A comparative study of microbial populations in sewage-impacted estuarine environments: mt. Hope bay, Massachusetts and lake pontchartrain, Louisiana
Linda Amaral-Zettler^1,4, Abby Laatsch¹, Jennifer Rocca^1,4, Leslie Graham¹, Benjamin Tully², Mark Dennett^3,4 and Rebecca Gast^3,4
1The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Marine Biological Laboratory 3. Woods Hole Oceanographic Institution 4. The Woods Hole Center for Oceans and Human Health, Woods Hole, MA 02543 USA
²Rutgers, The State University of New Jersey, NJ USA

Coastal marine environments have been impacted by human activity for several centuries, including shoreline alteration, nutrient introduction, sedimentation, toxic compound release, and thermal modification. Our studies of human pathogens in the marine environment include a temperate New England Bay - Mt. Hope Bay in Massachusetts and Hurricane Katrina-impacted Lake Pontchartrain in New Orleans, Louisiana. Mt. Hope Bay, Massachusetts is an ideal site to base a study of human pathogen presence and distribution because it has several important sources of human impact, including sewage disposal sites and the thermal output of a coal-burning power plant within a mile of each other. The pumping of sewage-contaminated floodwaters into Lake Pontchartrain presents a more extreme scenario for studying contamination of coastal waters. The elevated temperatures like those present in the Gulf Coast, on their own or in conjunction with nutrient addition, may also stimulate the growth of microbes associated with human diseases, and their subsequent impact on humans could be significant. We report our combined (eukaryal, bacterial, archaeal) data from small-subunit ribosomal RNA gene clone libraries for samples collected near the thermal plume and underlying sediments of the Brayton Point Power Plant, as well as data from canal and transect sites near or in Lake Pontchartrain. We also present bacterial diversity data from a high-throughput tag sequencing strategy known as Serial Analysis of Ribosomal Sequence Tags (SARST -V6). Not surprising, our findings reveal a highly diverse consortium of the three domains including relatives of sludge and waste-water associated bacteria, polyaromatic hydrocarbon-degrading bacteria, and representatives related to the genera Staphylococcus, Streptococcus, and Clostridium. Phylogenetic analyses will further unveil the relationships of many of these clones and determine whether they are related to known pathogens and may possibly represent undescribed taxa. It is clear that even limited knowledge about the overall microbial community composition can lead to important observations about the ecosystem as a whole. Furthermore, understanding whether free-living pathogens participate in relationships with other members of the microbial community will be important in understanding their distributions and persistence.

Marine algae-associated bacteria: diversity assessment and biotechnological exploration
Noopur Mandrekar, Roopesh Jain and Narsinh Thakur
Nicholas Piramal Research Center, Nicholas Piramal India Ltd., 1 Nirlon Complex, Off Western Express Highway, Goregaon (East), Mumbai 400 063, India

It was interesting to investigate the microbial diversity, associated with marine algae and to explore these bacteria for the production of novel bioactive metabolites. Three different marine algae, having same ecological habitat were assessed for microbial diversity by using classical and molecular biological approach. Algae-associated bacteria were isolated, enumerated, cultured and studied for morphological characteristics. Molecular phylogenetic analysis of these bacteria was executed using 16S rRNA gene sequences. Bacterial PCR products were digested with four-base-cutting restriction enzymes for Restriction Fragment Length Polymorphism (RFLP) studies, in order to evaluate the degree of polymorphism existing among these strains. These bacteria were assessed for antibacterial activity against each other by using ecologically relevant agar plug method to explore their bioactive potential. It was observed that the different algal species from same ecological niche showed diversity in their associated bacterial population. Algae-associated bacteria exhibited antagonism against their microbial neighbors by producing antimicrobial metabolites. In molecular biological analysis, these bacteria showed some prominent gene clusters in their DNA, which are possibly involved in the synthesis of structurally diverse bioactive metabolites of bacterial origin. In summary, this investigation highlights the importance of marine algae as a rich source of unusual microorganisms, having remarkable bioactive prospective.

