Exploring Diversity and Polymer Degrading Potential of Epiphytic Bacteria Isolated from Marine Macroalgae

The macroalgae surface allows specific bacterial communities to colonize, resulting in complex biological interactions. In recent years, several researchers have studied the diversity and function of the epiphytic bacteria associated with algal host, but largely these interactions remain underexplored. In the present study we analysed the cultivable diversity and polymer degradation potential of epiphytic bacteria associated with five different marine macroalgae (Sargassum, Ulva, Padina, Dictyota and Pterocladia sp.) sampled from the central west coast of India. Out of the total 360 strains isolated, purified and preserved, about 238 strains were identified through 16S rRNA gene sequence analysis and processed for polymer (cellulose, pectin, xylan and starch) degrading activities. Phylogeny placed the strains within the classes Actinobacteria, Bacilli, Alpha-proteobacteria, and Gamma-proteobacteria and clustered them into 45 genera, wherein Vibrio, Bacillus, Pseudoalteromonas, Alteromonas, Staphylococcus and Kocuria spp. were the most abundant with 20 strains identified as potentially novel taxa within the genera Bacillus, Cellulosimicrobium, Gordonia, Marinomonas, Vibrio, Luteimonas and Pseudoalteromonas. In terms of polymer hydrolysis potential, 61.3% had xylanase activity, while 59.7%, 58.8%, and 52.2% had amylase, cellulase, and pectinase activity, respectively. Overall, 75.6% of the strains degraded more than one polysaccharide, 24% degraded all polymers, while nine strains (3.8%) degraded raw sugarcane bagasse. This study showed great potential for seaweed-associated bacteria in the bio-remediation of agro-waste based raw materials, which can be employed in the form of green technology.


Introduction
Marine macroalgae/seaweeds that contribute to approximately half of primary sustainable productivity [1][2][3], inhabit the coastal intertidal regions. The micro-environment on algal surfaces is highly dynamic and complex due to colonization by planktonic microbes, such as bacteria, fungi, and diatoms, among other organisms [3][4][5][6][7]. They have emerged as a rich source of microbial diversity and biologically active secondary metabolites in recent years [8,9]. "Algal-microbes" interactions are facilitated through multiple and complex mechanisms involving novel bioactive compounds [10]. Physicochemical properties, metabolite composition, defense mechanism [11] and attractant patterns [12], among different groups of macroalgae (Phaeophyceae, Chlorophyceae and Rhodophyceae) prompt them to have specific and unique microbial architectures [3]. However, the majority of studies over the last decade have focused on an estimation of microbial diversity associated with macroalgae through metagenomics [6,13,14] along with a handful of culture-based approaches [9,[15][16][17][18].

Cultivation of Epiphytic Bacteria
About 1 g of macroalgal tissue was weighed and washed with sterile distilled water to remove loosely attached microbes and debris from its surface, and vigorously vortexed with 9 mL of sterile 75% artificial sea water (ASW) to re-suspend the epibiotic bacterial community in the diluent [18]. The original suspension was serially diluted (10 −1 to 10 −8 times) and 100 μL from each dilution was spread plated on six different microbiological media in duplicates, i.e., soyabean casein digest agar (TSBA; HiMedia ® ), soyabean casein digest broth diluted 100 times with distilled water and solidified with bacteriological agar (TSBAD; HiMedia ® ), Zobell marine agar (MA; HiMedia ® ), reasoner's 2 agar (R2A; HiMedia ® ), sea water complex agar medium (SWC; containing 6.05 g/L tris base, 12.35 g/L magnesium sulphate, 0.74 g/L potassium chloride, 0.13 g/L diammonium hydrogen phosphate, 17.50 g/L, sodium chloride, 0.14 g/L calcium chloride dihydrate, 1 g/L peptone, 5 g/L yeast extract, 3 mL/L glycerol, and 20 g/L bacteriological grade agar; HiMedia ® ), and Vaatanen nine salt solution agar medium (VNSS medium containing 17.60 g/L sodium chloride, 1.47 g/L sodium sulphate, 0.08 g/L sodium bicarbonate, 0.25 g/L potassium chloride, 0.04 g/L potassium bromide, 1.87 g/L magnesium chloride hexahydrate, 0.41 g/L calcium chloride dihydrate, 0.01 g/L strontium chloride hexahydrate, 0.01 g/L boric acid, 1 g/L peptone, 0.50 g/L yeast extract, 0.50 g/L glucose, 0.50 g/L soluble starch, 0.01 g/L ferrous sulphate heptahydrate, 0.01 g/L disodium hydrogen phosphate, and 20 g/L agar). The plates were incubated at 30 °C and the colonies were picked after every 24 hr for a period of four-six weeks to take care of slow-growers. The CFUs were estimated for a period of 7 days. Bacterial strains were purified by sub-culturing through streaking on the respective fresh medium and preserved at −80 °C in 2 mL cryoprotectant vials (Tarson, 523053) by using 20% (v/v) glycerol.

