Results and Discussion

Variations in Hydrological Parameters

Figure 4.a-d reflects the variability of physical parameters (AT, WT, pH, and Salinity) at sampling locations. Temperature is a critical physical factor that controls the rate of photosynthesis in addition to influencing many other chemical and biological reactions (Fatema et al., 2014). Temperature variations are influenced by prevailing precipitation and humidity levels. In this study, air temperature during pre-monsoon season was varying across stations with a mean temperature of (av. 31.36 ± 1.20 ^oC). During premonsoon, the estuary was characterized by warm (31.42 ± 0.66 ^oC) with high saline (30.20 ± 3.45 ppt) water in all stations (S1 to S6): lower (stations S5, S6), central (stations S3, S4) and upper reaches (stations S1, S2).

During monsoon season, the estuary had relatively lower variations of air and surface water temperatures (~ 1 to 3 ^oC), with air temperature of (29.06 ± 0.67 ^oC) and water temperature (28.11 ± 0.87 ^oC) respectively. The mean air and water temperature across the stations during post-monsoon season was (31.73 ± 0.82 ^oC) and (29.04 ± 0.56 ^oC). This drop-in water temperature during post-monsoon season could be attributed to heavy precipitation that resulted in the inflow of cold freshwater from the Ghats. Generally, in a tropical estuary, the temperature is inversely correlated with salinity and water transparency (Fatema et al., 2014).

Figure 3 Light Micrographs of Select Diatom Species a) Navicula forcipata b) Gomphonema sp. c) Cyclotella operculata d) Diploneis subovalis e) Aulacoseira granulata f) Bacillaria paradoxa g) Epithema gibberula h) Pinnularia sp. i) Navicula integra j) Melosira jurgensii k) Cyclotella meneghiniana l) Achnanthes brevipes m) Frustulia crassinervia n) Coscinodiscus radiatus o) Raphoneis amphiceros p) Amphora salina

The water quality results depicted the seasonal variability. Air temperature showed wider fluctuations primarily due to higher insolation during pre-monsoon seasons followed by reduced levels due to precipitation. Air and water temperature variations were comparable to earlier studies in tropical estuaries of the south-west coast of India (Madhu et al., 2007; Martin et al., 2008). pH values in water samples in all sampling locations across seasons were neutral to slightly basic (7.88 ± 0.04) with no significant fluctuations. pH of a waterbody is mainly influenced by induced anthropogenic disturbances in the form of domestic or industrial effluents. Variations in pH were minimal as the study locations were mainly oligotrophic to mesotrophic. The salinity during monsoon season in central and upper reaches (stations S1 – S4) was lower (5.6 ± 2.58 ppt) compared to lower reaches (stations S5 and S6) with (18.1 ± 2.9 ppt). A marked wider salinity gradient was observed in all the stations during different seasons. The upper (S1 and S2) and the middle reaches (S3 and S4) with an influx of freshwater during monsoon had freshwater regime. However, the salinity levels peaked to 30 ppt with the onset of the summer season (pre-monsoon). The salinity levels were distinctly different across stations during post-monsoon with low salinity levels (6.25 ± 2.47 ppt) in upper reaches, mid salinity levels (18.12 ± 2.29 ppt) in central reaches and high salinity (25.37 ± 1.59 ppt) in lower reaches of the estuary.

Nutrient levels across the stations were more of temporal dilutions due to anthropogenic inputs. There was no trend in nutrient variation across seasons among the stations and the results were highly location specific and prone to non-point source pollutions. Higher nitrate levels were observed (5.12 ± 0.57 µg/L) during pre-monsoon season followed by post-monsoon (4.875 ± 1.11 µg/L) and monsoon (4.36 ± 0.58 µg/L). Phosphates showed fluctuations within stations across different seasons from (1.43 ± 0.09 µg/L) to (2.43 ± 0.54 µg/L). Silicate levels showed distinct variability (Figure 4.b-d) across seasons among stations, with higher silicate levels observed during monsoon (6.40 ± 1.82 µg/L), followed by post-monsoon (5.04 ± 2.66 µg/L) and pre-monsoon (av. 3.90 ± 2.34 µg/L). The variation in silicate range was both stations as well as season-specific and comparable to earlier studies (Madhu et al., 2007; Srinivas et al., 2003) in the two tropical estuaries along the Indian west coast.

