Introduction

Estuaries are complex coastal ecosystems with constant dynamic fluxes of fresh and marine water, which influences the microalgae communities - important primary producers of intertidal, shallow and subtidal sediments (Underwood & Paterson 1993, Badarudeen et al. 1996). Microalgae, mainly diatoms (class: Bacillariophyceae), ubiquitous in estuaries, are mostly of bilaterally symmetrical - pennate forms, which are usually attach to a substrate (plants, sediments, pebbles, etc.), whereas the radially symmetric centric diatoms are predominantly planktonic(Werner 1977). Coastal environments in general, especially estuaries, characteristically rich in dissolved organic matter, account for most of pennate diatoms, in their mud and sandflats, tidal pools and marshes, etc. The centric diatoms are predominant in open waters, characterized by lower concentrations of dissolved organic matter, and to a lesser extent occur in benthic habitats. Diatoms adhering to substrates (known as benthic diatoms) are a well-known bio-indicators of health of an aquatic ecosystem. Over the past few decades, community ecology of diatoms through environmental monitoring has gained momentum. The spatial distribution and composition of diatom species are mainly influenced by climatic, geological and anthropogenic factors such as land uses in the catchments and varying levels of nutrients (Pan et al. 1996, Townsend & Gell 2005). Diatoms are sensitive to even the slightest changes in its habitat and are aptly being used as bio-indicators of aquatic ecosystems. Community composition of benthic diatoms is an outcome of complex interactions between abiotic and biotic factors (Stevenson 1997). Diatom community structure, ecology and distributional patterns in relation to environmental gradients (Soininen & Eloranta 2004) has already been explored and the current focus is on its exploration as an ideal biofuel feedstock (Graham et al. 2012) under varying nutritional modes from autotrophic to heterotrophic or mixotrophic. Doing so, would help in minimizing the uncertainties associated with cultures under fluctuating qualities of water. Moreover, relating environmental conditions and species composition would help in optimizing the growth strategy for higher lipid productivity by mimicking the actual field conditions.
Many studies are under-way in utilizing diatoms – both planktonic and benthic forms as viable biofuel feedstocks. Biofuel derived from unicellular microalgae are popularly known as third generation biofuels. About 3000 microalgal strains were isolated, screened and tested for its lipid productivity and potential towards biodiesel production under the Aquatic Species Program (ASP), a pioneering effort of US Department of Energy (DOE), during 1980 to 1996. The ASP program in its close-out report in 1998, recommended 50 promising strains attributed with higher growth rate, lipid productivity and survival capability under harsh environmental conditions. Sixty per cent of those selected 50 promising microalgal species were diatoms demonstrating its potential as promising third-generation biofuel feedstocks (Hildebrand et al. 2012, Sheehan et al. 1998). Despite the rigorous investigations involving a large number of microalgae for favorable lipid productivity, ASP apprehended likely higher costs compared to conventional fossil fuels. Biofuel research in 2000’s on microalgae mainly focused on determining and optimizing algal growth, lipid estimation and characterization of single axenic monocultures of marine and fresh water microalgae (both green microalgae and diatoms) procured from culture banks/repositories or random isolation of a single local strain apart from nutrient parameters optimization under lab conditions (Yusuf 2007, Widjaja et al. 2009, Liu et al. 2011, Tang et al. 2011). Efforts on increasing the biomass productivity by reducing the predation or contamination due to pests, lead to innovative designs of photobioreactors (PBR’s). Variants of PBR’s designed for improved biomass productivities are now available (Grobbelaar 2009, Tang et al. 2012, Sforza et al. 2012 and Huesemann et al. 2013). But the major hindrance in fulfilling the dream of biofuel, a promise to reality, hovers around the critical dependence on obtaining higher algal productivity in a less sophisticated, low cost sustainable model which would reduce the burden of fixed and operational costs that would eventually improve the economic viability with the technical feasibility of algae-based biofuels. NASA’s Offshore Membrane Enclosures for Growing Algae (OMEGA project 2010 – 2012), while being innovative, also revived the scope on algae-based biofuel