Physicochemical parameters of samples
Sambhar Lake (SLW) and Bichitrapur (BPW) wood particles had significant differences in their salinity and pH. While both values for SLW were high with 21.54% total salinity and pH 9.0, the same for BPW were 2.18% and 6.5. Organic carbon content in both wood particles was 43.25% and 41.98%, respectively. On the other hand, available nitrogen of these two samples was 0.83% and 0.59%, respectively. Thus, although, the wood particles were collected from two drastically different saline environments, their total organic carbon and available nitrogen content were comparable.
Mangrove environment have more cellulolytic bacteria than Hypersaline Lake
To get an idea about the cellulolytic bacterial load associated with the two degraded wood samples, ten thousand colonies each were randomly picked from respective heterotrophic plates. Each colony was replica plated onto respective agar medium for screening endoglucanase and β-glucosidase activity using Congo red assay and esculin hydrate assay, respectively. Interestingly, BPW had 58% and 33% endoglucanase and β-glucosidase positive colonies respectively, as compared to only 31% and 21% for the same phenotypes in SLW (Fig. S1). Although both samples were degraded wood particles, the extreme condition in Sambhar Lake might be the reason behind low cellulolytic bacterial count in SLW. Bichitrapur, on the other hand, is a mangrove area, with moderate climatic conditions and abundant organic matter that favors microbial growth.
Endoglucanase of SLW isolates are stable and active in wide range of NaCl concentrations
Ten best performing and distinct endoglucanase positive colonies (hydrolysis zone of 3 mm or above in Congo red assay) from both SLW and BPW samples were selected for further characterization. Optimum heterotrophic growth was obtained for SLW isolates with 10–15% NaCl and pH 9.0 maintained in culture media, while BPW isolates require NaCl concentration (1–5%) and pH 7.0–8.0 at relatively lower amounts (Table S1). Thus, optimum growth conditions of all twenty isolates are in sync to their respective environmental parameters.
All twenty isolates produced endoglucanase extracellularly and activities were detected in spent media. Enzymes of SLW isolates were active in wide range of NaCl (0–25%) (Fig. 1a). On the other hand, endoglucanase of BPW isolates had optimum activity at 5% NaCl, but in higher concentrations, activity reduced drastically (Fig. 1b). Bacteria living in Sambhar Lake have to employ adaptive mechanisms and thus high salt requirements by SLW endoglucanases are justified. In a previous study by our laboratory, an endoglucanase from Sambhar Lake isolate, Salisediminibacterium halotolerans EN1, exhibited optimum activity at 25% NaCl (Sar et al. 2021). Another endoglucanase encoded by Paenibacillus tarimensis, which was isolated from an inland saline system in Tunisia, exhibited optimum activity at 29% NaCl (Raddadi et al. 2013). Endoglucanase from Yuncheng Salt Lake, China was highly active in wide range of NaCl concentration (7.5–17.5%) (Yu and Li 2015). On the other hand, endoglucanase secreted by Bacillus agaradhaerens, which was isolated from seawater showed optimum activity at 1.2% NaCl (Hirasawa et al. 2006). Altogether, the obtained results suggest that the optimum requirements by bacterial enzymes, particularly the extracellular ones correlate with the respective natural environments.
[Bmim][Cl] tolerance potency correlates with NaCl tolerance potency
Endoglucanase from all twenty isolates were assayed in presence of different concentrations of [Bmim][Cl]. Among the twenty selected isolates, only SL1, SL2, SL8, SL14 and SL24 retained more than 50% of its activity in 10% (v/v) [Bmim][Cl] (Fig. 2a) with maximum 72.9% retention by SL1. At 30% (v/v) [Bmim][Cl] concentration, SL1, SL8 and SL24 again retained a maximum of 33.89%, 38.43% and 38.88% activities respectively. SL14 and SL48 also retains 17.69% and 12.42% activity in 30% [Bmim][Cl]. Interestingly, all BPW endoglucanase lost their activity at 30% (v/v) [Bmim][Cl], and even with 10% of the IL, only BP6 and BP31, retained 49.39% and 45.64% activity, respectively (Fig. 2b). Thus, our results further support the idea of correlation of salt and ionic liquid tolerance of bacterial cellulase. A recent report by our group on Sambhar lake showed that bacteria which can grow in wide range of salt, also exhibit high ionic liquid tolerance potency (Pal et al. 2019). Recombinant endoglucanase NMgh45, with salt stability in 4M NaCl, also showed excellent ionic liquid tolerance with residual activities of 90% and 43% at 10% and 20% [Bmim][Cl], respectively (Zhao et al. 2018). Another ionic liquid tolerant cellulase CelA10 (Pottkamper et al. 2008) exhibited salt tolerance. Paenibacillus tarimensis, isolated from Tunisian salt lake, has ability to maintain 40 and 90% of its hydrolysis efficiency in 40% (v/v) [Bmim][Cl] and 20% (v/v) [Emim][Ac], respectively (Raddadi et al. 2013). It is logical to have ionic liquid tolerance properties of salt tolerant enzymes, because like inorganic salts ionic liquids also gets dissociated in respective ions in aqueous solution. However, based on the present results, it cannot be said that any halophilic bacteria or their extracellular enzyme would be tolerant to the IL.