Cultivation of Epiphytic Bacteria
About 1 g of macroalgal tissue was weighed and washed with sterile distilled water to remove loosely attached microbes and debris from its surface, and vigorously vortexed with 9 mL of sterile 75% artificial sea water (ASW) to re-suspend the epibiotic bacterial community in the diluent [18]. The original suspension was serially diluted (10 −1 to 10 −8 times) and 100 µL from each dilution was spread plated on six different microbiological media in duplicates, i.e., soyabean casein digest agar (TSBA; HiMedia ® ), soyabean casein digest broth diluted 100 times with distilled water and solidified with bacteriological agar (TSBAD; HiMedia ® ), Zobell marine agar (MA; HiMedia ® ), reasoner's 2 agar (R2A; HiMedia ® ), sea water complex agar medium (SWC; containing 6.05 g/L tris base, 12.35 g/L magnesium sulphate, 0.74 g/L potassium chloride, 0.13 g/L diammonium hydrogen phosphate, 17.50 g/L, sodium chloride, 0.14 g/L calcium chloride dihydrate, 1 g/L peptone, 5 g/L yeast extract, 3 mL/L glycerol, and 20 g/L bacteriological grade agar; HiMedia ® ), and Vaatanen nine salt solution agar medium (VNSS medium containing 17.60 g/L sodium chloride, 1.47 g/L sodium sulphate, 0.08 g/L sodium bicarbonate, 0.25 g/L potassium chloride, 0.04 g/L potassium bromide, 1.87 g/L magnesium chloride hexahydrate, 0.41 g/L calcium chloride dihydrate, 0.01 g/L strontium chloride hexahydrate, 0.01 g/L boric acid, 1 g/L peptone, 0.50 g/L yeast extract, 0.50 g/L glucose, 0.50 g/L soluble starch, 0.01 g/L ferrous sulphate heptahydrate, 0.01 g/L disodium hydrogen phosphate, and 20 g/L agar). The plates were incubated at 30 • C and the colonies were picked after every 24 h for a period of four-six weeks to take care of slow-growers. The CFUs were estimated for a period of 7 days. Bacterial strains were purified by sub-culturing through streaking on the respective fresh medium and preserved at −80 • C in 2 mL cryoprotectant vials (Tarson, 523053) by using 20% (v/v) glycerol.

Polymer Hydrolysis and Raw Substrate Degradation
The strains were screened for the hydrolysis of polymer substrates (xylan from beechwood, pectin at 5 g/L; starch and cellulose (α-cellulose; crystalline) at 2 g/L; HiMedia ® ) in their respective growth media (TSBA, TSBAD, MA, SWC, VNSS and R2A) by incubation at 30 • C for 4-7 days followed by visual confirmation of zone clearance. For xylan and pectin, hydrolytic zone was visualized by flooding the plate with 0.1% (w/v) Congo red for 5 min, followed by decanting and washing with 5M NaCl and 1% (v/v) acetic acid. Positive strains for amylase activity (starch hydrolysis) were selected by flooding with gram's iodine (3 g/L iodine + 2 g/L potassium iodide). Cellulose degradation (through discoloration of medium around growth) was studied in cellulose Congo red agar medium (0.5 g/L monopotassium phosphate, 0.25 g/L magnesium sulphate, 2 g/L cellulose, 0.2 g/L congo red, 2 g/L gelatin, and 15 g/L agar at pH 7.2 ± 0.2 [40,41]. The ability of strains to degrade raw substrates, i.e., sugarcane bagasse (SB) was assayed as described earlier [42]. Briefly, SB was procured from the local market and dried at 65-70 • C for 5 days in a hot air oven followed by grinding in mixer grinder (Philip). About 2% (w/v) of the SB was added in the respective agar media before autoclaving and plates incubated for 4 days were observed for halos after flooding with iodine solution (zone enhancer). For quantitative analyses, broth medium was used for determining the reduced sugar using DNS method [43].

