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Graduation date: 2005

Textile industries are the major users of dyes in the world. A huge fraction of dyes are discharged out from the textile industries, causing serious damage to the environment. Bioremediation based technologies has been proved to be the most desirable and cost- effective method to counter textile dye pollution. The ability of the microorganisms to decolorize and metabolize dyes can be employed to treat the environment polluted by textile dyes. In this work, a total of 84 bacterial strains were isolated from Kelambakkam Solar Salt Crystallizer ponds (or salterns) and screened for their ability to produce extracellular tannase and laccase enzymes and eventually to decolorize three widely used textile dyes- Reactive Blue 81, Reactive Red 111 and Reactive Yellow 44. Of these 84 strains, 18 strains exhibited tannase activity and 36 strains showed positive laccase enzyme activity. The 11 bacterial strains that displayed both tannase and laccase enzyme activity were screened for their ability to decolorize the three textile azo dyes (100 mg/L). Out of 11 strains only 2 strains i.e., AMETH72 and AMETH77 showed best decolorization (%) in all the three dyes under static condition at room temperature. Repeated- batch immobilization study used to select the most efficient bacterial strain revealed that, isolate AMETH72 was efficient than AMETH77 in decolorizing the dyes. The 16S rRNA sequencing of AMETH72 showed 99% phylogenetic similarity to Halomonas elongata. The dye degradation products analyzed by FTIR and UV-Vis techniques displayed complete disruption of azo linkages and biodegradation of dyes to simpler compounds. The treated dyes also improved growth and total chlorophyll content in Wheat and Green gram seedlings, as compared to the untreated dyes. This indicated the non- toxicity of the biologically degraded dye products. Thus the entire study concluded that halotolerant marine bacteria from the salterns can be effectively used to bioremediate the textile dyes. / Program: MSc in Resource Recovery - Industrial Biotechnology

This thesis addresses time-series analyses of microbial (i.e. marine heterotrophic bacteria and phytoplankton) and microbially relevant ecosystem variables at two ocean time series stations - Palmer Station in the coastal Western Antarctic Peninsula (WAP) and the Bermuda Atlantic Time-series Study (BATS) site in the Sargasso Sea. Using a diverse spectrum of statistical analyses and models, the aim of this thesis is to gain the better insight into 1) variability of microbial and ecosystem processes across varying time scales, from seasonal to interdecadal, and 2) how each process is influenced by variability of surrounding local physical forcing factors as well as regional and global-scale climate variability along the study region. Chapter 1 provides an introduction to the two study sites as well as a brief history of the ocean time-series programs there. Chapter 2 deals with phytoplankton and nutrient drawdown variability over an interdecadal (1993-2013) period using seasonal time-series variables collected at Palmer Station during full 6-months of Austral growing seasons (October-March). Specifically, the linkage between large-scale climate modes relevant to the WAP area and phytoplankton and nutrient patterns is explored to establish the underlying mechanisms of the observed ecosystem variability, which is ultimately triggered by climate conditions via mediatory physical forcing factors. Chapter 3 addresses a decadal (2002-2014) variability of heterotrophic bacterial variables collected at Palmer Station in Antarctica. This Chapter 3 provides an insight into why bacterial activity was shown to be restricted in this very productive ecosystem from diverse aspects gained using different statistical approaches. Furthermore, the linkage between bacterial properties and surrounding environmental conditions is explored. Finally, Chapter 4 concerns an event-scale phenomenon - the frequency of winter storms - and its impact on bacterial dynamics and ecological processes at the BATS site. Using a previously developed storm tracking algorithm, this study benefits from establishing a mechanistic connection between storm forcing and bacterial processes via storm-induced variability of physical environments - the extent of wind-mixing and entrainment of cold water into the upper mixed-layer. The finding of Chapter 4 is novel in the aspect that prevalent negative North Atlantic Oscillation (NAO) conditions, which lead to frequent arrivals of winter storms over the BATS region, in part, explain a significant decreasing bacterial trend over the past 24-year period. Overall, my thesis, in conjunction with work performed by fellow microbial oceanographers, aims to provide evidence of microbial responses to physical forcings across varying time scales in the strongly coupled climate-biogeochemical systems at two contrasting ocean sites based on a variety of statistical approaches.

