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Fungal Lipase Production by Solid State Fermentation-An Overview

Devarai Santhosh Kumar^1* and Suman Ray^2*

¹Department of Chemical Engineering, Ordnance Factory Estate, Yeddumailaram, Indian Institute of Technology Hyderabad, Telangana, India

²CSIR-National Institute of Science Technology and Development Studies, Pusa Gate, K.S. Krishnan Marg, New Delhi, India

*Corresponding Author:

Devarai Santhosh Kumar
Department of Chemical Engineering
Ordnance Factory Estate, Yeddumailaram
Indian Institute of Technology
Hyderabad, Telangana, India.
Suman Ray
Scientist, Room No:206
CSIR-National Institute of Science
Technology and Development Studies (CSIR-NISTADS)
Pusa Gate, K.S. Krishnan Marg, New Delhi, India
Tel: +91 011 2584-6064/3227
Fax: +91 11 25846640;
E-mail: sumanitrc@gmail.com

Received Date: October 27, 2014; Accepted Date:December 12, 2014; Published Date: December 17, 2014

Citation: Kumar DS, Ray S (2014) Fungal Lipase Production by Solid State Fermentation-An Overview. J Anal Bioanal Tech 5:230. doi: 10.4172/2155-9872.1000230

Copyright: © 2014 Kumar DS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Importance of enzymes is ever-growing specifically microbial lipases which are of great industrial significance because of their applications in detergent, food, pharmaceutical, chemical and leather industry. Solid state fermentation (SSF) is an economical alternative for large scale production of enzymes that are produced by fungi. Therefore, production of lipases by solid state fermentation is a good and preferred option than submerged fermentation (SmF). The important factors in fermentation are carbon concentration, nitrogen concentration, pH, growth temperature, fermentation time and moisture content. This review mainly focuses on production of fungal lipase by solid state fermentation using various fungal strains, substrates and fermentation conditions. Enzyme characteristics, industrial application and assay methods of lipase, biomass estimation, enzyme extraction methods and engineering aspects of fermentation are also dealt with briefly. The main aim of the review is to give an overview of advancements in solid state fermentation for production of fungal lipase hitherto.

Keywords

Lipase; Solid State Fermentation (SSF); Submerged Fermentation (SmF); Fungal Sp; Agricultural Substrates

Introduction

(SSF) is an economical alternative technique to submerged fermentation (SmF) for large scale production of industrial enzymes, where raw materials and processing is cheap with reduced chemical cost and less processing risk. It is defined as ‘the growth of microorganisms (mainly fungi) on moist solid materials in the absence of free flowing water’ [1]. This technique uses agricultural substrates with various carbohydrates, nitrogen sources, salts as well as nutrients like starch, cellulose, pectin, lignin, fibers and minerals. SSF technique has the potential to produce desired microbial products more efficiently than submerged fermentation (SmF) and has practical and economical advantages over SmF [2]. It has been reported that SSF with fungal strains results in greater productivity than submerged fermentation [3,4]. Substrates that have been traditionally fermented by solid state fermentation for enzyme production include agricultural wastes such as rice husk, wheat bran, beans, corn steep dry, sugar cane bagasse, coffee pulp, lemon peel and apple pomace [5-11]. Currently, major countries in the world are implementing this technique for large scale production of enzymes due to its higher titer values [12]. The substrates used for SSF can be classified as (a) agricultural raw materials and waste products (b) industrial wastes and (c) synthetic materials [13].

Lipase

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) (Figure 1) are a group of enzymes which catalyze the hydrolysis of triglycerides to diglycerides, and monoglycerides which are further hydrolyzed to glycerol and free fatty acids (Figure 2). Although there is lack of knowledge of the intrinsic kinetics of microbial growth, lipases are assumed to interact at oil-water interface. However, the kinetics regarding the adsorption processes is known [14,15]. Lipases occur widely in bacteria [16-18], yeasts [19,20] and fungi [21,22]. These enzymes also display catalytic activity towards a large variety of alcohols and acids in ester synthesis provided that the water activity is low. They can hydrolyze the ester bonds, trans-esterified triglycerides, resolve raceme mixture and are used in the synthesis of ester bonds in nonaqueous media [23-25]. Fungi are widely recognized as the best lipase sources and used preferably for industrial applications. A few of the general fungal genus include Rhizopus, Aspergillus, Pencillium, Mucor, Ashbya, Geotrichum, Beauveria, Hunicola, Rhizomucor, Fusarium, Acremonium, Alternaria, Eurotrium and Ophiostoma. Fungal species which produces lipases are Candida rugosa, Candida Antarctica, T. lanuginosus, R. miehei, Pseudomonas, Mucor and Geotrichu. Fungal lipases are often reported as Aspergillussp, Candida rugosa, Candida antarctica, Thermomyceslanuginosus, Rhizomucormiehei, Mucor and Geotrichum [26-28]. Lipases play an important role in lipid metabolism in eukaryotes at various stages like fat digestion, reconstitution, adsorption, and lipoprotein metabolism. Lipases are also found in energy tissues of plants and the mechanism of their interactions with lipids at the interface is still a subject of investigation [29]. Lipases provide improved worldwide attention due to their diverse industrial applications and versatility in nature. The world’s enzyme demand is met by 12 major producers and 400 minor suppliers across the globe, 60% of the world’s supply of industrial enzymes is from Europe alone and strikingly 75% of these industrial enzymes include lipases [26,30]. The importance of lipase as biocatalyst is well understood. It is used to catalyze several unnatural and remarkable reactions in non-aqueous media that include bio-fuel production, production of value added products like esters, organic acids, food, beverage, cosmetics, and pharmaceutical materials [13].