Isolation, Identification and Phylogenetic Analysis
For the cultivation of a diverse group of bacterial taxa from all the macroalgal samples, identified as Sargassum polycystum, Padina antilarum, Dictyota sp. (Phaeophyta), Pterocladia musciformis (Rhodophyta), and Ulva sp. (Chlorophyta), a combination of six different media formulations were used which resulted in the isolation of 360 strains based on colony morphology and growth parameters, i.e., the time of the appearance of colonies with the perspective of including slow growers in the collection (Supplementary Table S1 and Figure S1). For the majority of the samples, the highest CFU was reported in MA medium followed by VNSS and SWC, though R2A retrieved the maximum numbers of isolates (91) followed by TSBA (67) and SWC (58) depending on several factors such as the growth of the colonies, cross contamination due to overgrowth/slime production, etc. TSBA100 had minimum CFU for most of the samples with no growth for two of the samples (GAMAL and LLKUN). This might be because of low nutrient and slow growth conditions of bacteria. Among a total of 360, 238 strains (Supplementary Table S2) were prioritized for further identification and enzymatic screening, keeping in perspective the strain details (isolation source, media used and subculture period) and logistics.
(2.5%) were cosmopolitan in distribution and abundant in the majority of the samples (at least three), whereas Gordonia (2.1%), Neobacillus (1.3%), Psychrobacter (1.3%) and Enterobacter sp. (1.7%) were moderately abundant in few of the samples ( Figure 3C).          Geography, sampling sites and host organism shape the abundance and occurrence of epiphytic bacterial communities [3,13,18,[44][45][46][47][48][49][50][51][52]. Several culture-based studies have highlighted that the members of Proteobacteria (Vibrio; class: Gamma-proteobacteria), Actinomyctota (Micrococcus; class: Actinobacteria) and Bacilliota (Bacillus and Staphylococcus) are shared between some groups of algae (Rhodophyta, Phaeophyta and Chlorophyta) [16,53,54]. Among these group of macroalage, Vibrio is abundant in the marine ecosystem and has important role in terms of organic matter mineralization [9], pathogenesis of marine organisms [55] and the protection of macroalgae from antifouling [56,57]. Furthermore, Bacillus and Micrococcus are known to have growth and morphogenetic effects [7] along with antibacterial activity, respectively [58]. Considering geography and climate distribution, the genera Alteromonas, Bacillus, Cobetia, Labrenzia, Microbacterium, Micrococcus, Pseudoalteromonas, Shewanella, Vibrio and Arthrobacter are cosmopolitan both in the tropical and temperate environments in all the major groups of algae (red, green and brown seaweeds) (Supplementary Table S4) [9,16,52,[59][60][61][62]. The genera Pseudoalteromonas and Alteromonas are involved in nutrient cycling processes through their ability to produce polysaccharide degrading enzymes [5,63]. Several Pseudoalteromonas and Alteromonas strains display algicidal activities and play an important role in protecting shellfish farms from toxic dinoflagellate blooms [9,64,65]. Pseudoalteromonas and Shewanella sp. are involved in antibacterial processes, including antifouling, and either stimulate or inhibit the settlement of zoopsores of Ulva sp. through the production of quorum sensing metabolites thus protecting the alga from pathogens, herbivores and fouling organisms and thereby making them an important part of the  Geography, sampling sites and host organism shape the abundance and occurrence of epiphytic bacterial communities [3,13,18,[44][45][46][47][48][49][50][51][52]. Several culture-based studies have highlighted that the members of Proteobacteria (Vibrio; class: Gamma-proteobacteria), Actinomyctota (Micrococcus; class: Actinobacteria) and Bacilliota (Bacillus and Staphylococcus) are shared between some groups of algae (Rhodophyta, Phaeophyta and Chlorophyta) [16,53,54]. Among these group of macroalage, Vibrio is abundant in the marine ecosystem and has important role in terms of organic matter mineralization [9], pathogenesis of marine organisms [55] and the protection of macroalgae from antifouling [56,57]. Furthermore, Bacillus and Micrococcus are known to have growth and morphogenetic effects [7] along with antibacterial activity, respectively [58]. Considering geography and climate distribution, the genera Alteromonas, Bacillus, Cobetia, Labrenzia, Microbacterium, Micrococcus, Pseudoalteromonas, Shewanella, Vibrio and Arthrobacter are cosmopolitan both in the tropical and temperate environments in all the major groups of algae (red, green and brown seaweeds) ( Supplementary Table