Spatio- temporal variation of environmental variables
The changes in environmental variables of Ashtamudi estuarine sediments is summarized in Table 1.0. The overall sediment temperature was higher during April 2016 than January 2017. The spatial variation of sediment temperature was statistically insignificant along the estuary whereas the temporal variation was significant (p=0.001) (Table 2). Previous study by Reshmi et al. (Reshmi et al 2015) in Ashtamudi estuarine sediment also points out similar results. During both sampling periods, Ashtamudi estuary exhibited brackish to marine conditions with regard to pH and salinity values (Jennerjahn et al 2008). Alkaline pH was observed all over the estuary during the sampling seasons with an average value of 7.97 and 8.26. However, the spatial variation of salinity was statistically significant (p<0.001) and temporal variation was insignificant. Ashtamudi estuary opens to the Arabian sea through a wide opening which obviously shows a marine influence all over the estuary (Vincent et al 2017). Maximum electrical conductivity was observed in the Kollam city part of the estuary during April 2016 and Kallada River part of the estuary during January 2017, which indicates the influence of dissolved nutrient load from the Kallada River (Jennerjahn et al 2008) during the post-monsoon season.
Spatio- temporal variation and source of organic matter
The quality and quantity of organic matter can regulate the composition and activity of microbial communities in aquatic sediments (Torres et al 2011; Cawley et al 2012). Particularly, the lignocellulosic biomass forms the substrate for cellulolytic anaerobes in the anaerobic sediment. Hence, it is important to analyze the quality and quantity of organic matter in the sediment. Significant spatial variations (p < 0.001) in the Corg values were observed, whereas the temporal variations were statistically insignificant in the Ashtamudi estuary. In both seasons, average Corg values was higher in Kollam city part as compared to the marine and river influenced part of the estuary. The sampling station S12 in the Kollam city region showed higher values during both seasons. Previous studies (Jennerjahn et al 2008; Vincent et al 2017) also showed the dominance of Corg in the Kollam city region. Lowest Corg values were observed in the open estuary portion particularly S9 and S10. That is related to the flushing of sea water to the open estuary during the tidal cycles and high rate of microbial degradative activities (Jonathan et al 2004; Hussain et al 2020). Stable isotopic δ13 Corg study by Jennerjahn et al. (Jennerjahn et al 2008) points out the different source and diagenesis of organic matter, in which open estuary had highest value of δ13 Corg indicating the presence of marine derived organic matter (Thimdee et al 2003) and lowest value of δ13 Corg in the upper part, which is attributed to the presence of river derived organic matter. LOM and BPC values showed significant spatio- temporal variations (p<0.001). The higher LOM and BPC values were observed in Kallada River region during April 2016 and in Kollam city region of the estuary during January 2017. The LOM to TOM value can be used as an index of organic matter lability (Gonsalves et al 2011). In Ashtamudi estuarine sediments, higher LOM of TOM values were observed during January 2017 (21.82%) than during April 2016 (13.22%), which indicates that the organic matter was more labile during January 2017. LOM was dominated by protein followed by lipid and carbohydrate for both seasons. Similar result was observed in Kerala coast (Nair and Sujatha 2012), Galician coast (Cividanes et al 2002) and Ross sea, Antarctica (Fabiano et al 1995). Low carbohydrate values were also observed in cochin estuary (Joseph et al 2008) of Kerala. High protein concentration in the sediment is due to allochthonous inputs (Danovaro 1996) of organic matter. Usually, proteins can be readily utilized by the bacteria than carbohydrate (Newell and Field 1983) and the dominance of protein concentration indicates the presence of fresh organic matter. Contrastingly, high values of lipid were reported from decayed organism (Danovaro et al 1993). Nevertheless, carbohydrates are important fraction of Corg contributed by living organisms (Børsheim et al 1999; Burdige et al 2000; Bacic et al 2012) and low carbohydrate values show the refractory nature of organic matter (Danovaro et al 1993). Protein: carbohydrate ratio was observed to be higher in Kallada River region during April 2016 and in Kollam region during January 2017. The high protein to carbohydrate ratio also indicates that the organic matter is fresh and recently generated (Danovaro et al 1993; Fabiano and Pusceddu 1998; Joseph et al 2008; Joy et al 2019), whereas higher lipid carbohydrate ratio was observed in the open estuary during both seasons.