Can bacteria modulate diatom community structure?
Priya M. D'Costa and Arga Chandrashekar Anil
Marine Corrosion and Materials Research Division, National Institute of Oceanography, Dona Paula 403 004, Goa, India

This study focuses on the interaction of bacteria with diatoms, the major phytoplankton group in coastal waters. In an attempt to study whether bacteria affect benthic diatom community structure, sediment samples collected from a tropical, intertidal, sandflat were incubated in nutrient-enriched media (f/2) supplemented with three different antibiotics (penicillin, streptomycin, chloramphenicol), individually and in combination. This study is based on the approach that treatment with antibiotics suppresses bacterial growth and the effect of bacteria on emerging diatoms can be observed and compared to diatom emergence in media without antibiotics. The antibiotic treatments, except streptomycin, showed lower diatom emergence compared to the control. Least diatom emergence (61% reduction) was observed in the antibiotic combination treatment. Among the individual antibiotics, chloramphenicol was the most potent and streptomycin, the least potent with an increase of 44% in diatom emergence compared to the control. The abundance of culturable bacteria was reduced up to 98% in the different antibiotic treatments. Bacteria were most sensitive to the antibiotic combination treatment (no survival), followed by streptomycin, chloramphenicol and penicillin. Since there is no known direct effect of antibiotics on diatoms, it is evident that bacteria can modulate diatom community structure. This aspect plays an important role in trophic dynamics and thus needs to be investigated in detail.

Monsoon-related periodicity in diversity and abundance of estuarine and non-estuarine foraminifera in the west coast of India
Subhara Devi Gadi and K.P. Rajashekhar
Department of Applied Zoology, Mangalore University, Mangalore 574 199, India

Global climate and agricultural productivity are strongly influenced by the Indian Ocean Monsoon system. In an effort to accurately predict the behaviour of Monsoons, paleomonsoons are being investigated. Paleomonsoonal data is often interpreted from foraminiferal proxies from sea-bed core samples. Foraminifera (Protista, Foraminiferida) are an important component of the marine ecosystem and are sensitive to changes in the environment. Due to the steep gradient of Western Ghats and a narrow coastal plain that is 60 km wide, the monsoons have a strong influence on the physico-chemical properties of the Arabian Sea off Indian West Coast. The fluxes in the diversity and distribution of foraminifera due to Monsoons need to be known for better analyses of paleomonsoons through foraminiferal proxies. As such data is lacking, the present study has been undertaken to analyse the influence of Monsoons on foraminiferal diversity.

As estuarine and non-estuarine systems differ in their dynamics of physicochemical properties during Monsoons, the diversity, abundance and test morphology of inter-tidal foraminifera has been studied at River Sal estuary (estuarine), and Utorda (non-estuarine) sites of coast of Goa during Pre- and Post-Monsoon seasons of 2004-2006. The observations were correlated with various sedimentological and hydrological parameters.

At the estuary, lesser diversity of foraminifera as shown by Shannon Index, represented by 25 species (23 benthic and 2 planktonic species) belonging to 15 genera, 9 families and 2 sub-orders were found. In contrast, 59 foraminiferal species (55 benthic and 4 planktonic species) were recorded at non-estuarine site. They belong to 27 genera, 13 families and 4 sub-orders. Spiroloculina tricarinata, Quinqueloculina vulgaris, Rotalidium annectans, Rotalinoides papillosus, Ammonia beccarii, A. dentata, Elphidium discoidale, Amphistegina radiata, and Poroeponides lateralis were found in abundance at both the locations. Total foraminiferal number (TFN) was higher at non-estuarine site. Onset of monsoons reduces the angular asymmetrical forms during monsoon indicating influx of fresh water through river discharge. Subsequently they increase during post monsoon. Clear periodicity is seen in the number of live foraminifera/ sediment, with foraminiferal density increasing during post-monsoon period. This is indicative of discharge from rivers decreasing the TFN during monsoon but subsequently TFN increases following monsoons. Species-specific responses were also observed in relation to rainfall. While Rotalidium annectans shows negative correlation with Monsoon, Nonion boueanum shows increase in abudance. The observed fluctuations in foraminiferal diversity during monsoon and post monsoon permit better understanding and analyses of proxy data.

