1Energy and Wetlands Research Group, Centre for Ecological Sciences [CES],
Indian Institute of Science, Bangalore – 560012, India.
2 Centre for Sustainable Technologies [CST], Indian Institute of Science.
3Centre for Infrastructure, Sustainable Transport and Urban Planning [CiSTUP],
Indian Institute of Science, Bangalore 560 012.
*Corresponding author:
trv@iisc.ac.in
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.