Introduction

Major commercial energy sources such as oil, coal and natural gas are extracted from fossil fuels. Burning of fossil fuel results in the escalation of CO₂ in the atmosphere- a major cause for global warming, price volatility, air pollution and environmental degradation (Adenle et al., 2013; Naik et al., 2010). Surging demand in these sectors has led to increase in oil production from the finite source of fuel reserves. Continuous exploitation is depleting these reserves at staggering speed and will no longer suffice the world’s energy demand (del Río et al., 2020; Goli et al., 2016; Hirsch et al., 2005; Raheem et al., 2018) leading to global energy crisis. Hence, fossil fuels are regarded as unsustainable and questionable from economic, ecology and environmental point of view (Naik et al., 2010). Therefore the quest for an economical, renewable, sustainable and environmentally benign source of energy is underway (Hahn-Hägerdal et al., 2006; Tripathi et al., 2016). Biomass energy in the form of cow dung cake, firewood, agriculture residue and other natural feedstock for cooking and heating have been prevailing from ages which contributes to 80% of rural energy in developing countries like India (Kumar et al., 2015; Ramachandra, 2010 , 2000, 2004). Biofuels from biomass such as plants, algae or organic waste are emerging as promising alternative renewable energy sources to liquid fuels ((Jambo et al., 2016). Different technologies have evolved towards the conversion of biomass into fuels and other value added products that have the advantage of mitigating global warming by cutting down the carbon dioxide emission, as CO₂ is fixed by the biomass via photosynthesis, making it a carbon neutral emission (del Río et al., 2020) and also ease the dependency on oil reserve (Bhattacharyya, 2006; Kumar et al., 2015; McKendry, 2002; Naik et al., 2010). Biofuels are of two types bioethanol and biodiesel; bioethanol are produced from carbohydrate rich algal biomass (e.g. macroalgae) whereas, biodiesel is produced from lipid rich algal biomass (e.g. microalgae). Dependence on fossil fuels (gasoline) in transport sector can be reduced by bioethanol, as it is effective in replacing or blending with gasoline. Development and commercialization of bioethanol is largely achievable due to the availability of feedstock in large quantum (Jambo et al., 2016). Bioethanol feedstocks are categorised into first, second and third generation based on the feedstock’s carbon source. Bioethanol from first generation feedstock (1G) involved food crops like corn and sugarcane, which encountered resistance due to the arable land, freshwater source for its cultivation and competition with food crops (Naik et al., 2010). The lacunae of 1G bioethanol in supplementing the growing energy demand led to the exploration of alternate feedstock involving agricultural residues and woody biomass rich in lignocellulose (second generation (2G) bioethanol feedstock). However, 1G and 2G bioethanol production failed, due to process technology involving the cost-intensive delignification process and difficulty in scaling up (Zhu and Pan, 2010). Bioethanol potential from 1G and 2G feedstock marginally complies with various other sustainability criteria’s such as; conversion of ecologically vulnerable wetlands, extensive usage of fertilizers, soil erosion, rainforests, peat lands, savannas into energy crop lands, disruption of global food supply contributing to several magnitude of CO₂ (Gasparatos et al., 2013; Maeda et al., 2015). Bioethanol production from third generation feedstock (3G) involves algal biomass that are grown in fresh water, wastewater (Ramachandra et al., 2013) and marine waters with zero nutrient input and more importantly, non-interference with the lands required for food production (Demirbas, 2008; Odum and Heald, 1972). At present, the research focus is currently on bioethanol production from 3G feedstock due to higher photosynthetic efficiency (6-8%), productivity (~13.1 kg dry weight/m² over 7 months), ease of cultivation, low consumption of fertilizers, no alteration with food supply, and high absorption of CO₂ (8-10 tonne CO­₂ per hectare) (Kraan, 2013)_, potential to obtain high value added products (pigments, cosmetics, food additives etc.). Algal biomass has emerged as one of the ideal feedstock for achieving sustainable biorefinery having immense potential for commercialization (Jambo et al., 2016).

