Abstract: A comparative study on the extra cellular lipase production in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with various mixed substrates has been made. Different parameters such as moisture content, carbon level and nitrogen level of the medium were optimized. The maximum lipase activity of 9.3 units per gram of dry fermented substrate (U g ds-1) was observed with mixed substrate of sugarcane bagasse and wheat bran in seven days of fermentation.
INTRODUCTION
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are one of the most important classes of industrial enzymes. They hydrolyse triglycerides into diglyceriods, monoglycerides, glycerol and fatty acids. In recent years, there has been an increasing interest in the study of lipases mainly due to their potential applications as medicines (digestive enzymes), food additives (flavour modifying enzymes), clinical reagents (glyceride-hydrolysing enzymes) and cleaners (detergent additives) (Sharma et al., 2001). Additionally, a promising application field for lipases is in the biodegradation of plastics such as Polyhydroxyalkanoates (PHA) and Polycaprolactone (PCL) (Jaeger and Reetz, 1995; Mochizuki et al., 1995).
Solid state fermentation is defined as the fermentation of solids in the absence of free water, however, the substrate must possess enough moisture to support the growth and metabolism of microorganisms. Recently, several reports have been published indicating the application of this culture in upgrading food and industrial wastes and in the production of fine chemicals and enzymes. The utilization of by-products and wastes from food and industrial sources has several advantages over submerged fermentation such as superior productivity, simple techniques, reduced energy requirements, low wastewater output, improved product recovery and the reduction in production costs, since they supply the microorganisms with some nutritive substances (Ashok, 2003). In SSF, any type of substrate could be used to enhance the production of enzymes because many food and industrial wastes are rich in fatty acids, triacylglycerols and/or sugars. The use of cheap raw materials would diminish the operating costs of the process. Moreover, total capital investment for lipase production has been reported to be significantly lower in solid state fermentation than in submerged fermentation (Castilho et al., 2000).
Most studies on lipolytic enzymes production by bacteria, fungi and yeasts have been performed in submerged fermentation; however, there are only few reports on lipase synthesis in solid state fermentation. In recent years, increasing attention has been paid to the conversion of industrial wastes to lipase by solid state fermentation. There are several reports dealing with extra cellular lipase production by fungi like Rhizhopus sp., Aspergillus sp., Pencillium sp. on different substrates (Christen et al., 1995; Kamini et al., 1998; Cordova et al., 1998; Gombert et al., 1999; Miranda et al., 1999).
Sugarcane bagasse, wheat bran and rice bran are produced abundantly in India. The present research was undertaken to optimize process conditions: substrate level, moisture content, carbon level and nitrogen level of the medium, for the production of lipase using agricultural residues (sugarcane bagasse, wheat bran and rice bran) by SSF.
MATERIALS AND METHODS
Substrate
Sugarcane bagasse, wheat bran and rice bran were used as the substrates.
They were procured from a local market of Visakhapatnam, India and were dried
at 60°C for 72 h to reduce the moisture content and ground to the desired
size.
Microorganism
Yarrowia lipolytica NCIM 3589 obtained from National Chemical Laboratory,
Pune, India, was used throughout the study.
Growth Conditions
The culture was maintained on MGYP slants having the composition (%): Malt
extract 0.3, glucose 1.0, yeast extract 0.3, peptone 0.5 and agar agar 2.0.
The pH of the medium was adjusted to 6.4-6.8 and culture was incubated at 30°C
for 48 h. Subculturing was carried out once in every 2 weeks and the culture
was stored at 4°C.
Inoculum Preparation
The yeast Yarrowia strain was cultivated in a medium containing peptone
5 g, yeast extract 3 g and sodium chloride 3 g per liter of distilled water.
The cells were cultivated in this medium at 30°C on a shaker at 200 rpm
for 24 h (Oswal et al., 2002).
Media Preparation
Ten grams of substrate was weighed into a 250 mL Erlenmeyer flask and to
this a supplementing salt solution was added to the desired moisture level.
The composition of the salt solution was as follows (% w w-1): Urea,
0.2; KH2PO4, 0.1; MgSO4.7H2O, 0.05;
CaCl2, 0.01; NaCl, 0.01; H3BO3, 0.00005; CuSO4.5H2O,
0.000004; KI, 0.00001; FeCl3.4H2O, 0.00002;
ZnSO4.7H2O, 0.00004; MnSO4.H2O,
0.00004; myo-inositol, 0.00000004 and d-biotin, 0.00000008 (Corzo and Revah,
1999). The substrate medium without vitamins was sterilized at 121°C for
15 min. After cooling, the vitamins previously sterilized by filtration were
added to the substrate medium.