technology by extracting clean energy from microalgae through cultivation in the open Sea using wastewater in flexible plastic sheets which would effectively sequester CO2, treat waste water while producing biofuel (Wiley et al. 2013). Currently, there is a renewed interest in diatom-based biofuels especially after unraveling the multiple benefits including scope for higher lipid accumulation (Ramachandra et al. 2009, Hildebrand et al. 2012, Levitan et al. 2014Levitan et al. 2014, Fu et al. 2015, Vinayak et al. 2015). Many diatoms species are prudent to be promising due to their ability to grow on non-arable lands, ability to sequester CO2, higher nutrient hoarding capabilities, shorter cycling period than higher vascular plants (Hildebrand et al. 2012) and most importantly, their nutrient removal efficiency from urban domestic wastewater (Kumar 2008, Marella et al. 2017) or aquaculture discharge water (Venkatesan et al. 2006). Recent researches in this direction (d’Ippolito et al. 2015, Marella et al. 2017) focused on screening and growing diatoms using waste/saline waters or domestic/municipal sewage (Ramachandra et al. 2013, Mahapatra et al. 2014, Ramachandra et al. 2015) and even industrial effluents (Chinnasamy et al. 2010, Abdel-Raouf et al. 2012, Kamyab et al. 2016, Idris et alr. 2018). Such screening of microalgae technically called as “Phyco-prospecting” (Chu 2017) coupled with phyco-remediation would help in isolating a strain that can withstand extreme environmental conditions (Abomohra et al. 2017) while reducing the burden of using fresh water and expensive synthetic chemicals/ fertilizers for growing diatoms at the large scale. Also, for a scaled-up production system running for a good portion of the year, having a strain that is consistently productive under a variety of environmental conditions is far more desirable than strains having higher productivity under optimal conditions for a shorter period of time (Hildebrand et al. 2012).
A recent estimate indicates that out of 72,500 strains of microalgae identified so far, only 44,000 species have been described with characteristics (De Clerck et al. 2013, Chu 2017). This shows the quantum of new species left unexplored. Thus, prioritizing diatom strains for sustainable biofuel production are prioritized based on abundance, productivity with higher resilience to fluctuating environmental conditions. This approach necessitates effective screening mechanism involving ecological distribution studies with habitat mapping under diverse ecological zones with varied environmental conditions. Hence as a preliminary step, field investigations of spatial distribution and community structure of estuarine benthic epipelic diatoms was carried out with respect to varying water quality. The variation in water quality or the local nutrient input in each habitat is heavily influenced by flora and fauna specific to a habitat. The community structure of benthic diatoms with varied levels of organic loadings would provide insights on a diatom species tolerance to different nutrient levels. The species abundance data from ground conditions along with respective lipid profiles would help in the successful design of economically viable bioreactors. A thorough review of literatures pertaining to the lipid content of diatoms grown in nitrogen replete conditions was carried out. Multivariate statistical analyses were performed to understand the functional relationship between varying nutrient levels and environmental parameters like light intensity, salinity and pH recorded under laboratory conditions. The integration of actual field parameters consisting of nutrient and physico-chemical parameters to the lipid content was done to determine the decisive environmental variables in the accumulation of lipid. This is a first of its kind study to relate diatom tolerances and its associated nutrient loadings under varied habitat conditions with lipid productivity potential. This could be a less time-consuming screening mechanism for exploiting mixed diatom consortia especially towards phyco-prospecting using conventional wastewaters as a means of integrated decentralized phycoremediation and energy production systems.
The current study was carried out with the following objectives:
1. To understand the diatom community assemblages and their preferred habitats in relation to hydrological and environmental parameters across different lentic and lotic ecosystems of the Aganashini estuary.
2. To understand relationship of diatom species composition with the environmental variables through multivariate statistical analyses.
3. To evaluate the biofuel prospects of prioritized algal strains