Fig.1. Bioethanol production process from biomass

Bioethanol production from algal biomass

Production of bioethanol from algal biomass involves three steps; pretreatment, saccharification and fermentation which are discussed in detail in the subsequent sections. Algae are of two types - micro and macroalgae. Microalgae are explored for production of biodiesel (Ramachandra et al., 2009; Saranya et al., 2018), whereas macroalgae rich in carbohydrate are suitable for production of bioethanol (Borines et al., 2013; John et al., 2011; Ramachandra & Hebbale, 2016; Ramachandra & Hebbale, 2020; Roesijadi et al., 2010; Wei et al., 2013; Yanagisawa et al., 2013). Macroalgae (commonly known as seaweeds) are multicellular, photosynthetic algae growing in marine environment and lesser extent in brackish waters. Photosynthetic pigments in seaweeds imparts characteristic range of colors such as red (Rhodophyta), green (Chlorophyta) and brown (Phaeophyta) algae (Abbott et al., 1992; Smith, 1938; Van Den Hoek, 1984). Green seaweeds are euryhaline i.e. tolerating a wide range of salinity whereas, red and brown seaweeds are strictly marine dwelling. Seaweeds have a wide range of distribution from tropics, temperate, polar, tidal pools, estuaries, deep waters and rocky shores whereas, brown seaweed species belonging to order Laminariales occur mostly in temperate regions (<24^oC) (Abbott et al., 1992). Seaweeds grow by attaching to a substrate (natural or artificial); for need of stable anchorage large seaweed beds are restricted to rocky substrate (Abbott et al., 1992; Speight and Henderson, 2013). Macroalgal tissues lack specialized translocatory systems and structure of the “higher plants” (Abbott et al., 1992). Macroalgal body is rootless, stemless and leafless entity called thallus, although many have superficially leaf-like blades, stem-like stipes and often having attaching organs called holdfast or haptera (Lobban et al., 1994; Smith, 1938). Most algae lack these structures owing to their morphological adaptations and modifications (Abbott et al., 1992). Seaweeds reproduce either asexually or sexually (Lobban et al., 1994). Asexual reproduction is a common mode of reproduction in seaweeds.

Fig.2. Macroalgal biochemical composition profile

Generally, the range of biochemical composition of seaweeds are; carbohydrate: 25-77% dry weight, proteins: 5-43% dry weight, lipid: 1-5% and ash content: 9-50% dry weight followed by higher water content of 70-90% fresh weight (Jung et al., 2013; Praveen et al., 2019). Seaweeds consist of varied profiles of structural and storage carbohydrate (Daroch et al., 2013; Kostas et al., 2016) based on the respective intercellular spaces and cell wall (Pereira and Neto, 2014) (Fig 2).

Seaweed polysaccharides show a range of structures and fulfil a variety of functions similar to neutral sugars and sugar acids of terrestrial plants. Certain seaweeds also contain acidic half ester sulphated groups attached to hydroxyl groups of sugars. Hexose sugars such as glucose, galactose and mannose found in these polysaccharides have identical chemical composition. Carbohydrate reserves of red algae are usually stored in the form of small grains that lie in the cytoplasm outside the algal plastids, the chromatophores. The insoluble carbohydrate reserve of red algae has been called floridean starch (intermediate between true starch and dextrin) (Yu et al., 2002). Polysaccharides such as starch and cellulose in green algae are similar to that of terrestrial plants. Macroalgal biomass lacks lignin in their composition (Jung et al., 2013) except in few red seaweed species. Apart from higher content of carbohydrates in seaweeds, protein and ash contents are also relatively higher; however, lipid fraction is considerably low.

An effective bio-refinery process is achieved by characterization of the feedstock employed such as large variety of carbohydrate (mono-, di-, polysaccharides) serves as raw materials for bioethanol production. Quantification of carbohydrate content (Table 1) in the biomass is an essential step in biorefinery process as it is directly proportional to ethanol yields in biochemical conversion process and facilitates overall process efficiency calculations as well as mass balance (Aden et al., 2002; Kostas et al., 2016). Seaweeds accumulate large concentrations of carbohydrates (polysaccharides) made up of various monosaccharides such as xylose, glucose, galactose and fructose. These sugars are converted to bioethanol through fermentation via appropriate microorganisms.

Availability of macroalgal feedstock

Macroalgae occur along the nutrient rich coastal zones by attaching on hard substratum. Global seaweed distribution is highest between 60^oN and 60^oS latitude with 900-1100 species, least species are recorded >60^o in both hemispheres, in these regions, mostly cold water desiring macroalgae are recorded such as Laminaria, Undaria etc. (Hurd et al., 2014). The most prominent macroalgal genera explored for bioethanol potential is indicated in Fig 3. along the coastal regions of India.