Sphingopyxis (formerly Sphingomonas) alaskensis, a numerically abundant species isolated from Alaskan waters and the North Sea represents one of the only pure cultures of a typical oligotrophic ultramicrobacterium isolated from the marine environment. In this study, physiological and molecular characterization of an extinction dilution isolate from the North Pacific indicate that it is a strain of Sphingopyxis alaskenis, extending the known geographical distribution of this strain and affirming its importance as a model marine oligotroph. Given the importance of open ocean systems in climatic processes, it is clearly important to understand the physiology and underlying molecular biology of abundant species, such as S. alaskensis, and to define their role in biogeochemical processes. S. alaskensis is thought to proliferate by growing slowly on limited concentrations of substrates thereby avoiding outright starvation. In order to mimic environmental conditions chemostat culture was used to study the physiology of this model oligotroph in response to slow growth and nutrient limitation. It was found that the extent of nutrient limitation and starvation has fundamentally different consequences for the physiology of oligotrophic ultramicrobacteria compared with well-studied copiotrophic bacteria (Vibrio angustum S14 and Escherichia coli). For example, growth rate played a critical role in hydrogen peroxide resistance of S. alaskensis with slowly growing cells being 10, 000 times more resistant than fast growing cells. In contrast, the responses of V. angustum and E. coli to nutrient availability differed in that starved cells were more resistant than growing cells, regardless of growth rate. In order to examine molecular basis of the response to general nutrient limitation, starvation and oxidative stress in S. alaskensis we used proteomics to define differences in protein profiles of chemostat-grown cultures at various levels of nutrient limitation. High-resolution two-dimensional electrophoresis (2DE) methods were developed and 2DE protein maps were used to define proteins regulated by the level of nutrient limitation. A number of these proteins were identified with the aid of mass spectrometry and cross-species database matching. The identified proteins are involved in fundamental cellular processes including protein synthesis, protein folding, energy generation and electron transport, providing an important step in discovering the molecular basis of oligotrophy in this model organism.

The marine bacterium Pseudoalteromonas tunicata produces target-specific inhibitory compounds against bacteria, algae, fungi and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. This study examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water-channels. Interestingly, a repeatable pattern of cell death in the centre of microcolonies was observed. The antibacterial and autolytic protein, AlpP, produced by P. tunicata was found to be involved in this biofilm killing and a

The objective of this project was to use ribosomal RNA genes, cloned from natural populations of Sargasso Sea bacterioplankton, as markers for picoplankton diversity. It is widely recognised that a majority of microorganisms have yet to be cultivated, and therefore much of extant microbial diversity remains unknown (50). The method described here for analyzing natural bacterial communities circumvents this problem by utilizing ribosomal RNA, found in all life forms, for defining and enumerating the components of natural populations. Two different clone libraries of eubacterial 16S rRNA genes amplified from a natural population of Sargasso Sea picoplankton by the polymerase chain reaction (11) have been phylogenetically analysed. The analyses indicate the presence of a wide variety of novel microorganisms, representing members of the α and γ proteobacteria and the oxyphototroph (13, 47) eubacterial phyla. One group of novel clones, represented by SAR 83, were found to be most closely related to Erythrobacter, a genus of aerobic bacteriochlorophyll~ containing organisms. The results imply that many closely related 16S rRNA lineages, or clusters of lineages, coexist within bacterioplankton communities. The significance of these clusters is uncertain. One interpretation, that they represent clonal structure within bacterial species, suggests that populations of marine bacteria are very ancient. / Graduation date: 1991

