Exploring Ecological Roles of Bacterioplankton Communities: Implications in Nutrient Cycling Within a Mangrove Ecosystem

Ghosh, Anwesha (2019) Exploring Ecological Roles of Bacterioplankton Communities: Implications in Nutrient Cycling Within a Mangrove Ecosystem. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

This thesis work was aimed towards understanding the ecological roles of bacterioplankton communities in mangrove ecosystems. With the introduction of the concept of microbial loop by Azam and colleagues in 1983, there has been a surge in research to elucidate the role of bacterioplankton in mediating biogeochemical cycling of key elements such as carbon and nitrogen in aquatic ecosystems. Functions of bacterioplankton communities in different aquatic ecosystems including coastal ecosystems are known to be influenced by hydrological parameters. Variation in hydrological parameters directly affects the structure of resident bacterioplankton communities which in turn is reflected at ecosystem level functions such as turn-over rates of biogeochemical cycles of elements in coastal ecosystems. Estuaries represent a unique ecotone where freshwater mixes with saline water, witnesses rapid changes in hydrological parameters such as salinity and dissolved nutrients. Freshwater flow from river results in addition of nutrients from the adjoining areas such as mangroves into estuaries. Moreover, litterfall from mangroves could act as additional sources of nutrients to resident bacterioplankton communities. At the same time, changes in hydrological parameters are significant on a seasonal scale as it alters river flow and ocean circulation patterns. These rapid changes act as stressors on the resident bacterial communities. This thesis has focused on understanding the role of environmental parameters in shaping bacterioplankton communities and their functioning within a mangrove ecosystem. In objective 1 (Chapter 3), bacterioplankton community structure was elucidated from two predesignated stations, Stn1 and Stn3, of Sundarbans Biological Observatory Time Series (SBOTS) over monsoon (June to September, 2014) and post-monsoon (October to December, 2014) seasons. These stations lie in close proximity to each other but have distinct hydrological conditions such as differences in salinity. Both the stations are fed by freshwater from the swiftly flowing Mooriganga River and saline water entering from the coastal Bay of Bengal on a diurnal basis. The bacterioplankton communities were elucidated from both stations using 16S rRNA clone library and Illumina MiSeq approaches. Out of 544 sequences from clone library data covering both stations over monsoon and post-monsoon seasons, Proteobacteria was found to be most abundant phylum (373 sequences). Two major classes, namely, Gammaproteobacteria and Alphaproteobacteria were dominant across both monsoon and post-monsoon seasons in both stations. Several sequences belonging to Sphingomondales, Chromatiales, Alteromondales and Oceanospirillaes were encountered that are known to play important roles in coastal carbon cycling. Some sequences showed identity with published uncultured Planctomycetes and Chloroflexi highlighting their role in nitrogen cycling. The detection of two novel clades revealed the existence of indigenous group of bacterioplankton that may play important ecological roles in this ecosystem. The eubacterial V3-V4 region from environmental DNA extracted from the above two stations, followed by sequencing in Illumina MiSeq platform was also targeted in the study. A congruency between the clone library followed by Sanger sequencing and Illumina sequencing approaches were observed. Strong variability in bacterioplankton community structure was encountered at a seasonal scale in link with precipitation. Drastic increase in sediment associated bacteria such as members of Firmicutes and Desulfovibrio were found in monsoon hinting possible resuspension of sediment dwelling bacteria into the overlying water column. Both sequencing methods showed Vibrio to be the most abundant genus in both the stations of Sundarbans. Vibrio showed a strong seasonal variation and could be a critical player in carbon and nitrogen cycling in Sundarbans. Principal Component Analysis (PCA) revealed dissolved ammonium and dissolved nitrate to account for maximum variation observed in the bacterioplankton community structure. Overall this chapter showed that a strong interplay exists between hydrological parameters and observed variability in bacterioplankton communities as a result of precipitation which can ultimately influence processes and rates linked to coastal biogeochemical cycles. It was important to establish the bacterioplankton community structure from other estuaries of Sundarbans. Mooriganga estuary represented the westernmost of the seven estuaries located across Indian Sundarbans. In objective 2 (Chapter 4) bacterioplankton community structure from Mooriganga estuary was compared to other global estuaries. Bacterioplankton community structure from three other estuaries of Sundarbans, namely Thakuran, Matla and Harinbhanga were elucidated using Illumina sequencing. The information on the bacterioplankton communities of Sundarbans was then compared against available Illumina sequence datasets from Columbia, Delaware, Jiulong, Pearl and Hangzhou estuaries to identify broad biogeographic patterns of bacterioplankton communities. All nine estuaries were dominated by Proteobacteria. Other abundant phyla included Bacteroidetes, Firmicutes, Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes and Verrucomicrobia. The abundant bacterial phyla showed a ubiquitous presence across the estuaries. At class level, the overwhelming abundance of Gammaproteobacteria in the estuaries of Sundarbans and Columbia estuary clearly stood out amidst the high abundance of Alphaproteobacteria observed in the other estuaries. Abundant bacterial families including Rhodobacteriaceae, Shingomonadaceae, Acidobacteriaceae, Vibrionaceae and Xanthomondaceae also showed ubiquitous presence in the studied estuaries. However, rare taxa including Chloroflexi, Tenericutes, Nitrospirae and Deinococcus-Thermus showed clear site-specific distribution patterns. Such distribution patterns were also reinstated by nMDS ordination plots. The clustering patterns may hint toward hydrological parameters and substrate specificity which could result in the development of distinct bacterioplankton communities at specific sites. The ubiquitous presence of abundant bacterioplankton groups along with their strong correlation with SWT and