Fig. 3. Distribution of macroalgal species along the coastal regions across the India, explored for bioethanol production potential ( Ramachandra & Hebbale, 2020)

Pretreatment

Bioethanol production from macroalgae requires extraction of fermentable sugars, several studies have reported (Table 2) of different pre-treatment techniques including chemical, physical or biological or combination of these techniques through which higher sugar concentration can be obtained (Feng et al., 2011; Kim et al., 2014; Meinita, Kang, et al., 2012; Park et al., 2012; Yoon et al., 2010). Pretreatment of biomass is carried out to reduce the size and alter or remove structural and compositional impediments prior to subsequent enzyme hydrolysis. Pre-treatment needs to be cost effective and release higher quantum of sugar with the minimal inhibitor formation.

Most commonly used chemical pretreatment method for obtaining higher fermentable sugars from macroalgal biomass is dilute acid pretreatment method, that employs mineral acids such as H₂SO₄ and HCl at milder concentrations of 0.3-0.9N (Meinita et al., 2012a; Park et al., 2012). During dilute acid pretreatment process, reaction parameters such as reaction time, acid concentration, and substrate concentration is involved for efficient sugar release from algal feedstock (Table 3). Pre-treatment with dilute- H₂SO₄ at optimal concentration and temperature is reported effective for cell-wall depolymerization. The advantage of dilute acid pretreatment method is lower energy consumption as compared to other pre-treatments. However, disadvantage of dilute acid pretreatment is formation of fermentation inhibitors such as 5-Hydroxymethyl furfural (HMF) and levulinic acid (LA) with the degradation of hexose sugars and furfurals from pentose sugar degradation. Hence, enzyme hydrolysis has been opted as sustainable option for hydrolysis as it does not involve formation of any inhibitors because enzymes do not cause degradation of monosaccharides (Yanagisawa et al., 2013).

Enzyme saccharification

Biological pretreatment method employs substrate specific enzymes (Fig 4). Major portion of macroalgal cell wall is composed of cellulose, which is made up of glucose subunits, in order to break the cellulose structure cellulase enzyme is commonly used. Similarly, agarases is used for agar, carageenase for carrageenan, alginase for alginate and laminarases for laminarin. Pretreatment is a prerequisite prior to enzyme saccharification, as it opens up the cellulose fibrils and maximizes the enzymatic conversion of cellulose (Harun, 2011; Jeong et al., 2013; Kang et al., 2013; Kim et al., 2014). Commercial enzymes as well as enzymes extracted from bacteria or fungi are reported for enzyme saccharification of macroalgal biomass (Table 4).

Fig 4. Schematic representation of enzyme action on substrate

Saccharomyces cerevisiae, is the predominant microorganism utilized in ethanol fermentation in industrial bioethanol production processes. Ethanol is produced via homoethanol pathways, by Embden-Meyerhof-Parnas (EMP). The EMP (glycolytic) pathway, summarized below (Walker & Walker, 2011):

Saccharomyces cerevisiae reoxidizes the reduced co-enzyme NADH to NAD⁺ in terminal fermentative step reactions emanating from pyruvate:

The intermediate compound, acetaldehyde, acts as the electron acceptor

NAD+ is re-generated by alcohol dehydrogenase which requires zinc as an essential co-factor for its activity. Fermentation thus maintains the redox balance by regenerating NAD and keeps glycolysis proceeding. In doing so, yeast gets energy for its own maintenance by generating 2ATP. The theoretical (stoichiometric) conversion to ethanol from glucose is as follows:

Fig.5. Glucose and Xylose metabolism and conversion to ethanol

For each kilogram of glucose fermented, around 470 g of ethanol can be produced (i.e., <50%) representing a yield of 92% of theoretical maximum. In industrial fermentation practice, however, the best yields are only around 90% of this theoretical conversion due to the diversion of fermentable carbon to new yeast biomass and minor fermentation metabolites (organic acids, esters, aldehydes, fuel oils etc). Bioethanol production from macroalgal biomass is carried out either by Separate Hydrolysis and Fermentation (SHF) or Simultaneous Saccharification and Fermentation (SSF) process. In SHF process, dilute acid hydrolysis/enzyme saccharification and fermentation are carried out separately. This process involves higher operating cost, energy consumption and more reaction time. Not all the sugars in the medium is utilized at the end of this process. In SSF process, enzymatic saccharification and fermentation is achieved in the same reactor. This process is favourable as it requires slower process time, lower energy consumption and yield higher ethanol. However, the process time required for both enzyme and yeast microorganisms are different which results in slower release and consumption of sugar. Lower concentrations of inhibitors are formed in SSF process.