Sulphate and possible microbial anomalies from an Antarctic ice core
Laluraj C.M¹, K.P. Krishnan², A. Rajakumar¹, Rahul Mohan¹, M. Thamban¹, A. Chaturvedi², S.S.Naik¹ and W. Dsouza¹
¹National Centre for Antarctic and Ocean Research, Vasco da Gama, Goa
²Geological Survey of India (Antarctica Division), Faridabad, India

A 62.2 m long ice core was drilled from Central Dronning Maud Land (CDML), East Antarctica during the 22^nd Indian Antarctic expedition. The high-resolution glacio-chemical analysis of this ice core was reported by Thamban et al. (2006). The presence of high non-sea salt sulphates revealed the existence of several outstanding peaks of sulphate aerosols at different depths indicating its volcanic origin. Particulate matter of size >0.22µ from depths of high non-sea salt sulphate was subjected to scanning electron microscopic analysis. Microbial cells were encountered at depths of 2.5 and 9.4m, which is considered to have been influenced by the volcanic eruption of Agung (1963) and Karakatau (1883) respectively from Indonesia. This occurrence of microbes in conjunction with high non-sea salt sulphate indicates a common origin, probably volcanic. The geochemistry of particles and its association with the microbes needs to be elucidated.

Heterotrophic bacterial biodiversity from the continental slope of Bay of Bengal
Surajit Das, P.S. Lyla and S. Ajmal Khan
Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, India

Much of human's exploitation of the ocean has been confined to a relatively narrow band round the coast on the continental shelf. But beyond the shelf area, the sea bottom constitutes a most typical environment for its diverse communities. Biodiversity and community structure are now recognized to be important determinants of ecosystem functioning and this ecosystem functioning is dictated to a large degree by biodiversity and the community structure, that results from factors such as the richness and evenness of the diversity. Based on rRNA trees, the main extent of earth's biodiversity is microbial and about half the biomass on earth is microbial but our knowledge of the extent and character of microbial diversity has been limited. Microorganisms are ubiquitous in the marine environment and are truly the 'unseen majority'. But in India studies on invisible entities are meager from the continental slope of Bay of Bengal. In this backdrop, studies were carried out to estimate the culturable fractions of heterotrophic bacterial population from the Bay of Bengal. Sediment samples were collected during cruise#225 of FORV Sagar Sampada in 2004 from 33 stations between 10°36'N-20°01'N and 79°59'E-87°30'E along 11 transects at three different depths i.e. ca 200m, 500m, 1000m in each transect. Total heterotrophic bacterial population ranged between 0.42 - 37.38x10⁴ CFU g^-¹ dry sediment weight. Totally 541 representative isolates were picked up and identified and categorized into eleven genera. Gram negative bacteria dominated than gram positive and the percentage of gram negative, gram positive and the unidentified bacteria were 60.65%, 38.61% and 0.74% respectively.

Communities can be analyzed and characterized in many different ways. One of the most common methods is by looking at community diversity. This is based on the relationship between the diversity of a community and it's stability- the more diverse and complex the community, the more stable it is. The study of marine microbial diversity is important in order to understand the community structure and the pattern of distribution. Therefore, diversity indices- Shannon-Wiener index (H'), Simpson index (D), Margalef index (d) and Pielou's Evenness Index (J') were calculated. Shannon index ranged between 1.915 and 2.323, Simpson index ranged between 0.8201 and 0.9118, Margalef index ranged between 2.072 and 2.643 and evenness varied from 0.8004 to 0.9689. The overall diversity analyses showed that1000m stations were more diverse followed by 200m and 500m depth stations. Because of the direct value as a major resource for biotechnology development, the conservation of microbial gene pool is a crucial issue. To conserve the microbial diversity it is imperative to study the extent of microbial diversity. Therefore, studies on marine microbial biodiversity and its functions are required by regular sampling and monitoring, to conserve and protect the microbial wealth.

