Materials and Methods

1. 1. Sample collection

E. intestinalis and U.lactuca seaweed samples were collected from Aghanashini estuary, Kumta taluk, Uttara Kannada district, Karnataka. Samples were thoroughly cleaned of epiphytes, then shade dried and powdered to obtain uniform size, and stored in polythene sachets for further analysis.

• 1. Dilute Acid hydrolysis

Pretreatment of macroalgal feedstock was carried out at different acid concentrations, substrate concentrations, reaction time, and temperatures. Initially, acid hydrolysis was carried out using H₂SO₄ and HCl with concentrations ranging from 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, and 1N and keeping other parameters constant. The concentration of acid required for optimal sugar production was assessed, and further optimization was carried out. The reaction temperature for pretreatment was carried out with 2% w/v substrate at 30, 60, 90, and 120^oC for 45 min. Reaction time pretreatment with 2% w/v substrate concentration was carried out for 30, 60, 90,105- and 120-min. Pretreatment for algal biomass at different substrate concentrations of 1, 2%, 3%, 5%, 7%, and 9% w/v was carried out at 120 ^oC for 45 min. After hydrolysis, residues were separated by filtration, and total sugar, and reducing sugar were determined by phenol sulfuric acid [40] and Dintirosalicylic acid (DNS) [41] methods, respectively. DNS method is widely used to determine reducing sugar content in fields of food, bioprocess, medicine, etc. [42, 43]. Neutralization was carried out for acid hydrolysate using Na₂CO₃, NaOH, activated charcoal, and Ca (OH)₂ [44, 45]_. The significance of the above factors in influencing sugar release was determined using ANOVA. Thin-layer chromatography analysis of algal hydrolysate obtained following optimized acid hydrolysis on silica gel plates using mobile phase butanol/ethanol/water (3:2:1 v/v/v). Later, these plates were dried at room temperature and dipped in AgNO₃ solution for 1 min, and when dried, plates were sprayed with ethanolic sodium hydroxide solution until dark brown spots appeared [46].

The efficiency of acid hydrolysis pretreatment (%) is calculated using equation 1.

E_p – Efficiency of acid hydrolysis pretreatment (%)

ΔS= disaccharide increase (mg) during acid hydrolysis pretreatment

TS= Total sugar (mg)

• 1. Response surface method

Response surface method (RSM) was used to evaluate the relationship between independent variables: reaction temperature (^oC, X1), reaction time (min, X2) and substrate concentration (% w/v, X3), and dependent variable: reducing sugar (mg g^-1, Y). The experimental data was analyzed and the probable relationship follows the second-order polynomial equation 2.

Y=βo+β₁ X₁+β₂ X₂+β₃ X₃+β₁₁ X₁²+β₂₂ X₂²+β₃₃ X₃²………… 2

Where Y is a response variable and X1, X2, X3 are independent variables, β₀ is the offset term; β₁, β_2, and β₃ are the linear coefficients as per least squares method; β₁₁, β_22, and β₃₃ are the first, second, and third linear coefficients, respectively [26]. The Student’s t-test was performed for the determination of the statistical significance of the regression coefficient [35, 47].

Crude enzyme production from Vibrio paraheamolyticus

Crude enzyme is extracted from Vibrio parahaemolyticus [48] using CMC as a sole source of carbon for screening cellulose-degrading bacteria. Endoglucanase was determined using the carboxymethyl cellulase method (CMCase)[49]; the endoglucanase enzyme cleaves the intermolecular β-1-4-glycosidic bonds present in cellulose. Cellulase was purified by centrifuging bacterial culture at 12000 rpm for 15 min at 4°C and supernatant was collected. Proteins were precipitated to 80% saturation with (NH₄)₂SO₄ at 4°C and pelletized through centrifugation. Pellet was dissolved in Tris-HCl (pH 7) and purified using Ion exchange chromatography, wherein the sample was applied to Superdex 200 column equilibrated with Tris HCl. Fractions were collected, and fractions with the highest enzyme activity were pooled and considered for other characterization. Enzymatic activity refers to the amount of enzyme that releases 1µmol of reducing sugar per minute.

Enzyme characterization

The effect of pH on the enzyme activities was estimated using a buffer of different concentrations; 50 mM citrate buffer (pH 3-4), citrate phosphate buffer (pH 5-6), Tris-HCl (pH 7), and Potassium phosphate buffer (pH 8). The effect of temperature on enzyme activity was determined by incubating the enzyme assay mixture of optimum pH at different temperatures ranging from 25 to 60 ^oC. Effect of salinity on enzyme assay mixture and salinity (NaCl) ranging from 4% to 20% was determined. Samples were incubated for 1 h with CMC as a sole source of carbon.

Efficient sugar release from macroalgal biomass through pretreatment - dilute acid hydrolysis and enzyme saccharification

Macroalgal feedstock Enteromorpha intestinalis (EI) and Ulva lactuca (UL) samples were subjected to dilute acid hydrolysis using an optimized acid concentration of 0.7N and 0.5N H2SO4 at optimal temperature and time of 121^oC and 45 min. Dilute acid pretreated macroalgal biomass EI and UL was subjected to Enzymatic hydrolysis at 55 ^oC pH 6 for 36 h. The reducing sugar released from the above processes was recovered through a centrifuge and was estimated using DNS method [41]_.

SEM analysis

Macroalgal biomass surface morphology (untreated, acid-treated, and enzyme-treated biomass) was qualitatively analyzed using SEM (JEOL-IT 300). Macroalgal samples were placed on an aluminum specimen mount using conductive carbon tape. Sputter gold coating was performed to prevent charging. Samples were then examined in SEM under vacuum condition at accelerating voltage of 10 kV.