Seventeen bacterial strains were isolated from 9 marine mud samples from the inshore environments of the East Sea. Four bacterial strains showed an inhibition against all tested microorganisms Staphylococcus aureus ATCC10832, Escherichia coli JM109, and Fusarium oxysporum. 16S rRNA sequences of four bacterial strains were obtained by PCR using specific primers. PCR products were cloned into E. coli DH5a using pJET1.2 blunt vector. The recombinant plasmids were sequenced and the lengths of these 16S rRNA sequences were ~930bp. The 16S rRNA sequence from the four bacterial DB1.2, DB1.2.3, DB4.2 and DB5.2 strain showed a high identity of 97 to 99% with the 16S rRNA sequence from Photobacterium sp., Oceanisphaera sp., Shigella sp., Stenotrophomonas sp, respectively. / Mười bảy chủng vi khuẩn đã được phân lập từ 9 mẫu bùn biển từ các vùng ven bờ biển Việt Nam. Bốn chủng vi khuẩn được ghi nhận có khả năng ức chế mạnh sự sinh trưởng và phát triển của các chủng vi khuẩn Staphylococcus aureus ATCC10832, Escherichia coli JM109, và thậm chí cả nấm Fusarium oxysporum. Trình tự gene 16S rRNA của bốn chủng vi khuẩn này đã được khuếch đại bằng PCR sử dụng cặp mồi đặc hiệu. Sản phẩm PCR được nối ghép vào vector pJET1.2 blunt sử dụng T4 ligase, hình thành plasmid tái tổ hợp và biến nạp vào E. coli DH5α. Khuẩn lạc có plasmid mang phân đoạn DNA chèn được nuôi cấy và tách plasmid. Trình tự 16S rRNA từ 4 chủng DB1.2, DB1.2.3, DB4.2 and DB5.2 chỉ ra có sự tương đồng 97 ÷ 99% so với trình tự 16S rRNA tương ứng của các chủng vi sinh vật biển trên ngân hàng gene thế giới là Photobacterium sp., Oceanisphaera sp., Shigella sp., và Stenotrophomonas sp.

Recent studies demonstrate that the dissolved iron in seawater is bound to strong organic complexes that have stability constants comparable to those of microbial iron chelates (siderophores). We investigated iron acquisition by 7 strains of heterotrophic marine bacteria using siderophores as a model for the natural iron-binding ligands. Siderophores were detected in the supernatants of 4 strains. All strains utilized iron bound to siderophores regardless of whether they produced their own. The majority took up iron bound to the fungal siderophore desferrioxamine B (dfoB). Over half also utilized iron bound to strain Neptune's siderophore, nep-L, while iron bound to pwf-L was available solely to the producing strain, Pwf3. Uptake rates of Fe-siderophores were similar among iron-limited strains and among ligands. Transport of Fe-dfoB in Neptune was enhanced 20 times by iron limitation. The half-saturation constant of Fe-dfoB transport was 15 nM, the lowest reported for Fe-siderophore transport in microorganisms. In contrast, uptake of inorganic iron (Fe' ) by iron-limited Neptune did not saturate at the highest concentration tested and was not upregulated under iron stress. This suggests that Fe ' uptake occurs by simple diffusion through the outer membrane. / Strain Lmg1, the sole catechol producer, did not take up iron bound to exogenous siderophores (dfoB, pwf-L, or nep-L). However, it utilized iron bound to its own ligand and, possibly, iron bound to the synthetic chelator EDTA. Transport of Fe' by iron-limited Lmg1 was 10 times higher than in the other strains and was upregulated 46 times in low iron conditions. The results suggest iron transport in Lmg 1 may be mediated by surface-associated catechol siderophores that scavenge inorganic ferric species as well as iron bound to weaker complexes, such as EDTA. This study elucidates the importance of siderophores in iron transport by heterotrophic marine bacteria. (Abstract shortened by UMI.)