The seaweed (E. cottonii) used in this work was obtained from Semporna, Sabah. The materials used were Yeast Peptone Dextrose (YPD) broth, S. cerevisiae pow­der, potato dextrose agar powder, distilled water, ethanol 98%, sodium hydroxide (NaOH), sodium chloride (NaCl), sulphuric acid 99% (H2SO4), phenol, sodium sulphate anhydrous (NaSO4H2O).

The equipments used were gas chromatography GC (Agilent, California), UV — vis spectrometer (Jasco UV-vis 650, Germany), conical flasks (250 mL), graduated cylinder (1 L), beakers (250 mL and 500 mL), blue cap bottles (1 L), pipettes (0.1 mL and 1 mL), pipette tips (0.1 mL and 1 mL), micro-centrifuge tubes 1.5 mL, cotton, loop, aluminium foil, sterile petri plates, balance, pH meter, autoclave (HICLAVE HVE-50, Japan), incubator, oven, fridge (5%), Bunsen burner, perma­nent marker pen, paper tape (for labelling), glass spreader, cuvette, oven, parafilm, filter paper 0.2 |rm, filtration unit, spectrophotometer, laminar flow cabinet, and 0.45 |rm Durapore (PVDF) syringe-driven filter.

Before fermentation of the E. cottonii, the broth media and agar medium was prepared and sterilised. Initially 20 g of potato dextrose powder was weighed and mixed with 1,000 mL distilled water in a sterile blue cap bottle. The pH was adjusted to pH 7 using 1 mol/L NaOH. A clean glass rod was flame sterilised to stir the medium. The nutrient agar did not fully dissolve until it was autoclaved at 121% for 20 min (HICLAVE HVE-50, Japan). Following that, the agar medium was allowed to cool to around 45%. Next, 20 mL of agar was carefully poured out into sterile plastic petri dishes. The YPD (yeast peptone dextrose) broth was mixed in the fol­lowing formula: 10 g yeast extract, 20 g peptone and 20 g dextrose (glucose). Following that, the broth was mixed with H2O to make up 1 L. The pH was adjusted to pH 7 using 1 mol/L NaOH and then autoclaved at 121% for 20 min (HICLAVE HVE-50, Japan). This preparation was conducted in an aseptic environment.

Subsequently, the yeast suspension was prepared. Firstly, 5 g of dry yeast powder was dissolved in 100 mL of warm water at 30%. The solution was incubated in a rotary incubator for 30-60 min prior to use. A portion of the culture suspension was used for inoculation and the remainder was kept as stock in glycerol (2:1 v/v glyc­erol and yeast solution at -82%).

The innoculum was prepared by aseptically transferring the yeast suspension into 100 mL of sterilised medium (YPD) and cultured in a rotary incubator. The incubator was set to 200 rpm and 32%. Yeast population (CFU/mL) was monitored periodically every 3 h for 36 h. The spread plate technique was used to obtain the colony forming units per mL, CFU/mL. After 48 h at 30%, yeast growth was moni­tored by measuring the optical density (OD) at 620 nm (OD620) after every 3 h. A calibration curve was constructed based on the population cell number and OD620 measured.

Next, the fresh substrates (E. cottonii) were prepared by cutting the samples into ~0.5 cm x 0.5 cm. The substrate was then mixed with water in 1:4 volume ratios and further size reduced using a blender for about 10 min, producing finely shredded seaweed slurry. It was stored in a refrigerator at 5% for future use.

Following that, the three types of fermentation media (YPD, YP and distilled water) were prepared. The YPD broth powder was prepared aseptically with the following formula: 1% yeast extract 10 g, 2% peptone 20 g and 2% dextrose (glu­cose) 20 g. This formed 50 g of YPD powder, which was mixed with 1,000 mL of distilled water. The YP broth was prepared using the following formula: 10 g of yeast extract and 20 g of peptone were mixed with 1,000 mL distilled water. The pH of the fermentation media were adjusted to pH 7 using 1 mol/L NaOH. Then 1,000 mL of distilled water was also prepared as addition to the E. cottonii as fer­mentation medium. After that, the media were autoclaved at 121% for 20 min to sterilise the medium.