S4) [9,16,52,[59][60][61][62]. The genera Pseudoalteromonas and Alteromonas are involved in nutrient cycling processes through their ability to produce polysaccharide degrading enzymes [5,63]. Several Pseudoalteromonas and Alteromonas strains display algicidal activities and play an important role in protecting shellfish farms from toxic dinoflagellate blooms [9,64,65]. Pseudoalteromonas and Shewanella sp. are involved in antibacterial processes, including antifouling, and either stimulate or inhibit the settlement of zoopsores of Ulva sp. through the production of quorum sensing metabolites thus protecting the alga from pathogens, herbivores and fouling organisms and thereby making them an important part of the epiphytic community [5,[66][67][68][69]. In fact, Pseudoalteromonas tunicata is a model organism for antifouling and displays activities against algal spores, larvae, diatoms, bacteria, fungi, protists and nematodes [63,[70][71][72]. However, contrary to several previous reports, we could not identify any member of the phylum Bacteriodetes. One of the reasons could be increasing the sequencing depth to cover more strains and collection of fresh algal samples attached to the intertidal rocks, unlike Barbato et al., [61] and Ihua et al., [54] who had used decaying algae as the starting material, different culture conditions and algal species which targeted the isolation of algal polysaccharide degrading bacteria. Interestingly, culture independent analyses of the same samples (unpublished study) identified the classes Gamma-proteobacteria, Bacteroidetes, Alpha-Proteobacteria, Beta-Proteobacteria, Bacilli and Actinobacteria with the families Pseudoalteromonadaceae, Vibrionaceae, Flavobacteriaceae and Bacillaceae as the core community, as supported in other metabarcoding surveys [4,63,64].
Analyses of the culturable diversity identified a total of 20 strains as putative novel taxa at genus and species level indicating that marine macroalgae harbour a vast reservoir of unexplored bacterial diversity. Also as compared to terrestrial isolates, the marine counterparts represent a potential pool of novel metabolic capacities which are yet to be fully explored and exploited for industrial applications [73]. The criteria for selection of novel taxa were based on the assumption that strains sharing low 16S rRNA gene sequence identities (≤98.7%) with valid species names in the EzTaxon server. are potential candidates for novel taxa description based on correlation plot analyses between 16S rRNA gene sequence similarities and corresponding overall genome relatedness indices of GGDC and ANI [74,75]. These potential novel strains were affiliated to the phyla Firmicutes (11), Proteobacteria (7) and Actinobacteria (2) belonging to the families Bacillaceae, Oceanospirulaceae, Microbacteriaceae, Gordoniaceae, Pseudoalteromonadaceae, Micrococcaceae, Vibrionaceae and Lysobacteriaceae. Few strains have already been described as valid species names (SAB 38 T ; Domibacillus epiphyticus sp. nov., SAB 3 T ; Marinomonas epiphytica sp. nov., and CDRSL-15 T ; Luteimonas padinae sp. nov. [37,76,77] and the remaining are undergoing polyphasic taxonomic characterization including phylogenomic analyses to ascertain their exact taxonomic status (Supplementary Table S5 and Figure S2).

Analysis of Polymer Hydrolysis Potential
Algal-associated microbial communities are crucial for algal biomass degradation and mineralization [78,79] and can bio-remediate algal waste material [79]. In further pursuance with an intent to utilize the macroalgal-associated bacterial communities, screening for polymer hydrolysis was undertaken and, among a total of 238 strains, the majority (89%) were positive for hydrolysis of at least a single polymer with the highest activity for xylanase (61.3%) followed by amylase (59.7%) and cellulase (58.8%) (Figures 2 and 5A, B). Approximately 24% of isolates were able to degrade all the screened substrates and among those belonging to Alteromonas, Pseudoalteromonas, Psychrobacter, Shewanella and Vibrio sp. (Gamma-proteobacteria); Brevundimonas, Paracoccus, Roseomonas and Sphingomonas sp.   