Figure 4 Variation in Physico-Chemical Parameters across the Sampling Stations during pre-post and monsoon Seasons (4.a-d)

Figure 5.a-d reflect variations in DO and nutrient levels. DO was lower (6.33 ± 0.26 mg/L) during pre-monsoon (6.33 ± 0.26 mg/L) and post-monsoon (6.72 ± 0.88 mg/L) compared to monsoonal DO (8.44 ± 0.52 mg/L).

Higher DO levels during monsoons could be attributed to the influx of rainwater and subsequent riverine catchment run-offs reaching the estuary. The temporal variation of DO among the stations during monsoon was higher, evident from Figure 5.a. DO levels observed in this region were relatively higher compared to Cochin backwaters (Madhu et al., 2007).

Effect of hydrological parameters on Species Richness

Two-way ANOVA analysis revealed a significant variation in species richness with salinity, DO, and nutrients (nitrates, phosphates, and silicates) across stations (P=0.015) and seasons (P<0.05%).

Diatom Diversity, Dominance, and Species Composition

An estuary is a dynamic aquatic eco-system that resonates with spatial and temporal changes. A total of 85 different species (Appendix 1) of diatoms were recorded with similar species occurring at different stations in one season. Station wise overall species diversity indices (Shannon-wiener Diversity) and Simpson’s dominance indices were estimated and are given in Fig. 6. The Shannon’s diatom diversity index (H’) was maximum for the mesohaline, upper reach station (S2) of the estuary with H’ = 3.19 and the least diversity was observed at the polyhaline station (S5) in the lower reaches of the estuary with H’ = 1.73, while all other 4 stations had intermediate H’ values. Simpson’s dominance index (D) was the highest for S1 and S6 with values D = 0.9 and 0.86 respectively due to the outnumbering dominance of Coscinodiscus subtilis and Navicula lanceolata in station S1, Melosira sp. and Navicula spp. in station S6 over other species.

Figure 6: Diversity & Dominance Indices of the Sampling Stations

Substrata wise Species Richness and Abundance

The data recorded substrata wise for a period of one year were consolidated to understand the role of substrata in species assemblage (Figure 7). The results demonstrated the highest species abundance (36%) at S2 followed by S3 (31%). Station S6 showed the least species abundance (4%). Stations S4 and Station S5 contributed to 11% and 5% of species abundance respectively. Among the different substrata considered, sediments had maximum epipelic diatom species richness of 40, 36, and 28 respectively at stations S2, S3, and S4. Higher species richness in sediments was followed by stones/rocks (Epilithons) at almost all the stations. Epiphytons attached to plant substrata had the third-highest species richness in S2, S3, and S4 stations with species richness of 35, 21, and 23 respectively, which is similar to the earlier report (Townsend and Gell, 2005). Lower species richness and poor community structure are due to the grazing pressure. Episammons (diatoms growing on sand) had the least species richness in polyhaline lower reaches stations S5 and S6 with S = 12 and S = 18, due to persistent wave action, washing away the community leading to lower diatom affinity in that substrata.

Species Tolerance

The presence of a species, its abundance as well as its distribution is largely determined by their level of tolerance to abiotic environmental factors. Tolerance is the ability or endurance of a species to survive in hostile environmental conditions. When a species is exposed to prolonged unfavorable conditions, the species tends to build up resilience to withstand harsh environmental conditions and in turn evolve as a tolerant species having higher ability to survive under wider ranges of fluctuating conditions, which is observed in certain diatom species with the consistent presence in fluctuating environmental conditions (seasonal variability). These tolerant species abundances w.r.t different seasons at each station is illustrated in the form of a radar plot in Figure 8. The species present during all the three seasons with their relative abundances greater than 15% were considered as “tolerant species” capable of surviving fluctuating environmental conditions and their season-wise relative abundances were plotted, which illustrates the site-specific abundances of tolerant species in premonsoon, post-monsoon and monsoon. The graph was generated by clubbing the tolerant species to the genus level.

