Materials and Methods

Case Study: Bioethanol from Enteromorpha intestinalis

The above-mentioned challenges are addressed in this case study, which involves bioethanol production from green macroalgae; Enteromorpha (Ulva) intestinalis belonging to Ulvaceae family. They grow profusely and occupy intertidal zones under favourable nutrient, salinity, light and temperature condition. E. intestinalis is composed of 40.1% total carbohydrate, 20.4% Protein, 2.8% Lipid. Elemental analysis such as carbon 33%, nitrogen 4.36% and hydrogen 6.44% were recorded. Biochemical composition of E. intestinalis is comparable to earlier studies Cho et al., (2013) recorded 42.8% carbohydrate, 31.6% crude protein and 1.3% crude lipid. Bioethanol prospects from E. intestinalis is elucidated in this section (Fig 6).

Fig. 6. Bioethanol production from green macroalgae Enteromorpha intestinalis

Dilute acid hydrolysis of E. intestinalis at 0.7 N H₂SO_4­, 5% substrate concentration, 121^oC for 45 min produced 239.94±1.3 mg/g of reducing sugar. Enzyme extracted from marine bacteria Vibrio parahaemolyticus (Hebbale et al., 2019). Pretreated biomass of E. intestinalis subjected to enzyme saccharification at pH 6, 50^oC for 24 and obtained reducing sugar of 289.89±2.4 mg/g of reducing sugar. Acid pretreated macroalgal biomass was subjected to enzyme hydrolysis using enzyme and incubated for 24 h, 1.2-fold increase in reducing sugar was observed in E. intestinalis when compared to dilute acid pretreatment. Scanning electron micrographs of hydrolysed biomass indicates that the dilute acid pretreatment prior to enzyme saccharification is a prerequisite as it loosens the rugged surface of the biomass increasing the surface area and exposing more of internal cellulose as seen in Fig 7.

Fig 7. Scanning electron micrograph of E.intestinalis illustrating ultrastructural variations in the feedstock after pretreatment and saccharification.

Acid hydrolysate obtained subjected to SHF using Pichia kudriavzevii yeast strain isolated from Toddy juice at 35^oC, 100 rpm for 24 h. Ethanol of 0.16 g with 51.8% efficiency obtained. Pretreated biomass subjected to SSF process using enzyme from Vibrio parahaemolyticus and Pichia kudriavzevii yeast at 55^oC,100 rpm for 24 h. Ethanol of 0.10 g obtained with 65.1% efficiency. SSF exhibited higher efficiency than SHF process.

Mass-energy balance was carried for analysing ethanol production from P. kudriavzevii (TY) and the sugars obtained from both SHF and SSF processes. Results obtained were extrapolated to 1kg to make the study more comprehensive. Fermentation of E. intestinalis in SHF process, obtained 23.9 g/kg (30.4 ml/kg) ethanol with 55.9% conversion efficiency whereas in SSF process 28.9 g/kg (35.8 ml/kg) ethanol with 83.9% conversion efficiency was obtained (Table 6). Ethanol from SSF was estimated 1.18-fold higher than the ethanol obtained from SHF process indicating better efficiency. Similar results were obtained for fermentation of E. intestinalis using S. cerevisiae. SSF process achieved 30.5% efficiency when compared to SHF (26.9%) process indicating better performance regarding fermentation yield and faster process. Similar mass-energy balance study was reported with the various feedstock, 1kg of Saccharina japonica biomass yielded 23.1g (29.2 ml) of ethanol using SSF process achieving conversion efficiency of 67.41% (Lee et al., 2013). Fermentation of 1kg G, verrucosa produced 38g (48.1 ml) of ethanol from SSF process achieving fermentation efficiency of 86% (Kumar et al., 2013). Acid pretreatment of 1kg K. alvarezii followed by detoxification produced 80g of galactose which was fermented (SSF) to produce 43.7g (55.3 ml) of ethanol achieving 78.5% conversion efficiency (Hargreaves et al., 2013). Fermentation of 1kg of switchgrass (second generation feedstock) produced 178.4 g (226.1 ml) of ethanol using SHF process, while SSF process produced 183.5g (232.5 ml) of ethanol, but achieved lower conversion efficiency which is attributed to the presence of insoluble lignin in the biomass, that was treated using Ammonium Fibre Expansion (AFEX) (Jin et al., 2010). Higher ethanol in SSF process is due to the rapid consumption of glucose by yeast as they were produced during enzyme hydrolysis (Xiao et al., 2004). Acid hydrolysis of 1 kg of Lantana camara followed by delignification, enzymatic hydrolysis of the biomass and fermentation yielded 148.14 g (187.7 ml) of ethanol, whereas, fermentation of pentose rich hydrolysate yielded 51.6 g (65.3 ml) of ethanol (Kuhad et al., 2010). Bagasse pith (1 kg) (second-generation feedstock) produced 46.2 g (58.5 ml) and 66.4 g (84.1 ml) of ethanol by SHF and SSF process respectively. In this study, commercial enzyme cellulase and β-glucosidase were employed for enzyme hydrolysis and fermentation was carried using P. stipitis JCM 10742 (Sritrakul et al., 2017). Notable advantages were observed from SSF over SHF process, as SSF process is amenable to enzyme hydrolysis with the rapid ethanol production and occurrs in a single reactor, thereby reducing the operation and investment costs for setting up a biorefinery.