Marine yeasts in the slope sediments of Bay of Bengal
Sreedevi N. Kutty and Rosamma Philip
Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, Cochin 682 016, Kerala, India

Marine yeasts population in the slope sediments of Bay of Bengal was estimated from Karaikkal to Paradip. Sediment samples were collected from 200, 500 and 1000m regions at 12 transects. The yeast population ranged from 0 to 59.3 cfu/g dry weight of sediment. Population was found to be maximum at 200m depth zones followed by 500m and 1000m depth zones. Generally the yeast population was found to be more in the northern regions at all the depths. All the yeast isolates (69 Nos.) were tested for hydrolytic potential. All the strains were found to be lipolytic (100%) 62.24% were lignolytic, 42.02% proteolytic and 26.08% amylolytic. Texture analysis showed that the bottom sediment was mostly silty sand in the southern region and clayey silt in the northern regions.

Association of labyrinthulomycetes with bacteria and TEPS in the equatorial Indian Ocean
Varada Damare
Marine Biotechnology, National Institute of Oceanography, Goa 403 004 India

Labyrinthulomycetes, a group of unicellular, obligately marine protists, comprising the thraustochytrids, aplanochytrids and labyrinthulids, are important in the marine microbial food web by virtue of their high contents of the omega-3 polyunsaturated fatty acid, DHA. Their abundance in the water column of the equatorial Indian Ocean, up to 120 m was examined during October 2004 along with that of bacteria. Both groups were distributed in patches, generally showing high numbers between 0 - 40 m and 100 - 120 m from equator northwards. A prominent peak at 20 m was observed in 3 out of 4 stations south of the equator. Labyrinthulomycetes and bacteria attained maximum densities at 120 m at 1° N, the former reaching 770 x 10³ cells L^-¹ and bacteria attaining 810 x 10⁶ cells L^-¹. The distribution of transparent exopolymeric particles (TEPS) in the water column was also studied and it displayed a pattern similar to that of the Labyrinthulomycetes and bacteria. TEPS are rich in organic matter and are a means of vertical flux of carbon to the ocean floor. TEPS ranged from 17 to 778 mg equivalent alginic acid L^-¹ in the water column. No significant correlation with nutrients was found and the patchy distribution of these organisms might be due to their association with TEPS. It is likely that TEPS sustained the growth of both bacteria and Labyrinthulomycetes. Studies on the enzymatic complex of Labyrinthulomycetes responsible for the decomposition of recalcitrant TEPS might prove to be useful in biotechnology.

Actinomycetes from the coral reef environment of the Little Andaman Island, India
K. Sivakumar, Maloy Kumar Sahu, M. Murugan and L. Kannan
Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, Tamilnadu, India

Among the marine biota, microorganisms play a vital role in the dynamics of biogeochemical cycles of the sea including the coral reef environment. They mediate between the dissolved, particulate and gaseous states of chemical compounds, thereby regulating the distribution of organic and inorganic nutrients through out the ocean. They are efficient in these processes because they penetrate the water and sediments most intimately by their number of individuals. Occurrence and distribution of such microorganisms particularly bacteria and fungi have been well studied in the reef environment. But, till date, there is no such work on actinomycetes in the reef environment. Therefore, the present study was undertaken for the first time in eight stations of the coral reef environment of the Little Andaman island to assess the population density of actinomycetes.

Mean population density of actinomycetes recorded from the water samples varied from 0.29 to 0.45 x 10³ CFU/ml with the minimum (0.29 x10³ CFU/ml) at station 2 (Naval Area) and the maximum (0.45 x 10³ CFU/ml) at station 4 (Chandra Nalla Coast). While in the case of sediment samples, population density ranged from 1.21 to 3.29 x 10³ CFU/g with the minimum (1.21 x 10³ CFU/g) at station 2 (Naval area) and the maximum (3.29 x 10³ CFU/g) at station 6 (Buttler Bay). During the present investigation, a total of 41 isolates that were growing well in the media were randomly selected, sub-cultured and identified upto the generic level. The genera encountered were Streptomyces (90%), Micromonospora (6%) and Nocardia (4%). Thus, the present study indicates that the coral reef environment could be the important habitat for actinomycetes which may help in nutrient recycling thereby enhancing productivity. Further studies are required to know the specific role of actinomycetes in the reef environment.