Table S2) suggesting impact of the isolation source for the induction of enzymatic activity. The highest frequency (n = 58) of polymer degraders were observed in the CDRSL samples, followed by SAB (n = 51), and GAMAL (n = 27). Several researchers have isolated bacteria belonging to the phyla Bacillaeota, Bacteriodetes, Sphingobacteria and Actinomyctota from live and decaying macroalgae and screened for the degradation of algal biomass and associated polysaccharides [18,61,80]. Barbato et al., [61] isolated and tested 634 bacterial isolates from decaying Rhodophyta and Phaeophyta for algal polysaccharide degrading activity wherein approximately 65% strains were capable of degrading at least one algal polysaccharide. Furthermore, Ihua et al., [54] reported that among 800 isolates, 7% of bacteria had polysaccharidase producing activity (cellulase, lichenase and pectinase) and Rodrigues et al., [18] reported 71% bacteria with ulvan lyase, 11.5% carbohydrate sulfatase, 32.3% cellulase and 29% with glucosidase activity. Martin et al., [10] identified association of the genera Maribacter, Algibacter, Celluophaga, Pseudoalteromonas, Vibrio, Cobetia, Shewanella, Marinomonas and Paraglaceciola sp. of the classes Flavobacteria and Gamma-proteobacteria with Ascophylum nodosum along with the degradation of marine polymeric carbon associated with macro-and microphytes. Furthermore, some other studies [13,[15][16][17][52][53][54]81,82] have shown that the phyla Proteobacteria (genera Vibrio, Alteromonas Pseudoalteromonas), Bacillaeota (Bacillus, Alkalihalobacillus), Actinobacteria (Streptomyces) and Bacteroidetes (Algibacter, Zobelia, Maribacter) predominate the marine environment and can degrade agar, alginate, xylan, carrageenan, cellulose and chitin (Supplementary Table S4). Congruent to the above reports, our study retrieved a similar pattern in bacterial diversity profiles with the genera Bacillus and Vibrio sp. as the predominant taxa, except Bacteroidetes, which was entirely absent despite using media conditions (MA and seawater agar) suitable for their isolation. This could be attributed to the fact that reports highlighting their abundance used degrading algal biomass as the starting material (substrates) for isolation, therefore, the sequencing depth (total strains identified) was insufficient to cover the range of strain diversity and culture conditions relative to previous culturable diversity reports [17,75,76]. Interestingly, compared with earlier findings, the frequency of polysaccharide degraders was much higher in our work (Supplementary Figure S6). One of the aims of culture-dependent studies has been to focus on the ability of the isolated marine bacterial communities associated with sediment, water, molluscs, sponge and seaweed, etc., to bio-remediate algal waste material [17,61,79,83]. Imran et al., [82] reported that the multiple polysaccharide (cellulose, chitin, fucoidan, pectin, laminarin, pullulan, xylan, agar, alginate and starch) degraders (Microbulbifer and Sacchrophagus) were capable of decomposing red seaweed thallus. Although most of the reports targeted algal specific sulphated polymers, such as alginate, agar and carrageenan, we screened for hydrolysis of non-sulphated plant polymers (cellulose, starch, pectin and xylan) for testing our hypothesis that algal-associated bacterial communities can be exploited for the bioremediation of agricultural wastes because of similarities in structure of the two types of polymers. Incidentally, we did recover several strains with multiple polysaccharides degrading activities (56% of the total strains screened, Figure 2 and Supplementary Table S2).

Conclusions
In the present studythe macroalgal samples were examined for their bacterial diversity, as well as their polymer degrading potential in the context of their application related to agriculture wastes bioremediation. The highest average CFU and isolates were recovered on MA and R2A medium, respectively. Among all the macroalgal samples, the maximum and minimum diversity with respect to diversity richness and evenness was observed in Dictyota sp. and Sargassum polycystum respectively. The most frequently isolated bacteria (≥5) belonged to Vibrio, Bacillus, Pseudoalteromonas, Staphylococcus, Alteromonas, Kocuria, Micrococcus, Shewanella, Microbacterium, Streptomyces, Brevibacillus and Gordonia sp. in descending order of abundance. The genera Bacillus and Vibrio were shared among all the algal species whereas Brevibacillus and Pseudoalteromonas sp. were cosmopolitan for S. polycsytum (constituting ≥ 50% of the total community) collected from different coastal locations. The genera Micrococcus and Catenococcus were specific to S. polysystum collected from Anjuna beach (LLAB) while Gracibacillus, Alteromonas and Microbacteriun were specific to Kunkeshwar (LLKUN) and Kocuria, Enhydrobacter and Cellulomonas were uniquely observed in S. polysystum collected from the Malwan region (MBA) emphasizing that host specificity and biogeographic conditions play an important role in the selection of microbial community. About 20 novel taxa were identified in our study with three strains published as novel species, i.e., Marinomonas epiphytica sp. nov. Out of the total strains screened, 54% (n=129) were positive for degrading at least three substrates, 24% (n=56) degraded all the four polymers, and the majority (89%; n=212) degraded at least one polymer and species in the genera Alteromonas (4), Cellulomonas (1), Cellulosimicrobium (1), Catenococcus (1), Pseudoalteromonas (6), Shewanella (1), Vibrio (13)

