Alkaline Lipase production from Enterobacter aerogenes by solid-state fermentation of agro-industrial wastes

Abstract

Lipases that are stable at alkaline pH are highly desirable for detergent formulations. In the present study, an alkaline lipase was produced from solid-state fermentation of agro-industrial wastes by lipase producer strain of Enterobacter aerogenes. Use of such wastes as substrates instead of commercial synthetic media was done to develop a cost-effective method for lipase production. Fermentation conditions were optimised for maximising enzyme production. The lipase was found to be stable at higher pH 9-11 and temperature 45-60°C ranges. Its incorporation in detergent formulation enhanced the washing efficiency.

Full text

Int. J. Environment and Waste Management, Vol. x, No. x, xxxx 1
Copyright © 200x Inderscience Enterprises Ltd.
Alkaline lipase production from Enterobacter
aerogenes by solid-state fermentation of
agro-industrial wastes
Sanghamitra Mitra and S.K. Khare
Department of Chemistry,
Indian Institute of Technology,
Hauz Khas, New Delhi
E-mail: skkhare@chemistryiitd.ac.in
E-mail: khare_sk@hotmail.com
Rajni Singh*
Amity Institute of Biotechnology,
Amity University,
Sector-125, Noida, Uttar Pradesh, India
Fax: 0120-4392295
E-mail: rajni_vishal@yahoo.com
E-mail: rsingh3@amity.edu
*Corresponding author
Abstract: Lipases that are stable at alkaline pH are highly desirable for
detergent formulations. In the present study, an alkaline lipase was produced
from solid-state fermentation of agro-industrial wastes by lipase producer strain
of Enterobacter aerogenes. Use of such wastes as substrates instead of
commercial synthetic media was done to develop a cost-effective method for
lipase production. Among various agro-wastes screened, soybean flour was
found to be best utilised by Enterobacter aerogenes as compared with
cottonseed oilcake and mustard oilcake. Fermentation conditions were
optimised for maximising enzyme production. A total 12 units (U) lipase
per gram substrate was obtained. The lipase was found to be stable at higher
pH 9–11 and temperature 45–60°C ranges. Its incorporation in detergent
formulation enhanced the washing efficiency.
Keywords: alkaline lipase; detergent application; Enterobacter aerogenes
lipase; solid-state fermentation; soybean oil cake.
Reference to this paper should be made as follows: Mitra, S., Khare, S.K. and
Singh, R. (xxxx) ‘Alkaline lipase production from Enterobacter aerogenes
by solid-State fermentation of agro-industrial wastes’, Int. J. Environment and
Waste Management, Vol. x, No. x, pp.xxx–xxx.
Biographical notes: Sanghamitra Mitra has completed her Master Degree in
Chemistry from IIT, Delhi and during her Master Degree she had got research
experience in lipase production using industrial waste.
Author: Please
supply e-mail id
for the author
‘Sanghamitra
Mitra’.
Author: Please
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2 S. Mitra et al.
S.K. Khare has done his Doctoral Degree from IIT Delhi. He has 15 years
of teaching and research experience and published more than 50 papers in
peer-reviewed national and international journals. He is reviewer of several
international journals. At present, he is an Associate Professor of Biochemistry
in the Department of Chemistry, IIT Delhi.
Rajni Singh has Masters and Doctoral Degree in Microbiology from GB Pant
University of Agriculture and Technology, Pantnagar, India. She has expertise
in the fields of microbial enzymes and biotechnology. She has more than
six years of teaching and research experience and has published 14 papers in
peer-reviewed national and international journals. At present, she is Lecturer
at Amity Institute of Biotechnology, Amity University, Noida, India.
1 Introduction
Lipases are important class of industrial enzymes having wide-ranging applications from
oleochemicals processing to food and pharmaceuticals designing (Bosley, 1997; Khare
et al., 2000, 2001; Singh et al., 2006). The major commercial use of lipases was in
detergent formulations, contributing 32% of their total sales (Jaeger and Reetz, 1998).
Typically, a detergent lipase should possess
• low substrate specificity to meet high variation in the triglyceride content
of oil stains
• stability in alkaline environment (pH 9–11) and moderately higher temperatures
30–60°C encountered during washing
• stability in presence of surfactants and detergents
• low cost. Lipases, in general, are not stable under these conditions (Sharma et al.,
2001).
Hence, there have been extensive studies on cost-effective production of stable lipases
suitable for detergent applications. Most of the detergent preparations are patented viz.
commercial recombinant ‘Lipolase’ of Novo Nordisk, ‘Lumafast’ and ‘Lipomax’ from
Genencor International (Jaeger and Reetz, 1998).
Commercial lipases are produced by fermentation process wherein fermentable
substrate and downstream processing govern the cost of enzyme production. The carbon
source used in fermentation itself accounts for major cost of bioprocess (Arbige and
Pitcher, 1989; Pandey, 2003). Use of agro-industrial wastes as substrate, therefore,
has been a promising approach especially in countries where abundance of biomass/
agro-industrial residue is available and poses problem of their safe disposal. Other
advantages of lipase production by solid-state fermentation such as higher yield, better
controls and cheap recovery have also been reported (Kamini et al., 1998; Gombert et al.,
1999; Pandey, 2003). However, in most of these cases, known microbial strains have
been used and lipases suitable for fat and oil processing have been obtained (Adinarayana
et al., 2003; Cavalcanti et al., 2005; Hadj-Ali et al., 2007). Suitability of SSF processes
for production of alkaline detergent lipases has not been assessed. We have previously
reported isolation of an Enterobacter aerogenes strain producing alkaline lipase under

