128 S. S. Aulakh et al.
Nitrogen sources included in the study were
di-ammonium
((NH4)2HPO4)
35 ꢀC with constant stirring. Alcohols (propanol,
butanol, pentanol, hexanol, heptanol, octanol, non-
anol, or decanol) were added stepwise at an interval
of 6 h, with total reaction time of 36 h. The reactions
were carried out in triplicate and the data are
represented as mean (ꢂSD). The samples obtained
from above experiments with each of the different
acyl acceptors were pooled and independently
analyzed using TLC and proton NMR.
hydrogen
and
ortho-phosphate
peptone
mycological
(HiMedia, India). Culture media used were mineral
salt medium, potato dextrose agar (PDA), potato
dextrose broth (PDB) (HiMedia).
Freshly
grown
Aspergillus
sp.
RBD01
(MTCC5436), earlier isolated from biocontami-
nated clarified butter (Prakash and Aulakh 2011),
was inoculated under sterile conditions into 500 mL
Erlenmeyer flask containing 200 mL of potato dex-
trose broth and incubated at 28 ꢀC, 120 rpm for 72 h.
The fresh biomass thus obtained was further used for
experimentation. The mineral medium comprising
magnesium sulphate (0.20 g Lꢁ1), calcium chloride
(0.02 g Lꢁ1), mono-potassium phosphate (1.0 g Lꢁ1),
di-potassium phosphate (1.0 g Lꢁ1), and ferric chlor-
ide (0.05 g Lꢁ1), was used for the growth of fungal
strain. The medium was supplemented (0.5% w/v)
with mycological peptone or/and di-ammonium
hydrogen ortho-phosphate as nitrogen source.
The alkyl oleate separated from reaction was
analysed using thin layer chromatography with
silica gel G as stationery phase and hexane:ethyl
acetate:acetic acid (90:10:1) as a mobile phase and
the chromatogram was developed in the iodine
chamber (Samukawa et al. 2000). The ester was
further quantified using proton nuclear magnetic
resonance spectroscopy (1H NMR) (400 MHz,
Bruker-Advance II-400 with 5mm BBO probes).
CDCl3 (deuterated chloroform) was used as solvent
and tetra methyl silane as internal standard.
1H NMR spectra were recorded with pulse duration
of 2.72 s with a relaxation delay of 1 s and 16 scans.
Alkyl ester produced by esterification was quantified
by using the derivation given by Satyarthi et al.
(2009).
In case of cell suspension, the esterification
reaction was carried out with cell suspension in
500 ml Erlenmeyer flask, containing 200 ml of oleic
acid: mineral medium (50:50) with oleic acid acting
as carbon source, peptone, and bi-ammonium
hydrogen ortho phosphate (0.5%) as nitrogen
sources, and pH of the medium set at 7.0 ꢂ 0.2.
Inoculum from fresh biomass of Aspergillus sp. was
added and incubated for 72 h at 28 ꢀC and 120 rpm.
Following incubation, the influence of chain length
of alcohols on the esterification process was studied
by addition of various alcohols (methanol, ethanol,
propanol, butanol, pentanol, hexanol, heptanol,
octanol, nonanol, and decanol) in the ratio of 1:2
(oleic acid:alcohol). Marginal additional amounts of
alcohols were supplemented to avoid reversible
reaction. The addition of alcohols was carried out
stepwise at an interval of 12 h. After completion of
the reactions, biomass was recovered by simple
filtration. The upper layer of ester was separated by
using separation funnel and used further for analysis.
Similarly, to examine the esterification process
with powdered biomass of Aspergillus sp. freshly
cultivated biomass generated as discussed previously,
was separated from reaction mixture using Whatman
filter paper and washed with n-hexane followed by
water to remove adhering oil. This was then air-dried
overnight to remove excess water and crushed to
powder in liquid nitrogen. Esterification of oleic acid
was carried out with varying chain length of alcohol
(methanol, ethanol, propanol, butanol, pentanol,
hexanol, heptanol, octanol, nonanol, and decanol)
for generation of ethyl oleate. To examine the
esterification process, 2 g of biomass and 10 g oleic
acid were taken in round bottom flask and kept at
% of FFA ðfree fatty acidsÞ
¼ ð½4 ꢃ area of unmerged peak of / CH2
of fatty acidꢄꢅ½total area of / CH2
of fatty acid and esterꢄÞꢃ100
To draw a comparison between the activities of
wet and dry forms of biomass, lipase activity was
determined following method outlined by
Sigurgisladottir et al. (1993). The enzyme activity
was determined by adding 1 g of biomass to a
reaction mixture containing 0.9 ml of 0.05 M phos-
phate buffer (pH 7.0) and 0.1 ml of 0.005 M pNP
(p-nitrophenol laurate) in ethanol. The mixture was
incubated at 60 ꢀC for 30 min, followed by addition
of 0.25 ml of 0.1 M Na2CO3 on cooling to room
temperature. The activity was determined at 420 nm.
One unit of lipase activity is defined as the amount
of enzyme that liberates 1 mg p-nitrophenol (molar
extinction coefficient 1.336 ꢃ 107 cm2/mol at
420 nm) with pNP-laurate as substrate under stand-
ard assay conditions in 30 min.
Result and discussion
The study compared the potential of the culture
suspension and dry biomass of Aspergillus sp. RBD01
to esterify oleic acid in the presence of alcohols of
varying chain length and polarity.