In situ saccharification of lignocellulosic substrate with potent endoglucanases
Three of the best performing endoglucanase, two produced by Sambhar lake isolates SL1, and SL24, and one from the Bichitrapur isolate BP6 were used for in situ saccharification of alkali and [Bmim][Cl] pretreated rice straw. Based on 16S rRNA gene sequence analysis, SL1 and SL24 respectively showed 99.90 % and 100% sequence similarity to the single NCBI entry Salipaludibacillus sp. AK99 (Accession no. LT882622.1) that again was isolated from Sambhar Lake. Members of the genera Salipaludibacillus, belonging to class Bacilli are rod-shaped, non-motile, and form endospores (ellipsoidal or oval) at the sub-terminal position. Cells are aerobic or facultatively anaerobic (Sultanpuram and Mothe 2016). BP6 showed 100% sequence similarity with Salinicola sp. strain SB01-L0, which belongs to class Gammaproteobacteria. The genus Salinicola was proposed by Anan’ina et al. and member of this genus are rod shaped, non-spore-forming and motile by a single polar flagellum (Anan’ina et al. 2007).
Alkali pretreatment of lignocellulosic biomass with 2% NaOH solution has been suggested for effective removal of lignin (Jeya et al. 2009; Zhang and Cai 2008). Till date, several studies on saccharification have been reported (Hari Krishna and Chowdary 2000; Jeya et al. 2009; Sukumaran et al. 2009; Zhang and Cai 2008) using different lignocellulosic substrates. Rice straw is most preferred substrate for in situ saccharification experiments because of its abundant and cheap availability. After alkali pretreatment, pretreated rice straw was distorted and swelled which supports better accessibility of cellulose and efficiency of pretreatment. No reducing end was generated in ‘no enzyme control’ and ‘no pretreatment control’ sets suggesting importance of the hydrolytic enzyme and the pretreatment process respectively for saccharification of lignocellulosic biomass (Fig. 3). Residual alkali strongly inhibited the activity of all three endoglucanases. Thus, washing off pretreated substrate with water to minimize further inhibition by residual alkali is needed. But global paucity of freshwater is another serious concern (Zhang et al. 2010), which can be overcome by replacing freshwater with seawater. Despite this considerable interest, no study has focused on enzymatic saccharification on seawater washed substrate. In distilled water and seawater washed condition, release of reducing end by all three endoglucanases was clearly detectable after 24 h of incubation and reached peak after 48 h. In all reactions, reducing end was not generated after 48 or 72 h incubation. It indicates that either reaction was completed after that particular time period or enzyme has lost its activity. After 48 h of incubation, reducing end generated from in situ saccharification of ‘water washed’ rice straw with SL1, SL24 and BP6 endoglucanase was 117.59, 114.9 and 124.12 µg mL− 1, respectively. Thus, these enzymes are excellent components for in situ saccharification of alkali pretreated biomass. In addition, reducing end generated by SL1 and SL24 endoglucanase from ‘seawater washed’ substrate was low, 106.06 and 110.2 µg mL− 1, respectively. Interestingly, BP6 endoglucanase exhibited 140% higher activity (173.7 µg mL− 1) in ‘seawater washed’ biomass as compared to ‘water washed’. Even seawater washing steps can be included which makes these enzymes promising candidates for industrial use.
Applicability of these endoglucanase on [Bmim][Cl] pretreated biomass, was also tested in different concentration of residual or added or washed samples enzyme activity were assessed. Reducing end generated in washed substrate by SL1, SL24 and BP6 endoglucanase were 112.8, 92.4 and 119.9 µg mL− 1, respectively (Fig. 4). But, in presence of residual [Bmim][Cl], reducing end yield dropped to 48.0, 27.7 and 67.6 µg mL− 1, while in 5% [Bmim][Cl] yield by three endoglucanase was almost none (~ 2 µg mL− 1). A few studies on enzymatic saccharification of IL pretreated lignocellulosic material have been reported previously (Li et al. 2010; Wang et al. 2011; Xu et al. 2015). Although the saccharification rates was inhibited in 5% [Bmim][Cl], hydrolysis in residual IL conditions is advantageous, as it eliminates substrate washing step.