prone to high nutrient input, N2-fixation is minimal and denitrification rather than Anx serves as an important mechanism for counteracting N loading.

Effects of composition of labile organic matter on biogenic production of methane in the coastal sediments of the Arabian Sea.

Coastal regions are potential zones for production of methane which could be governed by ecological/environmental differences or even sediment properties of a niche. In order to test the hypothesis that methanogenesis in most marine sediments could be driven more by proteins than by carbohydrates and lipid content of labile organic matter (LOM), incubation experiments were carried out with sediments from different environmental niches to measure methane production. The methane production rates were examined in relationship to the sediment biochemistry, i.e., carbohydrates, proteins, and lipids. The gas production measured by head space method ranged from 216 ng g( -1) day( -1) in the mangrove sediments to 3.1 µg g( -1) day( -1) in the shallow Arabian Sea. LOM ranged from 1.56 to 2.85 mg g( -1) in the shallow Arabian Sea, from 3.35 to 5.43 mg g( -1) in the mangrove estuary, and from 0.66 to 0.70 mg g( -1) in the sandy sediments with proteins contributing maximum to the LOM pool. Proteins influenced methane production in the clayey sediments of shallow depths of the Arabian Sea (r = 0.933, p < 0.001) and mangrove estuary (r = 0.981, p < 0.001) but in the sandy beach sediments, carbohydrates (r = 0.924, p < 0.001) governed the net methane production. The gas production was more pronounced in shallow and surface sediments and it decreased with depth apparently governed by the decrease in lability index. Thus, the lability index and protein content are important factors that determine methane production rates in these coastal ecosystems.

Nitrate levels modulate denitrification activity in tropical mangrove sediments (Goa, India).

A study to examine the short-term effect of nitrate and organic carbon addition on denitrification activity was carried out on sediments from a mangrove ecosystem prone to anthropogenic activities (Divar, Goa, India). Laboratory microcosms were prepared using sediment sectioned at every 2-cm-depth interval from the surface to 10 cm. The incubations were subjected to varying nitrate amendments at concentrations ranging from 0, 5, 10, 20, 40 to 60 µmol l(-1) (up to three times more than measured in field). Nitrous oxide production rates increased significantly (n=15; p<0.001) on addition of the nutrient at all depths investigated indicating that denitrification in mangrove sediments was NO[Formula: see text] limited. Incubations amended with organic carbon were prepared using glucose as a substrate with concentrations ranging from 0%, 0.1%, 0.3%, 0.5%, 0.75% to 1%. No significant increase in N2O production was observed on organic C addition. When both the substrates were in excess (1 mmol KNO3+1 mmol glucose), potential denitrification rates decreased with depth and were up to 38 times higher than the in situ denitrification activity varying from 81.26 to 304.09 µmol N2O-N m(-2) h(-1). These results reveal that mangrove sediments could act as a sink for nitrate and microbially mediated denitrification could effectively reduce N load controlling any adverse environmental impact in the adjoining estuarine system.

Cobalt immobilization by manganese oxidizing bacteria from the Indian ridge system.

Co immobilization by two manganese oxidizing isolates from Carlsberg Ridge waters (CR35 and CR48) was compared with that of Mn at same molar concentrations. At a lower concentration of 10 µM, CR35 and CR48 immobilized 22 and 23 fM Co cell(-1) respectively, which was 1.4 to 2 times higher than that of Mn oxidation, while at 10 mM the immobilization was 15-69 times lower than that of Mn. Scanning electron microscope and energy dispersive X-ray analyses of intact bacterial cells grown in 1 mM Co revealed Co peaks showing extracellular binding of the metal. However, it was evident from transmission electron microscope analyses that most of the sequestered Co was bound intracellularly along the cell membrane in both the isolates. Change in morphology was one of the strategies bacteria adopted to counter metal stress. The cells grew larger and thus maintained a lower than normal surface area-volume ratio on exposure to Co to reduce the number of binding sites. An unbalanced growth with increasing Co additions was observed in the isolates. Cells attained a length of 10-18 µm at 10 mM Co which was 11-15 times the original cell length. Extensive cell rupture indicated that Co was harmful at this concentration. It is apparent that biological and optimal requirement of Mn is more than Co. Thus, these differences in the immobilization of the two metals could be driven by the differences in the requirement, cell physiology and the affinities of the isolates for the concentrations of the metals tested.

Denitrification: an important pathway for nitrous oxide production in tropical mangrove sediments (Goa, India).

Net nitrous oxide production and denitrification activity were measured in two mangrove ecosystems of Goa, India. The relatively pristine site Tuvem was compared to Divar, which is prone to high nutrient input. Stratified sampling at 2-cm intervals within the 0- to 10-cm depth range showed that N2O production at both the locations decreased with depth. Elevated denitrification activity at Divar resulted in maximum production of up to 1.95 nmol N2O-N g(-1) h(-1) at 2 to 4 cm, which was three times higher than at Tuvem. Detailed investigations to understand the major pathway contributing to N2O production performed at Tuvem showed that incomplete denitrification was responsible for up to 43 to 93% of N2O production. Nitrous oxide production rates closely correlated to nitrite concentration (n = 15; r = -0.47; p < 0.05) and denitrifier abundance (r = 0.55; p < 0.05), suggesting that nitrite utilization by microbial activity leads to N2O production. Nitrous oxide production through nitrification was below detection, affirming that denitrification is the major pathway responsible for production of the greenhouse gas. Net N2O production in these mangrove systems are comparatively higher than those reported from other natural estuarine sediments and therefore warrant mitigation measures.