Figure 7 Pie chart representing SR and SA w.r.t Substrata

Overall tolerant species’ abundance was highest during pre-monsoon, followed by post-monsoon and the least abundance in monsoon season. During premonsoon, the species abundance of Cyclotella meneghiniana at station S2 was the highest (74.5%) followed by consortia of Navicula sp. (Navicula lanceolata, Navicula bicapitata, Navicula forcipata, Navicula mutica and Navicula amphisbaena) with 68.9% at station S3. Polyhaline (S5) station showed a dominance of Melosira spp. with corresponding species abundance of 72.7% during the premonsoon season. Though the abundance of these species showed a marked peak during pre-monsoon season of the year, the presence of those species was recorded at other seasons as well with varying abundance levels, proving the species to be tolerant with evolved ecological adaptations to season-based dynamism prevailing in real-time estuarine systems.

Season wise analyses of species’ relative abundances reveal higher relative abundance during premonsoon with >15% of tolerant species followed by post-monsoon and the least relative abundances were observed during monsoon season (due to influx of freshwater). As per the tolerance results primarily determined by their presence in almost all the seasons with their corresponding species-wise relative abundances to be >15%, the diatoms belonging to the genus of Achnanthes spp., Nitzschia spp., Navicula spp., Cyclotella spp., Melosira spp. were found to be tolerant with consistent availability at different stations as well as during different seasons.

Among the pennate ones, the genus belonging to Amphora and Nitzschia are known to have pronounced heterotrophic capabilities (Linkins, 1973; Werner, 1977). Most of the Nitzschia sp. is known to be obligate heterotrophs with their habitat preferences in strong heterotrophic environments like decaying piles of seaweeds, with little or nil light penetration. Cyclotella meneghiniana was also known to have strong heterotrophic capabilities (Horner and Alexander, 1972; Lewin and Hellebust, 1976; Lylis and Trainor, 1973). Another dominant taxon observed very frequently in lower reaches (S5 and S6) was known to be a taxon frequently encountered in higher insolation regions, especially in tidal pools and upper inter-tidal regions. This taxon was proven to be relatively tolerant of inter-tidal exposure to desiccation (Castenholz, 1963; Stevenson et al., 1996) which could have given Achnanthes sp. the ability to survive at higher insolation over large boulders subjected to prolonged intertidal exposure. Melosira sp. could dominate in varying pH conditions and warmer waters with higher light intensity. It is found that Navicula sp. which occurs mostly over the sediment surfaces and on other aquatic plants are facultative heterotrophs that utilize organic matter found in the substrate (Werner, 1977)., which justifies dominance of certain species across different seasons in more than one station.

Figure 8 Relative abundance of tolerant species w.r.t seasons

*spp. represents species composition with more than one diatom species at respective stations from S1 to S6

S2 Achnanthes spp. – Achnanthes brevipes, Achnanthes longipes

S2 Amphora spp. – Amphora salina, Amphora ovalis

S2 Nitzschia spp. – Nitzschia obtusa, Nitzschia sigma, Nitzschia apiculata, Nitzschia fasciculata, Nitzschia panduriformis

S2 Navicula spp. – Navicula forcipata, Navicula amphisbaena, Navicula permagna

S3 Nitzschia spp. – Nitzschia obtusa, Nitzschia sigma, Nitzschia fasciculata, Nitzschia acicularis, Nitzschia panduriformis

S3 Navicula spp. – Navicula lanceolata, Navicula bicapitata, Navicula forcipata, Navicula mutica, Navicula amphisbaena

S4 Melosira spp. – Melosira varians, Melosira jurgensii

S4 Nitzschia spp. – Nitzschia obtusa, Nitzschia fasciculata, Nitzschia sigma, Nitzschia acicularis

S5 Melosira spp. – Melosira lineatus, Melosira jurgensii

S5 Nitzschia spp. – Nitzschia obtusa, Nitzschia sigma, Nitzschia apiculata, Nitzschia fasciculata

S5 Achnanthes spp. – Achnanthes brevipes, Achnanthes longipes

S6 Cyclotella spp. – Cyclotella meneghiniana, Cyclotella operculata

S6 Navicula spp. – Navicula lanceolata, Navicula granulata, Navicula mutica

S6 Cyclotella spp. includes Cyclotella meneghiniana, Cyclotella operculata

Clustering based on Species Abundance

Month-wise species composition along with its relative abundance of diatoms was organized season-wise and subjected to agglomerative hierarchical clustering (Figure 9) to understand the species predominance and season sensitivity. Cluster analysis resulted in three distinct clusters based on their species abundance across different seasons. Cluster 1 formed the major group of diatom species that are sensitive and predominantly either freshwater species or euryhaline species.