Current Status

Kappaphycus, Gelidium, Gracilaria, Sargassum, Laminaria and Ulva, are largely cultivated macroalgal genera for hydrocolloid extraction and human food usage by China, Philippines and Indonesia. However in recent years these genera are regarded as potential feedstock for biofuel production in addition to the value added products for phycocolloids extraction, human food, cosmetics, fertilizer and other chemicals (Harun, 2011; Jang, Cho, Jeong, & Kim, 2012, Dhargalkar & Pereira, 2005; McHugh, 2003; Yanagisawa et al., 2013). Species from these genera have been chosen considering the availability and assessment of resources around the globe, ease of cultivation and harvesting. Shorter life cycles of seaweed are taken into advantage for largescale cultivation, which is cost-effective and involves environmentally friendly methods, zero input of fertilizers, and no changes in land use as they are exclusively grown in marine waters. Laminaria is most cultivated seaweed with average production of 5.14 million tonnes (Alaswad et al., 2015).

Bioethanol of 40 g/L is reported from green seaweed by the glucose subunits alone, whereas other sulphated polysaccharides such as, Ulvan is yet to be explored. In brown seaweeds, mannitol is fermented to produce 40 g/L of bioethanol whereas, techniques for conversion of alginate sugar to ethanol is still underway. Whereas, in red seaweeds 3,6 anhydrogalactose (composed of glucose and galactose) poses a hindrance for conversion to ethanol (Yanagisawa et al., 2013). Higher concentration of bioethanol is obtained by conversion of all the sugars present in the seaweed which can be achieved by developing methods appropriate to each seaweed species.

Challenges in bioethanol production

Following are the challenges to be addressed for successful bioethanol production from macroalgal biomass

• Major cost reductions need to be achieved by suitable biocatalyst and optimal processes.
• Microorganisms possessing enzymes, which have ability to convert polysaccharides to fermentable sugars needs to be screened or constructed.
• Commercial enzymes such as amylases, cellulases and proteases are available, but they are more efficient in depolymerizing polysaccharides from terrestrial sources. To produce, these enzymes for commercial use, microbial bioreactors are utilized by exploiting the microalgal strains to accumulate carbohydrate and directly utilize their enzymatic or anaerobic digestion systems to produce ethanol, resulting in a cost-effective bioethanol production process. In order to proceed with this procedure, screening of high carbohydrate accumulating seaweeds from natural water bodies based on their growth cycle is to be known.
• Large-scale production to be economical, needs to utilize all sugars present in macroalgal biomass to achieve 100% efficiency.
• Mannitol is a non-fermentable sugar alcohol produced from brown algae, most of the anaerobic bacteria are unable to carry out fermentation of mannitol as there is requirement of oxygen for regeneration of NAD+ for conversion of NADH to NADPH which is obtained from mannitol dehydrogenase during oxidation of mannitol to fructose and NADH. A facultative anaerobic bacterium, Zymobacter palmae ferments sugar alcohols, including mannitol from Laminaria hyperborea extracts. Pichia angophorae is also seen to consume both mannitol and laminarin and yield ethanol. Similar investigation to be carried out for ulvan, alginate, 3,6 anhydrogalactose conversions to bioethanol.
• Bioethanol is an intermediate product obtained during digestion of organic material, and is produced by specific microbial strains only which makes it an obvious practical constraint of keeping the microbial culture from getting contaminated by other microbes (Horn et al., 2000b, 2000a; Nguyen et al., 2017). Hence a controlled condition needs to be maintained.
• Setting up of decentralised biorefinery systems in coastal areas with supporting infrastructure (e.g. roads, utilities).
• Economically feasible algal bioethanol can be turned into a reality only through breakthrough technological innovations. Getting algae to produce bioethanol—in very large volumes, at a very low cost—is the grand challenge that young biotech firms have to shoulder.