Ethanol production from macroalgal biomass results in large quantity of spent biomass or waste products that are generally disposed. High value products are created from these wastes through the concept of biorefinery which aims to achieve no waste flow resulting in economic and environmental benefits (Balina et al., 2017).

Economic Prospects of Macroalgae- Biorefinery

Seaweeds were mostly restricted to domestic purposes such as food and feed, preparation of industrial gels, medicinal uses such as Laminaria sp. for dilation of cervix in difficult childbirth, Gelidium sp. used for intestinal afflictions etc. In recent times, macroalgal biomass are cultivated on largescale for production of more valuable commodity than food and feeds. These include, extraction of polysaccharides for agronomic applications, cosmeceuticals, nutraceuticals, pharmaceuticals, and bioenergy. Seaweed biorefinery approach extracts most valuable components from the macroalgal biomass without altering the residue for commodity purposes such as food, feed, fertilizers etc.(Balina et al., 2017; Buschmann et al., 2017).

Macroalgae is subjected to dilute-acid pretreatment and pretreated biomass is hydrolysed using enzyme. Enzyme hydrolysate is fermented to produce ethanol. Solid/liquid separation is carried out for the fermentation broth. The liquid fractions are rich in lipid, minerals and other unutilized sugars and are used as liquid fertilizers, which substitutes the conventional mineral fertilizers. The solid fraction is spray dried and rich with protein and minerals and is being used as fish feed, which serves as a substitute for soy protein (Fig 7). Selection of appropriate macroalgal feedstock accumulating higher carbohydrate fractions and nutrients can lower the CO₂ level and provide climate change and marine eutrophication mitigation services (Seghetta et al., 2016).

Fig 7. Seaweed biorefinery with probable constituents

Fatty acids content in the dried and canned macroalgae are of linear structures and are major sources of essential fatty acids such as palmitic acid and ω-3, 6 and 9. Agar from G. edulis, G. acerosa and Gracilaria sp. are extracted by boiling the seaweed and extract is filtered, freeze thawed and dried in sun and market as powder (Kaladharan and Kaliaperumal, 1999). Macrocystis pyrifera was harvested for production of acetone and potash (Roesijadi et al., 2010). Macroalgal biomass is composed of high amount of water-soluble potash, which is readily absorbed by the plants. Composting of seaweed along with shark liver sediments and fish offal (15:4:3 by weight) fetches high manure value with 2.4% N, 0.7% P and 3.5 % potash (Chennubhotla et al., 1981). Macroalgal biomass are regarded as ‘super foods’ for being rich in vitamins B12, A and iodine. Seaweed meal incorporated in poultry and animal feed increased the iodine content of the eggs and milk production in dairy cows (Hebbale et al., 2017; Holdt and Kraan, 2011; Torres et al., 2019). Discarded waste of algin extracted macroalgal biomass is estimated to contain 93-94% of iodine (Torres et al., 2019). Extracted protein fraction from Ulva increase illeal digestibility and rumen fermentation (Baeyens et al., 2015; Bikker et al., 2016). Apart from whole seaweed, residue obtained from industries; floating residues, spent biomass serves as feedstock for bioethanol production (Sudhakar et al., 2016). Therefore biorefinery approach is sustainable and environmentally friendly as it reduces the burden on environment.