Solid State Fermentation
The sterilized solid substrate was inoculated with 2 mL of inoculum. The
contents were mixed thoroughly and incubated in a slanting position at 30°C.
Enzyme Extraction
The crude enzyme from the fermented material was extracted by simple extraction
method. For this, the fermented substrate was mixed thoroughly with 50 mL of
50 mM phosphate buffer (pH 7.0) and then shaking the mixture in a rotary shaker
(200 rpm) for 60 min at 37°C, a temperature high enough to increase the
extraction efficiency without causing enzyme denaturation (Freire et al.,
1997). The raw extract was obtained by pressing the mixture and subsequent centrifugation.
The supernatant was used to determine enzyme activity (Gombert et al.,
1999).
Lipase Assay
The activity of lipase was determined as described in the literature (Winkler
and Stuckman, 1979) with the following modifications: 1 mL of isopropanal containing
3 mg of p-Nitrophenyl Palmitate (pNPP) was mixed with 9 mL of 0.05 M
Tris-HCl buffer (pH 8.0), 40 mg of Triton X-100 and 10 mg of gum arabic. Liberation
of p-nitrophenol at 28°C was detected at 410 nm. One enzyme unit
was defined as 1 μmol of p-nitrophenol enzymatically released from
the substrate per minute (Bruno et al., 2004).
Optimization of Medium Parameters
The strategy adopted was to optimize one particular parameter at a time
and then include it at its optimum value in the next optimization step, if found
beneficial. The parameters optimized were: substrate level, incubation time,
moisture content, carbon level and nitrogen level.
RESULTS AND DISCUSSION
Substrate Selection
Among all the substrates, the maximum lipase activity was observed for the
combination of sugarcane bagasse with wheat bran (Table 1).
These results were in accordance with the observed lipase production by Pencillium
restrictum (Gombert et al., 1999) and Asprgillus flaavus USM
A 10 on different substrates (Pau and Omar, 2004).
Optimization of Substrate Levels
Surface area occupied by the substrate was an important parameter in the
SSF. Figure 1 depicts that an amount of 10 g substrate yields
maximum production of lipase. The less yield at higher levels was due to the
low mass transfer rate and difficulty in the penetration of the organism (Rao
et al., 2003).
| Table 1: | Effect of different substrates on lipase activity |
| Fig. 1: | Effect of different substrate levels on lipase activity |
Effect of Incubation Time
The amount of lipase produced was observed daily during a period of nine
days. The maximum lipase activity was observed on seventh day as shown in Table
2. After seventh day, it was reduced due to the consumption of nutrient
materials.
Effect of Moisture Content
Moisture content of the substrate plays a vital role for microbial growth
and biochemical activities in SSF. The maximum yield was obtained at 80% moisture
content as listed in Table 3. In SSF processes, higher moisture
content would lead to decreased porosity, change in wheat bran particle structure,
development of stickiness, reduction in gas volume and decreased diffusion.
On other hand, the insufficient moisture leads to the reduction of solubility
of nutrients present in wheat bran (Babu and Satyanarayana, 1996).
Effect of Carbon Source
The imperative role of different carbon sources on lipase production by
this organism was elucidated by incorporating the selected carbon source (1
g) to the mixed substrate. Table 4 presents the results of
different carbon sources on lipase activity. Among all the carbon sources, glucose
had better impact on productivity.
Effect of Nitrogen Source
Nitrogen source mediated lipase production was well documented in submerged
fermentation (Corzo and Revah, 1999) and SSF (Dominguez et al., 2003).
Table 5 depicts the role of different nitrogen sources on
lipase production. Among all the nitrogen sources, urea yields maximum lipase
activity. Further studies were conducted by taking urea at different levels.
Figure 2 shows that 1 g of the urea yielded maximum lipase
activity. However, at higher levels the production was reduced due to the inhibitory
effects of urea.
| Table 2: | Effect of incubation time on lipase activity |
| Table 3: | Effect of moisture content on lipase activity |
| Table 4: | Effect of different carbon sources on lipase activity |
| Table 5: | Effect of different nitrogen sources on lipase activity |
| Fig. 2: | Effect of urea concentration on lipase activity |
CONCLUSIONS
Solid state fermentation of mixed substrate by Yarrowia lipolytica NCIM 3589 in the presence of sugarcane bagasse and wheat bran with moisture content of 80% yielded 9.3 U g ds-1 of lipase activity in 7 days. The mixed substrate use of sugarcane bagasse and wheat bran for lipase production may have the combined benefit of utilizing a low value waste material while producing a commercially valuable product.
ACKNOWLEDGMENTS
This work was carried out in the Center for Biotechnology, Department of Chemical Engineering Andhra University, Visakhapatnam during 2006 with the financial assistance received from the University Grants Commission (SAP, Phase-III), New Delhi, India.