Conclusions
In the present studythe macroalgal samples were examined for their bacterial diversity, as well as their polymer degrading potential in the context of their application related to agriculture wastes bioremediation. The highest average CFU and isolates were recovered on MA and R2A medium, respectively. Among all the macroalgal samples, the maximum and minimum diversity with respect to diversity richness and evenness was observed in Dictyota sp. and Sargassum polycystum respectively. The most frequently isolated bacteria (≥5) belonged to Vibrio, Bacillus, Pseudoalteromonas, Staphylococcus, Alteromonas, Kocuria, Micrococcus, Shewanella, Microbacterium, Streptomyces, Brevibacillus and Gordonia sp. in descending order of abundance. The genera Bacillus and Vibrio were shared among all the algal species whereas Brevibacillus and Pseudoalteromonas sp. were cosmopolitan for S. polycsytum (constituting ≥ 50% of the total community) collected from different coastal locations. The genera Micrococcus and Catenococcus were specific to S. polysystum collected from Anjuna beach (LLAB) while Gracibacillus, Alteromonas and Microbacteriun were specific to Kunkeshwar (LLKUN) and Kocuria, Enhydrobacter and Cellulomonas were uniquely observed in S. polysystum collected from the Malwan region (MBA) emphasizing that host specificity and biogeographic conditions play an important role in the selection of microbial community. About 20 novel taxa were identified in our study with three strains published as novel species, i.e., Marinomonas epiphytica sp. nov. ( Out of the total strains screened, 54% (n = 129) were positive for degrading at least three substrates, 24% (n = 56) degraded all the four polymers, and the majority (89%; n = 212) degraded at least one polymer and species in the genera Alteromonas (4), Cellulomonas (1), Cellulosimicrobium (1), Catenococcus (1), Pseudoalteromonas (6), Shewanella (1), Vibrio (13) (phylum Proteobacteria) Alkalihalobacillus (1), Bacillus (11), Lederbergia (1), Neobacillus (3), Planococcus (1), Planomicrobium (1), Priestia (1), Rossellomorea (1), Staphylococcus (2) [phylum Firmicutes], Kocuria (5), Micrococcus (1), Streptomyces (1) [phylum Actinobacteria] were the most potent degraders. In terms of degradation efficiency and scope, the genera Vibrio and Bacillus seemed best since they could hydrolyze several substrates. However, actinobacterial strains isolated in a lower frequency appeared to contain high potential, since the majority of the strains were able to degrade at least two polymers. From our preliminary screening procedures, we identified nine strains with the capability for degrading raw sugarcane bagasse (without any pre-treatment) in both plate assays and broth medium. The majority of the strains were identified as Bacillus sp. and related genera, phylogenetically clustering in three different lineages. Strains GAMAL-10 SWC (Rossellomorea marisflavi), LLKUN-4 TSBA (Bacillus pseudomycoides), LLAB-2 TSBAD (Neobacillus derentis) and GAMAL-8 SWC (Bacillus infentis) were able to maximally produce reduced sugar from raw sugarcane bagasse (0.1-0.3 g/L) in 24 h. Since these are marine strains with the ability to grow optimally at 35 ppt salinity, desiccation and moderately high temperatures, therefore high potential exists for the application of their enzyme systems as a bio-remediation option for agro-wastes in the form of green technology after process optimization. Further work is required in terms of pre-treatment and the formulation of microbial consortia/strain optimization.  Author Contributions: A.V. and S.K. collected the samples. P.K., A.V., S.S.S. and A.K.O. were involved in strain identification and screening process. Sugarcane bagasse hydrolysis was assayed by P.K. Result interpretation and draft preparation was done by P.K. and A.V. Writing review and editing was performed by P.K., A.V., S.S.S. and S.K. Funding, project administration, and supervision was taken care by S.K. All authors have read and agreed to the published version of the manuscript. had worked in the DST funded GAP0128 project "Study of Diversity of marine macroalgae associated epiphytic bacteria along the Central West Coast of India". Data Availability Statement: 16S rRNA gene sequence could be availed from NCBI server with the help of accession number mentioned in Supplementary Table S2. Other data can be found in the Supplementary File attached.