Bacterial contribution to mitigation of iron and manganese in mangrove sediments.

The Mandovi and Chapora are two tropical estuaries lying in close geographic proximity on the west coast of India. Seasonal changes in down core variation of Fe, Mn and Total Organic Carbon (TOC) in the mangrove sediments adjoining these estuaries were studied to assess their influence on some of the representative benthic bacteria belonging to heterotrophic and autotrophic groups. Heterotrophic bacteria (HB) cultured on different nutrient concentrations (0.01%, 0.1% and 25%) together with nitrifiers (NtB; representating autotroph) were chosen to assess the influence of the above-mentioned abiotic parameters on the former. The experimental site located along the Mandovi is under the influence of extensive ferromanganese ore mining, while the control site at Chapora is relatively free from such influences. Geoaccumulation index computed for Mandovi showed that sediments (0-10cm) were 'uncontaminated to moderately contaminated' by Fe during the pre monsoon and monsoon seasons, while in the post monsoon season the 4-10cm fraction was almost completely restored from contamination. Similar computations for Mn showed that in pre monsoon, sediments fell in the 'moderately contaminated' and 'moderately to strongly contaminated' categories, while in the monsoon and post monsoon seasons all the sections were 'Uncontaminated'. The difference observed in correlation between Fe and Mn with the various fractions of heterotrophs and nitrifiers indicated that though these two elements shared a similar chemistry in the environment, microbes involved in biogeochemical processes might prefer them differentially. The relationship between TOC and HB enumerated on 0.01% dilute nutrient agar remained at r=0.50, p<0.05 throughout the year. Hence, it could be apparently linked to their preferred concentration of organic carbon requirement. A relationship of r=0.61, p<0.01 between manganese concentration and heterotrophs recovered on different strengths of nutrient agar is suggestive of their response to the metal enrichment. They could thus contribute towards maintaining the level of Mn at par with reference levels at Chapora. A positive correlation between Mn with NtB (n=10, p<0.05, r=0.58) at the experimental site during the non-monsoon months is suggestive of the latter's contribution to regulation of the metal concentration in the sediment probably through anaerobic nitrification at the expense of manganese. The study therefore supports our hypothesis that both autochthonous autotrophs and heterotrophs work in tandem to mitigate concentration of Mn and related metals in mangrove sediments.

Manganese oxidation by bacterial isolates from the Indian ridge system.

The abundance and activity of culturable manganese-oxidizing bacteria were assessed from near-bottom water samples of the tectonically active Carlsberg Ridge. Retrievable counts as colony forming units (CFU) on dilute nutrient agar medium (dilNA=2 gm l(-1) nutrient broth+2% agar) and on dilNA supplemented with 1, 2 and 3 mM MnCl(2).4H(2)O were in the order of 10(6) CFU l(-1). Retrievability of heterotrophs ranged from non-detectable levels (ND) to 2.82 x 10(6) CFU l(-1). The retrievable counts on Mn amended dilNA ranged from ND to 3.21 x 10(6), 1.47 x 10(6) and 1.45 x 10(6) CFU l(-1) on 1, 2 and 3 mM, respectively. About 87% of the Mn tolerant isolates (n=39) showed taxonomic affinities to Pseudomonas I and II sp. Two representative strains CR35 and CR48 (CR-Carlsberg Ridge) isolated on manganese-supplemented media were tested for their ability to tolerate a range of Mn amendments from 1 nM to 100 mM in terms of growth and respiration. CR35 represents 66% of the total CFU (3.04 x 10(6) CFU l(-1)), while CR48 represented only 6% of the total CFU (1.05 x 10(6) CFU l(-1)). The colonies of these two isolates were dark brown in color suggesting precipitation of Mn as oxide. Tests for the effect on growth and respiration were conducted in media simulating heterotrophic (amended with 0.01% glucose) and lithotrophic (unamended) conditions. Maximum stimulation in growth and respiration of CR35 occurred at 100 microM Mn both in unamended and amended media. At levels of Mn greater than 100 microM the counts decreased steadily. Total respiring cells of CR48 were stimulated to a maximum at 1 microM Mn in unamended medium and 1 nM in amended medium. Total cells counts for the same decreased beyond 100 microM Mn in unamended and 1 nM in amended medium. The isolates were tested for their ability to oxidize Mn amendments from 1 microM to 10 mM Mn. At the end of a 76-day incubation period, there was evidence of manganese oxide precipitation at high Mn concentrations (>or=1 mM) as a dark brown coloration on the sides of culture tubes. Highest Mn oxidation rates were observed at 10 mM Mn(II) concentration with CR35 oxidizing 27 and 25 microM Mn day(-1) in unamended and amended condition, respectively. CR48 oxidized Mn at the rate of 26 microM Mn day(-1) in unamended medium and 35 microM Mn day(-1) in amended medium. Scanning electron microscope (SEM) observations of both isolates revealed free-living cells in clustered matrices approximately 2 microm diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30% Mn, while the cell aggregates of CR48 harbored 7-10% Mn. The relatively high specific activity of these mixotrophic bacteria under relatively oligotrophic conditions suggests that they may be responsible for scavenging dissolved Mn from the Carlsberg Ridge waters and could potentially participate in oxidation.