Abundance and distribution of cellulolytic anaerobes
The abundance of cellulolytic anaerobes was comparatively higher during January 2017 than April 2016 (Table 3). During both seasons, maximum cellulolytic population was observed with cellobiose and limited or slow growth with crystalline cellulose as substrate, which is attributed to the insolubility nature of crystalline cellulose (Leschine 1995). In S7 (open estuary) maximum growth was observed in April 2016 and S8 (open estuary) in January 2017 for cellobiose substrate. In case of crystalline cellulose substrate, S12 (Kollam city region) showed maximum growth in April 2016 and S9 in January 2017. The cellulolytic population of cellulolytic anaerobes were very low in Ashtamudi estuary as compared to the populations of other group of obligate anaerobes like methanogens and sulphate reducing bacteria (Reshmi et al 2015).
Cellulolytic enzyme activity
In natural lake sediments, most of the cellulose degradation occurs aerobically and only 5-10 % is available for anaerobic degradation (Leschine 1995). During April 2016, native enzyme activity (NEA) was predominant in the open estuary (Fig. 2). Whereas, during January 2017, NEA was more pronounced in the Kallada River region, that might be due the confluence of Kallada river and the availability of fresh organic load from the river as native substrate. In this study, the cumulative substrate induced enzyme activity (SIEA) (Fig. 3 & 4) was more than NEA. This reveals the potential of added substrates to induce cellulosic activity. Previous studies in Lake Gooimeer also showed increased activity of β –glucosidase by addition of cellulose (Boschker and Cappenberg 2006). During April 2016, thermophilic activity was predominant in the Kallada River region, whereas mesophilic enzyme activity was higher in the Kallada River and Kollam city region of the estuary. During January 2017, thermophilic cellulase activity predominated in the open estuary and mesophilic enzyme activity in the Kollam city and Kallada River regions. Overall cellulolytic activity was higher towards Kallada River region of the estuary. Biogeochemical analysis of Ashtamudi estuarine sediment by Jennerjahn et al. (Jennerjahn et al 2008) revealed that, most of the Kallada River load is deposited in the upper part of the estuary and middle and lower parts are subjected to strong marine influence. So, it is evident that the riverine dissolved organic load contains sufficient amount of labile fraction, that can be easily consumed by the microbial communities (Abril et al 2002).
Environmental controls on abundance and activity of cellulolytic anaerobe
The relationship between environmental variables and cellulolytic activity were analysed using principal component analysis (PCA) and redundancy analysis (RDA). The PCA generated three principal components (Table 3, Fig.5). During April 2016, the first PC accounted for 32.02% of total variance. Whereas PC2 and PC3 explained 22.03 and 17.68 of total variance respectively and also explained a cumulative variance of 71.74%. In the first PC, EC, sulphate Corg and TOM showed strong positive loadings and the factors were collectively referred as conductivity- nutrient factor. In PC 2 lipid:carbohydrate ratio showed strong positive loading and in PC3 carbohydrate showed strong negative loadings.
During January 2017, the first PC accounted for 31.62% of total variance. Whereas PC2 and PC3 explained 22.59 and 17.89 of total variance respectively and also showed a cumulative variance of 71.74%. In the first PC, Corg, TOM, protein, LOM and BPC showed strong positive loadings and was referred as nutrient factor. The composition of organic matter can influence extra cellular enzyme activities in lake sediments (Boschker and Cappenberg 2006). The second PC exhibited strong positive loading on protein:carbohydrate ratio and had a negative loading on carbohydrate. The third PC showed high positive loading on temperature and salinity. Hence, the factor was named as temperature- salinity factor.
During April 2016, RDA output showed that lipid followed by salinity, protein and Corg (explanatory variables) were the highest determining factors for explaining most of the variation in the abundance and activity of cellulolytic anaerobes (response variables) (Fig. 6). During January 2017, salinity followed by sulphate and organic matter were the highest determining factors. Energetic requirements of bacteria are associated with higher salinity and that can affect the abundance and activity of microbial groups (Gu et al 2012).