Materials and Methods

Materials and Methods

Isolation and screening of fermentative yeast

Yeast strains were isolated from various sources, namely fruits and fermented products (Supplementary Table S1). Fruit samples were cut into small pieces and incubated at room temperature overnight and 1ml of the fruit extract was serially diluted (10^-1 to 10^-6 dilutions) and plated on Yeast Extract Peptone Dextrose Agar (YEPDA) which consists of 2% Peptone, 2% Yeast extract, 5% Dextrose and 1.5% agar supplemented with streptomycin 30ug/ml, incubated for 24 h at 30 ^oC. After incubation, yeast colonies on agar were characterized based on size, shape, and pigmentation [32, 33]. Colonies were sub-cultured on YEPDA by streak plate technique and subsequent pure culture maintained on agar slants for further characterization. Screening of ethanologenic wild yeast strains for ethanol production was carried out in two steps (i) First, ethanol fermentation was carried out in Durham fermentation tube in six different sugar: 50 g/L of glucose, galactose, xylose, lactose, maltose and sucrose, 10g/L peptone, 5g/L NaCl and 0.5g/L phenol red and inoculated for 24h at 35^oC. The fermentation activity of yeast strains was confirmed by observing the volume of gas in Durham tube filled with CO₂, based on this positive yeast strains were selected for further studies [27]. Strains producing gas in glucose and xylose media were explicitly selected for the study. (ii) Next, biomass in glucose and xylose media were recorded by inoculating in 20 g/L yeast extract and 20 g/L peptone broth with 50 g/L of glucose and xylose separately and incubated at 35 ^oC on an orbital shaker at 100 rpm for 24h, and biomass was recorded at 600 nm. Fresh YEPD broth was prepared, and yeast organisms sample from the axenic culture was inoculated and incubated at 30 ^oC on an orbital shaker at 100 rpm. At every one hour, interval 5 ml sample was drawn, and the absorbance was measured at 600 nm. This experiment was carried out until the attainment of the stationary phase with the recurring values.

Characterization of yeast strain: Temperature, ethanol, and salt tolerance

The selected yeast strains were inoculated in glucose media at various temperatures from 30 ^oC to 50 ^oC at an interval of 5^oC, ethanol concentration 0-10% and salt concentration 0-14% with 2% interval and incubated for 24 h on an orbital shaker at 100 rpm and the absorbance was measured at 600 nm along with negative control (without yeast strain) and positive control using baker’s yeast (Saccharomyces cerevisiae) [27, 34]. Experiments were carried out in triplicates.

Identification of yeast strain using rDNA sequencing and molecular phylogenetic analysis

Genomic DNA was isolated, the quantity was measured using Nanodrop Spectrophotometer, and the quality was determined using 2% agarose gel. A single band of high-molecular-weight DNA has been observed. 18S rRNA gene was amplified by 18SrRNAF and 18SrRNAR primers. A single discrete PCR amplicon band of 1500 bp was observed when resolved on Agarose gel. The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with ITS1 (5`- TCCGTAGGTGAACCTGCGG-3`) and ITS4 (5`-TCCTCCGCTTATTGATATGC-3`) using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. A consensus sequence of 18S rRNA gene was generated from forward and reverse sequence. 18S rRNA gene sequence was compared to type strains in National Center for Biotechnology Information (NCBI). Based on the maximum identity score first ten sequences were selected and aligned using Clustal W, a phylogenetic tree was constructed using the neighbor-joining method with MEGA version 7.0 with a bootstrap number 1000 [35].

Fermentation

Efficacy of yeast strain to produce ethanol using synthetic sugars

Carbohydrates of Enteromorpha intestinalis and Ulva lactuca mainly composed of glucose and xylose, is the source of carbon for fermentation. Fermentation efficiency of the selected yeast strains was evaluated in a 250 ml Erlenmeyer flask containing 100 ml of 2 g/L yeast extract and 2 g/L peptones with 5 g/L glucose, 5 g/L xylose, glucose, and xylose 5g/L, 5% v/v yeast inoculum in 3 different flasks were subjected to fermentation at 35^oC, pH 4 for 24 h using prioritized yeasts strains in different combinations to determine its efficacy.

Efficacy of yeast strain to produce ethanol using macroalgal hydrolysate

Fermentation was carried out in a 250 ml Erlenmeyer flask containing 150 ml of clear hydrolysate. Macroalgal biomass (5g) Enteromorpha intestinalis and Ulva lactuca were acid pre-treated using 0.7N and 0.5N H₂SO₄ at 121^oC for 45 min to determine the efficacy of isolated yeast strain to ferment seaweed sugars. The acid hydrolysate was obtained and neutralized using Na₂CO₃. It results in lower sugar removal [7] and the fermentation medium adjusted to pH 4 and subjected to SHF using prioritized yeast (5% v/v) strains in different combination at 35^oC, 100rpm for 24h. For SSF, acid pretreated biomass (2g) were subjected to fermentation using an enzyme (5% v/v) extracted from S9 (V. parahaemolyticus) [14] and prioritized yeast (5% v/v) strains in different combinations at 55^oC, 100rpm for 24h. Macroalgal biomass contains abundant carbon sources and essential minerals for yeast growth; fermentation was carried out without exogenous nutrients.

Analytical method and data analysis

Reducing sugar obtained in both the process was estimated before and after fermentation by the DNS method [36]. Theoretically, 1kg of glucose produces 510g of ethanol, i.e., 51%. Theoretical yield is 51% of the fermented sugar by each of the yeast strain [Eq (2)], and fermentation efficiency is the percentage ratio of ethanol yield obtained from the experiment to theoretical yield as indicated in Eq (1)

The ethanol obtained was estimated using GCMS with an FID as a detector. The sample was injected using an Agilent gold standard syringe with an accuracy of ±1%. The analysis was performed under the following conditions: injector volume 1µl, inlet temperature 180 ºC, Mode was split-less, flowrate of1.2 ml/min, the runtime of 24.6 min by ramping method with a temperature of FID at 280 ºC. The gases used were Hydrogen with a flow of 30ml/min, Zero Airflow of 300 ml/min, and Helium flow of 10 ml/min. The identification of ethanol was done by MS at temperature: 230 ºC and Quadrupole temperature: 150 ºC. MS filament was switched on and off at 1.82 min and 2.82 min, respectively, to identify the ethanol ions in the sample, and the ethanol was identified through the NIST database

Agglomerative hierarchical clustering of screened yeast strains was carried out for the ethanol produced from the study and the literature-integrated data using R studio version 3.4.4. Multivariate analysis through Principal Component Analysis (PCA) was carried out using R studio software version 3.4.4, to determine strains responses to temperature, ethanol, and salt tolerance and provide an overview of similarities and differences among the yeast strains.

Screening for cellulolytic yeast and ethanol production by consolidated bioprocess

Prioritized yeast strains were screened for cellulolytic activity by inoculating on a 1% CMC plate supplemented with agar and incubated at 35^oC for 72h. Plates were flooded with Gram’s Iodine. Colonies producing zone of clearance were considered positive for cellulolytic activity. The hydrolytic activity of each strain was determined [14]. Macroalgal biomass was subjected to saccharification and fermentation using cellulolytic yeast strain in the consolidated bioprocess.