Alkaline lipase production from Enterobacter aerogenes 3
submerged fermentation conditions (Gupta et al., 2002). In the present work, feasibility
of producing this alkaline Enterobacter aerogenes lipase by solid-state fermentation of
soybean oil cake is explored. The washing efficiency and stability of this lipase in
presence of detergents is also described.
2 Materials and methods
The microbial media ingredients were purchased from Hi-media, Mumbai. p-Nitrophenyl
acetate and p-Nitrophenyl palmitate were obtained from Lancaster, England. All other
reagents used were of analytical grade. Different agro-industrial wastes to be used as
substrate viz., cottonseed oil cake, mustard oil cake and soybean cake were procured
from local market of Delhi and powdered before use.
2.1 Inoculum preparation
Bacterial strain 3HC (Enterobacter aerogenes) was isolated from the soil near oil
extraction unit (Gupta et al., 2002). The stock culture of Enterobacter aerogenes
(strain 3HC) was maintained on agar slant. The inoculum was prepared by transferring
stock culture into 25 mL Erlenmeyer flask containing 5.0 mL culture medium [consisting
of (g L
–1
): peptone, 5; yeast extract, 3; glucose, 5; NaCl, 0.25; MgSO
4
⋅ 7H
2
O, 0.5 and pH
adjusted to 7.5] followed by incubation at 30°C with constant shaking at 120 rpm for
16 h. Inoculum count was done by the plate count method.
2.2 Fermentation conditions
Oil cake substrate (10 g) was taken in a series of 250 mL Erlenmeyer flasks, moistened
with 20 mL of water and autoclaved. These were inoculated with 150 µL of inoculum
(1.26 × 10
13
cfu) and thoroughly mixed, followed by incubation at 30°C for five days.
Samples were aseptically withdrawn periodically and assayed for lipase activity.
2.3 Optimisation studies
One at a time strategy was used for optimisation.
Optimisation studies were carried out by varying: moisture content from 1 : 2 to 1 : 6
(substrate: water, w/v); inoculum concentration from 125 µL to 500 µL (1.05 × 10
13
and
4.2 × 10
13
; pH of substrate (from 7 to 10).
2.4 Extraction of the enzyme and lipase assay
Aseptically withdrawn fermented samples were homogenised with phosphate buffer
(0.1 M; pH 9.0) in the ratio 1 : 3 for extraction of the enzyme. The homogenate was
centrifuged at 10,000 g for 20 min and supernatant collected was used as crude lipase.
The crude had an average 4.0 U lipase activity/mL (corresponding to 12 units/g
fermentable substrate).
The lipase activity was assayed by the method of Montero et al. (1993) using PNPA
(p-nitrophenyl acetate) as substrate. For assay under alkaline conditions, procedure