dissolved nutrient concentrations indicate the role of such hydrological parameters in maintaining bacterioplankton community structures in estuaries. Overall, this chapter investigated the biogeographic patterns of bacterioplankton communities and provided interesting insights into their functioning with an emphasis on biogeochemical cycling as well as exploring health of global estuaries. Both objective 3 and 4 showed Vibrio was the most abundant genus in the estuaries of Sundarbans. In objective 3 (Chapter 5), a new species of Vibrio (annotated as SBOTS_Iso1) was isolated in August, 2014 from the Stn1 located in Chemaguri creek as part of SBOTS of Sundarbans mangroves using culture-based methodologies. Chemaguri creek exhibits typical estuarine conditions where it receives freshwater from Mooriganga River and saline water from the coastal Bay of Bengal. Phenotypic and genotypic characterization of the new species (SBOTS_Iso1) was performed using polyphasic approaches. Phenotypic analyses including biochemical tests and determination of doubling time indicated the typical estuarine characteristics of this new species. The bacterium was Gram negative, rod-shaped, oxidase and catalase negative and grows in the presence of NaCl. FAME analysis indicated 31.7% of the cellular fatty acids to be made up of 16:1 ω7c/16:1 ω 6c. Amplification and sequencing of 16S rRNA and multilocus sequence analysis (MLSA) of four loci (2040 bp; rpoA, topA, mreB, pyrH) and additional sequence data of ftsZ, atpD, ompW and rpoB genes showed this isolate to be a member of Harveyi clade of the genus Vibrio. The closest phylogenetic neighbour was Vibrio alginolyticus ATCC 17749T with 96.8% similarity between the above mentioned ORFs. Genotypic characterization showed 52 mol% G-C content and 68.48% similarity with Vibrio parahaemolyticus K12262 O1:K25, 67% with Vibrio natriegens ATCC 14088T, 54% with Vibrio campbelli ATCC 25920T and 55% with Vibrio alginolyticus DSM 2171 between the above mentioned ORFs. The isolate SBOTS_Iso1 has been named Vibrio chemaguriensis sp. nov. on the name of the site from where it was first isolated. Information on the bacterioplankton communities from the estuaries of Sundarbans hinted toward presence of bacteria that could be critical players in mediating biogeochemical cycling of carbon and nitrogen. Given mangrove litterfall constitute an important component, therefore the potential role of bacterioplankton in breakdown and utilization of litterfall components are essential towards understanding of remineralization of carbon and sustenance of microbial loop. Thus, in order to understand functioning of bacterioplankton communities specific to estuarine mangroves, a laboratory mesocosm experiment was set up. In objective 4 (Chapter 6), the influence of tannic acid, a major component of mangrove litterfall in shaping bacterioplankton community structure was analysed. Tannic acid is a secondary compound produced by vascular plants and is a major constituent of plant soft tissues including in mangrove plants. Tannins are water soluble compounds that leach out from mangrove litterfall and contribute to DOC and DOM pools of the adjacent estuaries. About 50% of the litterfall is believed to be exported out of the estuaries by tidal flushing. The remaining maybe degraded and channelized into the marine microbial loop. Tannins have been shown to be inhibitory to bacteria by a variety of mechanisms that primarily linked to bacterial cell growth. Some groups of bacteria such as Bacteroides are capable of degrading tannins by a specialized enzyme called tannase. The influence of tannic acid on bacterioplankton community structure was tested by setting up barrel experiments. Estuarine water from Stn3 of SBOTS was enriched with tannic acid and the change in bacterioplankton community structure was analysed on the start (Day 0), intermediate (Day 7) and end (Day 15) of the experiment. Bacterioplankton community structure was elucidated by sequencing the V3-V4 region of 16S rRNA on an Illumina MiSeq platform. Concentration of tannic acid and other dissolved nutrients such as nitrate and ortho-phosphate were determined using a UV-Vis Spectrophotometer. Additionally, concentration of some of the trace elements were also measured using ICP-MS approach. Tannic acid was shown to significantly affect the concentration of dissolved nitrate and trace elements in the barrels. Similar to information obtained previously, Proteobacteria was found to be the most dominant bacterial phylum in Control and tannic acid enriched barrels (Barrel 1 and 2) on Day 0. With the progression of experiment, the abundance of Proteobacteria decreased significantly in the Control barrel indicating the Proteobacteria may not be able to survive in high abundance in natural water without constant supply of nutrients. The abundance of Proteobacteria in the tannic acid enriched barrels remained high indicating that members of Proteobacteria may be capable of using tannic acid as a source of carbon and nitrogen. Tannic acid appeared to inhibit most of the other bacterioplankton phyla including Actinobacteria, Acidobacteria and Verrucomicrobia that existed in large abundance in the Control barrel on Day 15. At class level, Bacteroides was found to be present in highest abundance. This group is known to be capable of breaking down tannic acid using tannase as an enzyme. Tannic acid appeared to inhibit most bacterioplankton classes and families identified from the barrel mesocosm experiment. Degradation of tannic acid was tracked by decrease in concentration of tannic acid and generation of gallic acid. Gallic acid is one of the final products of tannic acid degradation. This experiment indicated that bacterioplankton communities of Sundarbans could harbour genes necessary for breakdown of complex components of litterfall and recycle them into the marine microbial loop. Breakdown of tannic acid, an important and abundant component of mangrove litterfall, could influence the marine nitrogen and carbon cycling by releasing DON and DOC respectively into the adjacent estuaries. An understanding of the breakdown of tannic acid and other components of mangrove litterfall and its influence on the resident biological communities of estuarine mangroves could be essential for our understanding of functioning of coastal ecosystems. Information of breakdown and remineralization of components of litterfall such as tannic acid would be also important for improved estimation of carbon and nitrogen budgets of coastal ecosystems such as mangroves.

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