Introduction

Energy is a pivotal resource for the economic development of a region [1], a resource that is confronted with far exceeding de- mands than supply at recent times due to speculated rapid indus- trialization and sophisticated living [2]. Adverse and irreversible effects due to global warming and consequent changes in climate make microalgae derived biofuels as the most attractive and sus- tainable energy options [3]. Algal biodiesel is renewable [4] with its feedstock having the ability to sequester atmospheric CO₂, which enhances the scope of sustainable energy option [5]. Algae as a biodiesel feedstock include an array of advantages like higherphotosynthetic efficiency (12.6%), efficient CO₂ sequestration capability, potential to bioremediate contaminated waters and non- arable lands [6,7], with the proven higher oil content [8] and algal biomass productivity. The maximum theoretical algal biomass productivity reported so far in a region of higher solar insolation is around 100e120 g/m²/day [9]. Algae stores fats and lipids in the form of triglycerides which is high quality and high-volume raw material. Most oleaginous microalgae accumulate 20% total lipids on dry cell weight and this increases up to 50% when algae are subjected to stress conditions [10]. Oil is being extracted from algae through transesterification process using chemical catalysis. There is a trade-off between the alkoxy group of an ester with a methyl group in most transesterification reactions [11]. Methanol or ethanol is used as a co-reactant (acyl acceptors) for algal derived oil but methanol is mostly preferred due to its lesser cost when compared to ethanol. Major catalysts that are in extensive use so far in the transesterification process are either of acid or alkali. The most commonly used acid catalysts are sulfuric acid and hydro- chloric acid in its diluted forms while alkali catalyzed reaction uses

sodium hydroxide or potassium hydroxide. The major drawbacks of acid- or alkali-based) catalyst assisted transesterification reactions are (i) low yield and purity due to unwanted side reactions, (ii) high energy requirements, (iii) higher costs involved in by-product (glycerol) separation, and (iv) need for neutralization and waste- water treatment, post-reaction completion [12]. The use of en- zymes as biocatalysts for transesterification [13] is an emerging technique compared to conventional acid/alkali catalysis for bio- diesel production. Enzymes synthesized by fermentation of bio- based materials [14] are naturally occurring biocatalysts,. Lipolytic enzymes play a crucial role in turn-over and mobilization of lipids, a major component of earth’s biomass from one organism to another [15]. Microorganisms such as bacteria and fungi produce bio- surfactants are known to solubilize lipids [16]. Moreover, when enzymes are used as biocatalysts during transesterification of algal oil, it renders a cleaner and an environmentally friendly option with an added advantage for third-generation (microalgal) biofuels. Among other enzymes, lipase (triacylglycerol acyl hydrolases, EC 3.1.1.3) and esterase (E.C. 3.1.1.1) are the two major classes of lipid hydrolytic enzymes belonging to a/b hydrolase family that are be- ing considered as promising industrial biocatalysts [17]. Esterase enzyme catalyzes the hydrolysis of shorter chain length fatty acid esters (<C8), while lipase catalyzes triacylglycerols which are of longer chain lengths (>C8) [18,19]. Lipase enzymes are categorized into three different classes based on the type of substrates: (i) lipase with regio- or positional specific lipolytic active sites, (ii) fatty acids specific lipases, (iii) highly specific to only certain acylglycerols present in oils [20]. These lipolytic enzymes are receiving consid- erable demand as potential industrial biocatalysts due to its manifold applications in dairy, food, detergents, fats and oil, organic synthesis, biodiesel, agro-chemicals, new polymeric materials [21], paper and pulp, leather, fine chemicals, cosmetics, pharmaceuticals [22e25] and various environmental applications including soil bioremediation and biodegradation of environmentally toxic pol- lutants such as phenolic compounds and endocrine disruptors [26]. Although lipases are ubiquitous and produced by most microor- ganisms, plants and animals, the most industrially exploited lipase sources are of microbial origin isolated commonly from bacteria and fungi [27,28]. Compared to other extraction sources, microbial lipases possess several advantages such as shorter cycling time, less expensive and are easily adaptable to grow and immobilize on any inexpensive solid media (substrates). They often fetch higher yields and also are compatible to genetic manipulations. Enzyme catalysts require milder ambient conditions, compared to chemical catalyzed reactions for its effective operation leading to a major cut-down in energy expenditure and hence the operational costs. Other ad- vantages of enzymatic reactions include high selectivity towards substrate with the capability to esterify triglycerides and free fatty acids in a single step, thus producing high-quality byproduct (glycerol) with no additional costs involved in byproduct separa- tion and recovery. Enzymes are highly specific to substrates, thus eliminating unwanted side reactions and the need for post-reaction byproduct separation. Moreover, enzymatic reactions are environment-friendly without posing any hazards during disposal [29]. There have seen enormous efforts during the past decade focusing on lipase enzyme production and characterization for diverse applications. Biodiesel production using lipase as a biocat- alyst is an emerging area of research with its application already standardized for first and second-generation biodiesel feedstocks such as sunflower oil [30], Jatropha oil [31], soybean oil [32], palm oil [33]. Recent researches focus on using lipase as biocatalyst [20,34e37] in the enzymatic conversion of microalgal oil into biodiesel.

1.1. Motivation for the study

Algae are primary producers in aquatic ecosystems and are emerging as promising biodiesel feedstocks due to the presence of proven higher oil content in the form of triglycerides. Algae based biodiesel have an array of advantages like viable replacement to fossil fuels, assured stock availability, efficient CO₂ sequestration capability, remediation and treatment of water etc. Fatty acid methyl ester (biodiesel) is derived from oil present in algae through transesterification. Catalysts of acids, base, supercritical fluids, etc., are being used to maximize the conversion of oil into biodiesel. These catalysts are corrosive and have been posing challenges of contaminating the environment necessitating environmentally friendly and biodegradable catalysts such as enzyme (lipase) based biocatalysts. Industrially important enzymes extracted from indigenous sources are least explored, especially for biofuel pro- duction. Exploitation of cellulase and lipase for biofuel production would greatly reduce the environmental burden imposed by con- ventional chemical catalysts. In the current study, extracellular lipase extracted from an indigenously isolated fungal strain Cla- dosporium tenuissimum was used as a biocatalyst to derive biodiesel from a salt-tolerant diatom Nitzschia punctata (microalga). Crude extracellular lipase was purified using gel filtration-based size- exclusion chromatographic system. The purified enzyme after characterization was used as a biological catalyst for trans- esterification of microalga derived oil. In addition, biodiesel (FAME) was derived using a conventional acid catalyst. Biodiesel derived from the acid catalyst and enzyme catalyst-based trans- esterification were assessed for FAME yields to understand the relative performances and efficiencies of the catalysts possessing different chemical properties.