4 S. Mitra et al.
of Lin et al. (1996) was followed. One unit of lipase activity (U) is defined as the amount
of enzyme, which catalysed the production of 1 µl of p-nitrophenol per minute under
standard assay conditions and lipase activity is expressed as units/g substrate.
Protein was estimated by dye binding method (Bradford, 1976).
2.5 Lipase stability
The crude enzyme was incubated at different temperatures 30–60°C. The aliquots were
withdrawn periodically and the residual activity was assayed.
2.6 Effect of detergent on lipase activity
Stability of lipase in detergents was studied by adding different detergents (Ariel, Fena
and Surf Excel) to the enzyme solution at a concentration of 1 mg/mL. The mixture was
incubated at 30°C for 1 h and residual lipase activity was determined.
2.7 Effect of lipase on removal of olive oil from cotton fabric
Two types of washing solution were prepared to check the washing efficiency of lipase:
i 10.0 mL of commercial detergent solution (0.1% w/v in 0.1 M phosphate buffer,
pH 7.0)
ii 5.0 mL lipase (20 Units) added to 10.0 mL of solution (i).
Final volume of all the solutions was adjusted to 20.0 mL with phosphate buffer.
The cotton fabric (5 × 5 cm
2
) spotted with 2.0 mL of olive oil–benzene solution
(500 mg/mL concentration) was washed with above-described washing solutions for
20 min at 37°C with constant shaking at 100 rpm. At the end of 20 min, the fabrics were
removed and rinsed three times with distilled water (100 mL), each for a period of 2 min
and then air-dried. Residual olive oil was extracted from the washed fabrics using hexane
for 12 h in a soxhlet apparatus. The hexane was completely evaporated, and olive oil thus
obtained was collected and weighed. The removal of olive oil was calculated by
determining the amount of olive oil before and after washing.
All the experiments were done in triplicate and the difference was in the range of ± 2.
3 Results and discussion
We have recently reported an Enterobacter aerogenes strain producing alkaline lipase
under submerged fermentation conditions (Gupta et al., 2002). Because of its alkaline
nature, its detergent application looks feasible. Its cost-effective production by solid-state
fermentation of agro-wastes and suitability for detergent application are explored in the
present work.
Some of the commonly available oil cakes were screened for their suitability
to support the growth of this E. aerogenes isolate vis-a-vis level of lipase production.
Figure 1 shows that soybean oilcake is best utilised as substrate by E. aerogenes,
resulting in maximum lipase production. Its composition, 40% protein, 24%
carbohydrates and 20% fat, indicates that it is wholesome for bacterial growth. Secretion

Alkaline lipase production from Enterobacter aerogenes 5
of extracellular lipase increases with time, reaching maximum in 96 h. Enterobactor
cloacae and E. hafniae are other species of Enterobactor reported to exhibit lipolytic
properties (Wessels et al., 1989). None of these have been studied in detail. Lipase from
closely related Serratia marcescens is well characterised (Winkler and Stuckman, 1979).
Figure 1 Time course of lipase production by Enterobacter aerogenes. 10 g of soybean cake was
mixed with varying amount of water (w/v), autoclaved and inoculated with 150 µL
(1.26 × 1013 cfu) following by incubation at 30°C for five days. Samples were
aseptically withdrawn at different time intervals for assaying lipase activity. Soybean
cake (▨), mustard cake (▦) and cottonseed cake (▤)
Soybean oil cake was selected as substrate for further studies. Defatted soybean cake is
used as one of the SSF substrate in the study. The total 4.07 million Metric tons soybean
cake is produced per annum in India (as per soybean information centre), which poses
a disposal problem. The SSF will generate a value added product from it. The residual
substrate will be free from anti-nutritional factors otherwise present in the cake. It can
therefore serve as a better-feed material.
Optimisation of fermentation conditions for lipase production using soybean cake as
substrate was carried out. The optimised conditions in terms of inoculum size, pH of the
substrate and effect of moisture content are shown in Figure 2. In fact, each of these
parameters are established to be critical for solid-state fermentation (Robinson et al.,
2001). Inoculum size of 2.1 × 10
13
cfu, pH 9.0 and moisture 1 : 3 (oilcake : water, w/v)
was found to give maximum lipase production. Under all the optimised conditions,
maximum 12 U/g lipase production was achieved. Similar level of lipase production is
reported from other microbial sources viz. 27.8–30 U/g in case of babassu oil cake solid-
state fermentation by Penicillium restrictum (Gombert et al., 1999; Palma et al., 2000);
37 U/g from rice bran fermentation using Candida rugosa (Rao et al., 1993). In all these
cases, the agro-waste has been supplemented with carbon and nitrogen sources
additionally. However, in our case no supplementation has been provided.
Some of the previously reported (by us) enzymatic properties of E. aerogenes lipase
(Gupta et al., 2002) are summarised in Table 1. It showed optimum pH and temperature
at pH 9.0 and 55°C respectively; and found to be stable at pH 9–11 and temperature at
60°C for 4 h (without any loss in activity). In general, microbial lipases are either acidic
or neutral and very few alkaline lipases have been reported. These are Novo’s ‘Lipolase’

