Algae and growth conditions
Marine microalga Nannochloropsis oculata (NNO-1 UTEX Culture LB 2164) belonging to Chrysophyta were obtained from Algal Biotechnology Unit, National Research Centre. Cultures were grown under conditions of F2 (Guillard 1975). According to the previously described structure (El- Sayed et al. 2020), a zigzag-shaped photobioreactor with a capacity of 1000 L was used to cultivate N. oculata grown on mixotrophic nutrition way by integrating both autotrophic and heterotrophic-machineries by absorbing provided organic substrates carbon source. Prior to cultivation, the unit was sterilized by hypochlorite solution before inoculation overnight, then washed by water till cleaning and then filled by 1000 L tap water containing 25% F2 artificial growth medium, 10% sugarcane bagasse aqueous extract (SBAE) filtrate, and enriched by commercial fertilizer compounds as described by El-Sayed et al. (2020). Night illumination was provided by the upper surface fluorescent lamp (6 lamps × 40 watts) along with the growth surface. When the alga reached its maximum growth dry weight (g/l), stress was induced with 10 Kg NaCl (1%), and samples were collected continuously from the bioreactor till the end of the experiments. Control treatment of N. oculata was grown by the autotrophic way of nutrition on the F2 medium.
Lipid-extracted alga biomass (LEAB)
Aeration of the culture was switched off at the end of the incubation cycle to allow algal cells to settle. The transparent supernatant was removed and concentrated the algal slurry by centrifugation using Thermo laboratory centrifuge (4,000 rpm/1800g). The lipids were extracted by using a hexane/isopropanol mixture (3:2) after drying the biomass. The extracted biomass was removed using the Soxhlet method, and the residual hexane was evaporated by bubbling with either nitrogen or carbon dioxide gas in a downflow mode. The procedure was repeated until no odor of hexane was detected. The LEAB was collected as dry biomass and held at 4°C until it was used.
Acid hydrolysis and preparation of defatted biomass hydrolysates
Single-stage acid hydrolysis was accomplished in 250 mL conical flasks with (1%, 2%, 3%, 4%, and 5%) of 98 percent H2SO4 for 15 minutes at 121°C. The solid/liquid ratio of defatted biomass content was set at 10% (w/v). As a control, no H2SO4 was used in the previous conditions, only distilled water (Miranda et al. 2012). The liquid fraction was isolated after hydrolysis by centrifugation at 14000 rpm (26342 RCF with radius of rotor (120mm)) for 15 minutes. The supernatant was neutralized to pH 7 with 5.0 M NaOH. The neutralized hydrolysates were then used to determine total carbohydrates and reducing sugars before being stored at 5–7oC until used to produce ethanol. All experiments were three times replicated.
Enzyme hydrolysis and preparation of defatted biomass hydrolysates
Under different conditions, the liquefaction process with diluted sulfuric acid was used for pretreatment of Nannochloropsis oculata biomass. In 250 mL flasks, 10g of defatted dry powder biomass were mixed in 1:10 (w/v) ratios with 0.0, 1.0, 2.0, and 3.0 percent H2SO4 solutions. The flasks were autoclaved for 15 minutes at 121°C.The autoclaved solutions were cooled, neutralized with 5 M NaOH, and subjected to commercially available hydrolytic enzymes α-amylase 1000U at 95oC with a pH of 6.0 for 6 hours, while the saccharification process was carried out with a mixture of commercially available enzyme mixtures from Trichoderma reesei ATCC 26921 and Trichoderma longibrachiatum C 9748 containing multiple enzyme activities mainly ; exoglucanase (1.6 FPU/mL), endoglucanase (33.3 U/mL), cellobiohydrolase (30.02 U/mL), xylanase (21.0 U/mL), and β-glucosidase content (12.4 U/mL).The hydrolysis was carried out by incubating the mixture in a water bath at 60 0C, pH 5.5, for 24 hours (pH was maintained using 0.05M citrate buffer). The residual materials were separated by centrifugation at 14,000 rpm for 15min. For complete free sugar analysis, the resulting hydrolysate has been used and then cooled up to 5-7oC until used for ethanol production (Mirsiaghi and Reardon 2015). All tests have been conducted three times. The commercial enzymes used in this study were procured from Sigma-Aldrich.
Ethanol production
Inoculum of Saccharomyces cerevisiae (NRRLY-2034) was obtained from department of Microbiology, Soils, Water and Environment Research Institute, Agriculture research center-Giza, Egypt. It was cultured on yeast extract peptone dextrose agar (YPD) and incubated at 30oC for 48 hours. After 24 hours of incubation at 30 °C and 150 rpm, a single colony was transferred into 50 mL YPD broth (20.0 g/L dextrose, 10.0 g/L yeast extract, and 20.0 g/L peptone). The cell growth in the inoculum was 5 X 107 cells/mL. It was used to inoculate the fermentation medium. The medium used for ethanol fermentation was composed of (%) 0.25 Yeast extract, 0.25 (NH4)2SO4, 0.1 KH2PO4, and 0.05 MgSO4.7H2O, which were added to 100 mL of the filtrate from saccharified biomasses and sterilized at 121°C for 15 min. The medium was cooled at room temperature after sterilization and then Saccharomyces cerevisiae was inoculated at 2.0 % V/V. For the first four days of fermentation, it was incubated under static conditions at 30°C. The produced ethanol was determined and the ethanol yield was calculated using the formula stated in Yoswathana et al. (2010). At the start and end of the 12-hour fermentation cycle, samples were taken for glucose and ethanol analysis. All the measurements were duplicated and the data reported are average of two replications. The following equation was used to calculate ethanol yield.:
Ethanol Yield = Measured Ethanol in the sample (g) / Theoretical Ethanol (g)
where theoretical ethanol (g) = Amount of initial sugar content (g) in fermentation solution X 0.5
Analytical procedures
The measurement of the total carbohydrate was performed by the method described by Dubois et al. (1956). Estimation for the amount of reducing sugars was carried out by the dinitro salicylic acid (DNS) method using glucose as standard (Miller 1959). The monosaccharides and hydroxymethylfurfural (HMF) in the hydrolysates were analyzed using high performance liquid chromatography (HPLC, Agilent, USA). The chromatographic conditions were waters amino chromatographic column with refractive index detector, elution influent. The mobile phase was deionized water that had been degassed in an ultrasonic bath under vacuum. At a temperature of 40oC and an injection volume of 10L, the flow rate was 0.7 mL/min. The repeatability relative standard deviation of this method is below 5% and the recovery is over 97%. A satisfactory result is obtained under these optimum conditions, and the whole separation can be finished within 25 min. The quantification was achieved by comparison with analytical curves using glucose, fructose, sucrose, fucose, ribose, arabinose, xylose, and mannose standards. HMF was determined using a diode-array detector and an ODS analytical column with detection at 280 nm. A filtered and degassed mobile phase was prepared at a flow rate of 1 mL/min with a sodium acetate buffer (0.08 M) and adjusted to pH 3.6 with glacial acetic acid. Gas Chromatography was used to determine the concentration of bioethanol. Purification by distillation is very low when the retention time of impurities is considered (below the boiling point of ethanol). The fermentation efficiency (FE %) was determined by dividing the average generated ethanol by the theoretically produced ethanol in the biochemical conversion of the sugars consumed.
Statistical analysis
Statistical analysis was done by ANOVA test using the Microsoft Excel program. The difference in values was indicated in the form of probability (p < 0.05) values.