Polyextremotolerant lignocellulolytic bacteria from contrasting halo-alkaline habitats as sources of extremozymes for rice husk saccharification and biofuel applications

Rationale Lignocellulosic agricultural residues represent an enormous and largely untapped feedstock for second-generation biofuels, yet their structural recalcitrance remains the central bottleneck for cost-effective bioconversion. Conventional mesophilic enzymes lose much of their activity under the high-salinity, alkaline and thermally stressful conditions that define industrial biomass processing- a gap that microorganisms from naturally halo-alkaline ecosystems are well positioned to fill. Rice husk, generated in massive quantities as a by-product of rice milling across South Asia, remains an underutilized substrate whose silica-rich, lignin-shielded architecture makes it an appropriate and demanding test case for halotolerant biocatalysts. Methods Three ecologically distinct saline habitats were sampled: Poovar Lake, a brackish mangrove estuary in Kerala (pH 6.92, salinity (chloride concentration) 1.90 g/L), Varkala Beach, a high-chloride concentration coastal site (pH 8.24, salinity (chloride concentration) 31.16 g/L), and Sanjay Lake, an anthropogenic inland lake in Delhi (pH 9.25, salinity (chloride concentration) 0.42 g/L). Using selective enrichment on halophilic media at 40 °C across a salinity range of 3–9% (w/v) NaCl, 29 halotolerant isolates were recovered and screened for xylanase, cellulase, lipase, amylase and protease activity using plate-based zone assays. The four most active cellulase- and xylanase-producing isolates, identified by 16S rRNA gene sequencing as Priestia flexa (XBL51), Bacillus proteolyticus (XBL61), Bacillus paralicheniformis (XBL66) and “ Vibrio neocaledonicus ” (XBL71), were then evaluated for their ability to hydrolyze alkaline-pretreated rice husk (1.5% (w/v) NaOH, 60 °C, 90 min) under saline-alkaline stress (4.5% (w/v) NaCl, pH 8.5, 40 °C). Growth kinetics and reducing sugar release were monitored over 6-days, and post-degradation structural changes in the biomass were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Results Alkaline pretreatment significantly disrupted the crystalline architecture of rice husk, reducing the crystallinity index (CrI) from 49.31% to 33.93%, thereby loosening the structure and substantially improving substrate accessibility for enzymatic attack. Among individual isolates, monoculture of XBL61 and XBL71, as well as XBL51 + XBL66 + XBL71 showed the strongest growth on pretreated rice husk as the sole carbon source. Reducing sugar release across the top-performing isolates and consortia ranged from 1.69 to 1.97 mg/g over the 6-day incubation. Monocultures XBL61 and XBL66 emerged as the most effective isolates, exhibiting sustained sugar production. Among the two-isolate consortium, XBL51 + XBL71 exhibited synergistic growth and hydrolysis activity. Post-incubation, XRD analysis showed that XBL61 alone raised the CrI marginally to 52.10%, suggesting reduction of amorphous regions, including non-cellulosic components such as hemicellulose and amorphous cellulose. By contrast, the XBL51 + XBL71 consortium reduced the CrI to 49.07%, reflecting more extensive and balanced structural deconstruction. SEM corroborated these findings, revealing a clear progression from the intact, tightly laminated surface of untreated biomass through the fissured, loosened layers of the pretreated material, to the deeply porous, honeycombed morphology left following consortium-mediated degradation. Conclusion These results demonstrate that halotolerant bacteria isolated from contrasting saline habitats, particularly when used as rationally designed consortia, can effectively deconstruct alkaline-pretreated rice husk under conditions that would substantially inhibit conventional mesophilic cellulases. The inherent stress tolerance of these isolates makes them promising candidates for lignocellulosic biorefinery processes where elevated salinity and alkaline pH are unavoidable operational realities. Enzyme-level characterization and fermentation coupling of these isolates represent logical next steps toward translating these findings into scalable platforms for second-generation biofuel production.