6 S. Mitra et al.
from Thermomyces lanuginosus, Genencor’s ‘Lipomax’ from Pseudomonas alcaligenes,
‘Lumafast’ from P. Mendocina, from Bacillus sp. (Wang et al., 1995), Proteus vulgaris
(Kim et al., 1996), and some lipases from Pseudomonas sp. (Lin et al., 1996; Kojima
et al., 1994).
Figure 2 Effect of fermentation conditions on lipase production: (i) 10 g of soybean cake was
mixed with varying amount of water (w/v), autoclaved and inoculated with 150 µL
(1.26 × 10
13
cfu) following by incubation at 30°C. Lipase was assayed after 96 h.
Cake : water (w/v): 1 : 2 (▤), 1 : 3 (▥), 1 : 4 (▨), 1 : 5 (▦) and 1 : 6 (□);
(ii) In similarly performed experiment, 10 g cake was mixed with 30 mL water,
autoclaved and inoculated with different inoculum size. 125 µL (1.05 × 10
13
cfu) (▤),
150 µL (1.26 × 10
13
cfu) (▥), 250 µL (2.10 × 10
13
cfu) (▨) and 500 µL
(4.20 × 10
13
cfu) (▦) and (iii) 10 g cake mixed with 30 mL water was adjusted to
different pH, autoclaved and inoculated with 250 µL inoculum (2.10 × 10
13
cfu). pH 7.0
(▤), pH 8.0 (▥), pH 9.0 (▨) and pH 10.0 (▦)
Table 1 pH and temperature stability of Enterobacter aerogenes lipase
Parameters Enterobacter aerogenes lipase
pH optima 9.0
pH stability 9–11
Temperature optima
55°C
Thermal stability
30–60°C (No loss in activity up to 4 h)
Although thermally stable lipases have been reported from many sources (Gowland et al.,
1987), a combination of thermostable alkaline lipase (combination of dual properties
required under harsh washing condition: temperature 30–60°C and pH 9–11) is reported
only in few cases viz. Bacillus sp. strain A 30-1 by Wang et al. (1995) and Rua et al.
(1998). Thus E. aerogenes lipase being stable in alkaline media vis-à-vis higher
temperature has potential for detergent application.
In the present investigation, its stability in presence of various detergent formulations
was examined. The lipase exhibited stability and enhanced activity in the presence of all
detergents (Figure 3). Surfactant provides more surface area required for lipase catalysis.

Alkaline lipase production from Enterobacter aerogenes 7
To examine efficiency in the removal of oil stain, a cotton fabric spotted with oil stain
was treated with commercial detergents in presence and absence of lipase (20 U). Results
are shown in Table 2. It can be seen that a combination of detergent and lipase removed
upto 85% oil. Oil stains removal efficiency increased by 35% in the presence of lipase.
Kamini et al. (1998) have also reported a detergent stable lipase from A. niger with 71%
oil removal efficiency.
Figure 3 Effect of detergent on lipase activity: Crude lipase (5 mL) was mixed with 0.1% (w/v)
detergent, incubated at 30°C with constant shaking at 100 rpm for 1 h. Lipase activity
was monitored in periodically withdrawn samples. Control (♦), SDS (■), Ariel (▲) and
Surf Excel (●)
Table 2 Oil removal efficiency of Enterobacter aerogenes lipase
Washing solution Commercial detergent used Oil removal (%)
Fena 30 Detergent solution*
Surf Excel 46
Fena 50 Lipase (20U) + Detergent solution*
Surf Excel 85
*Detergent solution consisted of 0.1% (w/v) detergent in 0.1 M phosphate buffer
(pH 7.0).
Cotton fabric (5 × 5 cm
2
) spotted with olive oil (1.0 g) was washed for 20 min 37°C with
100 rpm shaking.
4 Conclusions
Enterobactor aerogenes lipase meets all the criteria to suit detergent applications:
• stability under harsh washing conditions (pH 9–11, temperature 30–60°C)
• stability in presence of surfactants and other components of detergent formulation
• it can be produced as low cost enzyme by solid-state fermentation of agro-wastes.

8 S. Mitra et al.
Ackowledgement
S.K. Khare is grateful to UNU-Kirin, and UNUWA, Tokyo, Japan for their financial
support.
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