Then a carbohydrate extraction step was introduced (Table 13.3). Eight samples of seaweed (100 g seaweed each) in slurry form were mixed with 250 mL of dis­tilled water. Seven flasks were heated on a hot plate at 100% for 2 h to extract the polysaccharide (Lin et al. 2000). The last flask was left at room temperature without carbohydrate extraction. Subsequently, aliquots of 2 mL extracted solution were filtered using filter paper, followed by reducing sugar concentration analysis.

After the extraction step, the seaweed slurry was put through acid hydrolysis. Firstly, the extracted seaweed was cooled to 80%. Six flasks were then added with 70% sulphuric acid to a final concentration of 0.4 M. The other two flasks were added with 70% sulphuric acid to a final concentration of 0.1 M. One of these flasks at 0.1 M and five flasks at 0.4 M were heated at 100% for 3 h. The remaining flasks, one at 0.4 M and one at 0.1 M, were incubated at 30% for 3 h. The hydrolysate was neutralised with sodium hydroxide 5 M.

A reducing sugar concentration analysis was conducted to analyse the contents of sugar in samples without carbohydrate extraction, after carbohydrate extraction, without acid hydrolysis and after acid hydrolysis. The reducing sugar concentra­tions were analysed using the phenol-sulphuric acid method. Glucose was used as the standard for this analysis. A reducing sugar standard curve was generated as

follows: firstly, 2 mL of different concentrations of standard glucose solution (0-0.5 mg/mL) were prepared and 1 mL of the phenol reagent was added to each standard solution. Secondly, 5 mL of sulphuric acid was added, and UV absorbance was read at 495 nm using Jasco UV-vis 650. All the samples were then tested with this method for reducing sugar concentration. The concentrations of reducing sugar in the samples were estimated by comparison with the standard curve.

Following acid hydrolysis, the samples were fermented. The fermentation was initiated via inoculation of yeast culture added at 20% v/v of the fermentation vol­ume. The yeast inoculums should be harvested at the exponential phase. Flasks were sealed with parafilm and placed in a rotary incubator at 200 rpm and 30%. The fermentation process lasted for 72 h. Samples for analysis were taken at the end of fermentation.

Subsequently, the fermentation broth was distilled using a distillation unit Buchi K-350. Each distillation batch contained 50 mL of sample. The final volume of distillate was measured and recorded. Next the distillate samples were stored in 5-8% prior to gas chromatography (GC) analysis.

The alcohol content of the distilled samples was estimated using the relationship between specific gravity and percent of alcohol. According to Amerine and Ough (1974), the percentage of ethanol present in a mixture of water and ethanol (distil­late) can be obtained from the specific gravity of the distillate as a function of per­cent ethanol (volume/volume). From the specific gravity of the distillate which contains mainly ethanol and water, the volume percentage of ethanol in the distillate can be determined from Eq. (13.2).

%Ethanol = (7,982.69 x SG2) — (16,602.41 x SG) + 8,619.74. (13.2)

Further analysis using gas chromatography GC (Model 6890N, Agilent Technologies, CA) equipped with thermal conductivity detector and HP-5MS column, 0.25 mm x 30 m x 0.25 pm ID (Agilent, CA) was conducted to verify the presence of ethanol in the sample. Prior to the GC analysis, sodium sulphate anhy­drous (NaSO4H2O) was added to the distilled sample in order to remove the water in it. The NaSO4H2O was added slowly to the sample until there was no solid for­mation upon further addition. Then the samples were syringed through a 0.45 pm Durapore (PVDF) syringe-driven filter unit into 1.5 mL glass vials, sealed with a crimp cap and stored at 5-8% prior to the GC analysis. In order to get a clear and sharp peak for ethanol, trial and error was done to find the suitable setting for GC (Adams et al. 2009). Table 13.4 shows the setting of GC.