Fatty acids content in the dried and canned macroalgae are of linear structures and are major sources of essential fatty acids such as palmitic acid and ω-3, 6 and 9. Agar from G. edulis, G. acerosa and Gracilaria sp. are extracted by boiling the seaweed and extract is filtered, freeze thawed and dried in sun and market as powder (Kaladharan and Kaliaperumal, 1999). Macrocystis pyrifera was harvested for production of acetone and potash (Roesijadi et al., 2010). Macroalgal biomass is composed of high amount of water-soluble potash, which is readily absorbed by the plants. Composting of seaweed along with shark liver sediments and fish offal (15:4:3 by weight) fetches high manure value with 2.4% N, 0.7% P and 3.5 % potash (Chennubhotla et al., 1981). Macroalgal biomass are regarded as ‘super foods’ for being rich in vitamins B12, A and iodine. Seaweed meal incorporated in poultry and animal feed increased the iodine content of the eggs and milk production in dairy cows (Hebbale et al., 2017; Holdt and Kraan, 2011; Torres et al., 2019). Discarded waste of algin extracted macroalgal biomass is estimated to contain 93-94% of iodine (Torres et al., 2019). Extracted protein fraction from Ulva increase illeal digestibility and rumen fermentation (Baeyens et al., 2015; Bikker et al., 2016). Apart from whole seaweed, residue obtained from industries; floating residues, spent biomass serves as feedstock for bioethanol production (Sudhakar et al., 2016). Therefore biorefinery approach is sustainable and environmentally friendly as it reduces the burden on environment.

Scope for further research

Marine macroalgae have been explored worldwide for various application owing to their ability to accumulate large concentration of biomolecules (especially carbohydrates), which serve as raw material for bioethanol production and other value-added products. Bioprocess of bioethanol production involves three major steps; dilute acid pretreatment, enzyme saccharification and fermentation. The major future prospective for bioethanol production from macroalgal biomass includes (i) exploring enzymes having higher catalytic activity and stability at extreme conditions (ii) yeast microorganisms able to ferment broad range of sugars (iii) improved ethanol yield by process optimization (iv) Consolidated bioprocess involving cellulolytic yeast to hydrolyse cellulose as well as fermented subsequent glucose released during hydrolysis to ethanol. Biorefinery approach can be realised only with sufficient quantities of biomass. Largescale cultivation of macroalgae in open ocean is results is disease outbreaks and destruction of habitat (killing endemic corals) (Bindu & Levine, 2011; Patterson et al., 2008). In order to overcome this, Integrated Multi-Trophic Aquaculture (IMTA) (Fig. 8) concept is introduced, which farming macroalgae in close proximity to other species at different trophic level on land. Land-based seaweed cultivation with adaptation to much wider range of macroalgal genera offers raw materials for higher-value product development. Intertidal species like Ulva sp. and Enteromorpha sp. have high tolerance to temperature and irradiance ranges, which can be cultivated in IMTA system. Cultivation of seaweeds for biofuel productions needs to be encouraged to meet the future fuel demand as seaweeds have high potential as feedstock for biofuel production as part of the Nation’s strategic energy security program. This would also empower rural women with job opportunities. Development of seaweed-based industries at decentralized levels along coastal areas, where resources are abundantly available would enhance the job opportunities for the rural youth. Seaweed cultivation as a notable future enterprise can open up platform for establishing seed hatcheries, seeding units and processing units and enhance employment opportunities in rural coastal area

Fig. 8. Integrated Multi-Trophic Aquaculture Model