Materials and Methods

2.1. Macroalgal resource assessment and screening of potential feedstock for biofuel production

Macroalgal sampling has been done monthly during low tide period. Due to the immersion and emersion periods in rocky shore, a 0.25 m² random quadrats sampling was carried out. Algal biomass within the quadrat were handpicked, species-wise sorted and washed in seawater to remove adhering epiphytes. Collected samples were identified using standard identification keys [61,136] and standing biomass was determined, which is expressed in g/m². In areas, where seaweeds were not accessible, only qualitative data such as the presence of seaweed species were recorded with location details using high spatial resolution virtual remote sensing data (Google Earth).

• 1. Biochemical composition along Central West Coast of India

Species samples were washed thoroughly in seawater, followed by tap water to clean debris of extraneous material. Cleaned seaweeds were sun dried in the shade for 2-3 days, followed by drying in the hot air oven, below 60^oC till constant weight is attained. The dried material was pulverized and sieved to obtain a uniform particle size. The powder was preserved in a zip lock pouches for subsequent analysis of total carbohydrate [137], cellulose [138], protein [139], and lipid [140] contents. Carbon, hydrogen and nitrogen were estimated using CHN elemental analyser. These samples were analyzed in triplicates and the mean values were considered for further calculations and results are expressed in mean±SD.

2.3. Macroalgal pretreatment process

Sugar from macroalgae was extracted using boiling water, sonication, dilute acid and alkali pretreatment. Seaweed was boiled in a 10 ml water for 30 min, sonication was carried out using an ultrasonic bath (frequency 35 kHz) for 30 min. Dilute acid pretreatment involved 0.7 N H₂SO₄ and alkali pretreatment was carried out using 5N NaOH for 30 min. Neutralization was carried out for U.lactuca and E.intestinalis acid hydrolysate using Na₂CO₃, NaOH, Activated charcoal and Ca(OH)_2. The sugar removal (%) by these agents was calculated as per equation (Eq.1).

% Sugar removal = ((A - B)/A ) * 100 Eq1

Where, A is the amount of reducing sugar (mg/g) before treatment, and B is the amount of reducing sugar (mg/g) after treatment [141].

2.4. Enzyme hydrolysis

Enzyme was isolated from marine bacteria Vibrio parahaemolyticus [90] and hydrolysis was carried out for direct biomass and acid pretreated biomass for U.lactuca and E.intestinalis to demonstrate the effect of acid pretreatment in enzyme hydrolysis. Enzyme hydrolysis was carried out at 55 ^oC up to 48 h and reducing sugar was estimated every 6 h.

2.5. Bioethanol production from selected macroalgae

Dried biomass (5% w/v) was pretreated using dilute (0.7N) H₂SO₄ to release sugars and estimated for reducing sugars by 3, 5 dinitrosalicylic acid method [142]. Acid hydrolysate neutralized using Na₂CO_3. Released fermentable sugars were subjected to fermentation using cashew yeast (CY) and toddy yeast (TY) at 30^oC for 18 h in Separate hydrolysis and fermentation (SHF). Saccharification and Fermentation (SSF) was carried out using 2% (w/v) pretreated biomass and 6% (v/v) enzyme and yeast were added to the medium and fermented using CY and TY at 55^oC for 18h. Co-fermentation of algal hydrolysate was also carried out for SHF and SSF. Ethanol was estimated using GC-FID. Bioethanol production from all the macroalgal biomass was estimated based on published literatures and the probable relationship of ethanol yield and multivariate is determined through regression analyses.