The presence of which is observed only in specific sites at a particular season with its relative abundance <2%. Cluster 1 includes freshwater species such as Cocconeis placentula, Aulacoseira granulata and salinity tolerant euryhaline species such as Amphiprora alata, Eunotia flexuosa, Spermatogonia sp., Pleurosigma marcum etc., with least abundance recorded in one of the three seasons. Cluster 2 formed the second major group with a moderate species abundance of >5%. Cluster 3 formed the dominant groups with a higher relative species abundance of 8% - 15%. Out of 86 different species of diatoms, more than 80% of the population were either recorded only in upstream (less saline) regions during monsoon periods or only at downstream regions during pre/post-monsoon seasons forming cluster 1. Those species could be collectively termed as sensitive species, the presence or absence of which is influenced by one or more of the environmental parameters and even the slightest change in those parameters could have led to the species turnover by tolerant species. The species recorded in the upstream regions were found to exhibit predominant freshwater habitat characteristics. The species that exhibited such characteristics include Synedra ulna, Aulacoseira granulata, Bacillaria paradoxa, Cocconeis sp. Gomphonema sp., Cymbella sp., Pinnularia sp., and Diploneis sp. (Appendix 1). The presence of freshwater species was confined to upstream and middle estuarine portions in monsoon season. On the other hand, species like Biddulphia laevis, Licomophora sp., Amphiprora alata, Epithema argus were found only in downstream (S5 and S6) stations of the estuary. Hence these species could be considered as salinity loving/euryhaline species and their absence during monsoon and post monsoon could be attributed to overall low salinity of the estuary during those two seasons.

Figure 9 Agglomerative Hierarchical Clustering of Species w.r.t Species Abundance

Season-wise Variation in Species Composition

Non-metric multidimensional scaling analysis was performed to understand the dynamics of the diatom community structure among stations by considering season-wise species abundance across each station (Figure 10). NMDS is a rank-based approach in which the ordination is achieved through an iterative process based on pairwise dissimilarity in a low dimensional space. Here NMDS is plotted considering species abundance summed up season-wise to understand the commonness in species across stations. The plot shows ordination hulls formed with respect to pre- post- and monsoon seasons across stations (S1 PRM – S6 PRM), (S1 M – S6 M), and (S1 POM – S6 POM). The ordination hulls generated season-wise, which demarcates tolerant species that are present throughout the year from station-specific sensitive species. For example, diatom species like Melosira lineatus (MLI), Nitzschia obtusa (NIOB), Gomphonema gracile (GG), Cyclotella meneghiniana (CM), Coscinodiscus subtilis (CS) were present in the overlapping intersections of all three ordination hulls indicating its presence during all three seasons at different stations. Whereas species like Achnanthes brevipes (AB), Achnanthes longipes (AL), Amphora salina (AS), Navicula amphisbaena (NAM), Melosira jurgensii (MJ) were observed only during monsoon and pre-monsoon seasons at more than one study locations. Whereas Pleurosigma angulatum (PAN), Cocconeis scutellum (COS), Navicula ambigua (NAG), Coscinodiscus radiatus (CR) were present during pre- and post-monsoon seasons in one or two study locations. Whereas species like Frustulia crassinervia (FS), Aulacoseira granulata (AUG), Licomophora paradoxa (LP), Nitzschia sigma var. intercedens (NISV), Pleurosigma acutum (PAC), Gomphonema intricatum (GI), Cymbella parva (CP) were confined to one particular station with insignificant relative abundances and observed during only one of the three seasons.