Enumeration and characterization of microbes from aquaculture pond and mangrove ecosystem of Bhitarkanika, Orissa
H.N. Thatoi¹, R.R. Mishra¹, B. Dhal¹, B. Rath¹ and T. K. Dangar¹
¹P.G. Department of Biotechnology, North Orissa University, Baripada Orissa
²Division of Soil Science and Microbiology Laboratory, Central Rice Research Institute, Cuttack, Orissa, India

Bhitarkanika mangrove forests located in the Kendrapara district of Orissa is extended over 215 sq km is the second largest mangrove forest of India after Sunderbans. It has been declared as a National Park during 1998 because of its rich and diversified flora and fauna. However, a large number of aquaculture ponds have been developed in the periphery of the National park area, which poses threat to the mangrove ecosystem. Hence, in the present study attempt has been made to enumerate and characterize the microbes present in aquaculture pond and mangrove ecosystem of Bhitarkanika with a view to assess the environmental quality. The microbes isolated include algae, fungi and bacterial strain from water and soil samples. So far eight soil samples were collected out of which four samples were collected from aquaculture pond and rest of the samples were collected from mangrove environment during August-2005.The isolation of bacterial and fungal population was done by dilution plate technique. Enumeration of fungal and bacterial population form soil and water sample revealed more bacterial population in both soil and water samples than that of fungal population. Mangrove soils comparatively harbour more bacterial and fungal population than aquaculture pond. Identification and characterization of bacteria was done by of three tier screening processes such as morphological studies, microscopic studies and biochemical characterization. Identification of algae and fungi were done by microscopically. The identification of strains was done with the help of Bergey's manual of systematic bacteriology. In total four family of bacterial strains viz., Bacillus Spp, Methylococcus Spp, Pseudomonas Spp and Desulfotomaculum Spp were identified. It has been found that Methylococcus Spp and Pseudomonas Spp are abundant in soil of aquaculture pond where as Bacillus Spp and Desulfotomaculum Spp are present in mangrove soil. Apart from bacteria, fungal species were also isolates form soil samples. So far three fungi species have been identified viz. Penicillium, Aspergillus spp and Actinomycetes. These strains are further screened to asses the different biogeochemical processes of this unique and virgin mangrove ecosystem of Bhitarkanika. Study of halotolerance would provide the possibilities to exploit the applications of microbes for pollution control, industry, food and agriculture by genetic manipulation. In addition to that water samples were also collected from different locations and so far six algal species viz. Spirogyra, Anabena, Chlorella, Gleocapsa, Oscillatoria, Ulva etc. have been identified from Bhitarkanika mangrove area.

16S rDNA archeal diversity in a tropical estuary
S. K. Singh¹, P. Verma², Y.S. Shouche², A. C. Anil¹ anda N. Ramaiah¹
¹National Institute of Oceanography, Dona Paula, Goa 403 004,
²National Center for Cell Science, Pune University Campus, Ganeshkhand, Pune 411007, India

Cultivation independent methods have begun enhancing our knowledge about the presence and diversity of microbes in almost every conceivable ecosystem. Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured entities. Increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments is accruing and their widespread occurrence is being reported. To assess the existence, structure and diversity of the Archaea in Mandovi estuarine sediments we employed molecular, cultivation- independent phylogenetic techniques. The DNA was extracted from sediments collected from this typical tropical estuary and subjected to partial 16S ribosomal DNA library construction using the PCR amplicons by universal Archaea specific primers. Small-subunit ribosomal RNA genes of Archaea were amplified from the sediment and cloned. Phylogenetic analyses of these cloned small subunit ribosomal RNA gene sequences revealed that clusters of Archaea from this estuarine sediment are very divergent from those of cultured Archaea. The archaeal clones could be grouped into discrete phylogenetic lineages within the two subkingdoms Crenarchaeota and Euryarchaeota. With clones accounting to 78.21%, the Crenarchaeota dominated in our archaeal library and, Euryarchaeota represented 21.79% of the total 179 clones analyzed. Most clones of under within the dominating Crenarchaeota, were the members of Marine Archaea Group 1 (33.51% of total library analyzed) which appeared to cluster with the very recently cultured non-thermophilic autotrophic ammonia oxidizer Candidatus nitrosopumilus. Our phylogenetic study on Archaea, the first of its kind from Indian estuaries, is useful in ascertaining that the lineage of Crenarchaeota is predominant in this tropical estuary and the roles in biogeochemistry and organic matter biotransformation by Archaea are of greater ecological significance.