Tolerant Species for Bioremediation and Biofuel

Recent research on third-generation biofuel production is increasingly concentrating on using wastewater as a source of nutrients for the growth of microalgae. If a biofuel industry is to be set up based on open/raceway ponds using wastewater as a source of nutrients, running the industry at consistent productivities throughout the year proves challenging as contamination control becomes quite difficult in such open systems. The introduction of monoculture strains or sensitive species further complicates the scenario as the pond is continuously exposed to both diurnal and seasonal variations in weather as well as water quality used for biomass enrichment. Hence, phyco-prospecting consortia of tolerant species with year-round availability irrespective of microclimate/environmental conditions would be a better choice in averting the risks of contamination or crash encountered in monoculture sensitive strain-based cultivation systems. Thus, from the results of the present study, it is apt to consider tolerant diatom or consortia of tolerant diatoms capable of exhibiting robust growth despite fluctuations and variations in the environmental factors that greatly influence its growth. The species having potential for phyco-prospecting evident from the results of the present study are Amphora sp., Nitzschia sp., Navicula sp., Achnanthes sp., Melosira sp., Coscinodiscus sp., and Cyclotella sp. The above-mentioned species when chosen as a candidate strain or as consortia of strains for phyco-prospecting has better chances of survival in open pond/raceway pond systems exhibiting enhanced biomass productivities.

Figure 10: nMDS Plot based on Site-wise and Season-wise Species Abundance

_{*The acronyms of species names used in NMDS graph are in Appendix 1}

Lipid Potential of Select Tolerant Diatom Strains

Microalgae are widely considered as biodiesel feedstocks due to its better lipid productivity and shorter cycling period with quick multiplication rates when compared to conventional terrestrial oil seeds. Many kinds of research around the globe had focussed on lab-scale cultivation of monoculture strains to estimate lipid productivity. Studies so far on Achnanthes sp. under lab conditions showed a lipid content that varies from (19.6 – 27.7) % based on dry cell weight (Zhao et al., 2016). Lipid content studies on Nitzschia spp. showed values ranging between (37.5 – 46) % (Sheehan et al., 1998; Zhao et al., 2016). Melosira sp. exhibited lipid content varying from (10.52 – 33.01) % in two different studies (Chen et al., 2012; Renaud et al., 1994). Amphora sp. had shown the highest lipid content reported so far on diatoms with 49.95% (De La Peña, 2007). Different species of Navicula, experimented under varied conditions showed lipid content with 17.3 – 39.84% based on dry cell weight (Fields and Kociolek, 2015). These results corroborate the prospects of select tolerant species as potential biofuel strains.

Figure 11: Laboratory Experiments on Growth Rate and Species Composition

Laboratory Experiments to Elucidate the Lipid Potential of Indigenous Diatom strains

Benthic diatoms were collected from rocks from one of the study sites (S4) and immediately inoculated in F/2 media under sterile conditions. The nutrient enrichment provided by F/2 media facilitated a mixed consortium of diatoms to grow in the culture with a working volume of 250 mL. The light intensity was maintained at 90 µmol m^-2 s^-1 and the temperature was maintained at 29 ± 2^oC. The salinity of the cultures was maintained at 28 psu. The nutrient-enriched mixed cultures were then analyzed for species composition variations throughout the growth period as well as its lipid content once the cell reached its stationary phase. The consortium consisted of Coscinodiscus sp., Nitzschia panduriformis, Melosira sp., Navicula sp., Skeletonema sp., Amphora sp., Camphylodiscus sp., and Nitzschia obtusa. Figure 11 represents the changes in the relative abundance of each species in the consortium during the complete growth period of ten days. After ten days, the diatoms were harvested and subjected to lipid extraction using a modified Folch method.

The culture consortium was further sub-cultured as aliquots under various treatments such as nitrogen and silicon starvation to understand the percentage increase of lipids as dry cell weights with respect to no treatment (control) sample. Results on varying treatments revealed that nitrogen starvation had exhibited the highest lipid content (17 - 25% based in terms of dry cell weight). The biomass yield was found to be 80 – 160 mg/L. The nitrogen starved algal biomass extracted lipids were further subjected to acid catalysed transesterification to obtain FAMEs. The FAME thus obtained were analysed using GC-MS to understand the types of fatty acids produced by the diatom consortia.

Figure 12 Fatty acid Methyl Ester Profiles of harvested algal biomass

Figure 12 represents the peaks showing variations (%) in diatom derived FAMEs. Palmitic acid methyl ester was the dominant fatty acid recorded followed by palmitoleic acid, myristic acid, and trans-vaccenic acid methyl ester. These results elucidate the potential of indigenous diatom strains consortium in the production of third-generation biofuels.