Figure 1: Bio-action of lipase with nucleophilic targeted carbonyl function

Figure 2: Reaction scheme of lipase catalysis to free fatty acids and glycerol

Fungal Strains Producing Lipase

Several lipase producing fungal strains have been used for lipase production by SSF which include rhizopodiformis A13 and Rhizomucor pusillus A16 obtained from ORSTOM culture collection [31]. Other important fungi used are Aspergillus niger MTCC2594 [32], Penicillium restrictum [33], Rhizopus oligosporous [34], A.niger NCIM 1207 [35], Rhizopus delemer [36] and Aspergillus oryzae [37], Among these strains, Aspergillus niger is found to produce significant quantities of enzyme and is regarded as GRAS by Food and Drug Administration (FDA). A. niger MTCC 2594 cultured on gingelly oil cake has a lipase activity of 363.6 μ/g dry substrate [32], whereas maximum lipase activity of 630 IU/g dry solid substrate was observed using A.niger NCIM 1207 [35]. Fungal strains producing lipases by SSF are tabulated in detail in Table 1.

Table 1: Comparison of lipase production in various fungal microorganisms

Substrates used for lipase

Different types of agricultural wastes and oily substrates are used for the lipase because of their lipolytic nature. This includes rice bran, wheat bran, gingelly oil cake, almond meal, mustard oil cake, neem oil cake, groundnut oil cake, gingerly seed and groundnut kernel that are used routinely for lipase production by SSF. Lipase production from Aspergillus niger by SSF using gingelly oil cake as substrate is reported [32]. The feasibility of obtaining lipase with Rhizopus delemar growing on a polymeric resin has also been investigated [36]. Babassu oil cake is used as a substrate for the lipase production by Penicillium restrictum in SSF [33]. Among these substrates, almond meal is found to be the potential solid substrate [34], whereas wheat bran with olive oil as lipid substrates is suitable for the production of acidic lipase in SSF with maximum productivity [35].

Fermentation Conditions for Lipase Production in SSF

There have been numerous reports on the optimization of culture medium, agricultural substrates and nutrition factors for lipase production in SSF [38]. Production of lipases depends on different parameters like carbon and nitrogen concentrations, pH of the medium, temperature growth, moisture content and fermentation time required [39].

Substrates used for lipase production

Rice bran: In the case of Candida rugosa, addition of urea to rice bran enhanced lipase production during optimization [40]. Although addition of carbohydrates increase the production, but it causes contamination and this leads to overall reduction in the efficiency [32]. It has been reported that using maltose had only a marginal effect on lipase production with rice bran as substrate [41]. Fermentation time of 48 h at 30°C with an inoculum concentration of 1ml/g bran conducted by rice bran with a particle size of less than 250 μm results in a higher lipase production [42].

Wheat bran: Fungal Mucor javanicus IAM 6089 with wheat bran as substrate showed an activity of about 52 U/g dry bran after 6 days [43]. In the N-rich medium, lipase activity was increased by 4.3 times when it is compared to the basal medium [37]. This study further suggested that enriching the substrates had significant influence on lipase production compared to C/N ratio.

Almond meal: Ikram ul-Haq et al. [34] screened ten moulds to observe maximum lipase activity. Maximum activity of 48.0 ± 2.1 U/g was shown with almond meal, which contains required amount of carbon, nitrogen, sucrose, gum and proteins. In addition, a combination of 50% olive oil cake and sugarcane bagasse as substrate also showed improved activity. Rhizopus rhizopodiformis demonstrated a high activity of 79.6 U/g dry matter (DM) equivalent to 43.04 U/ml whereas, Rhizomucor pusillus showed an activity of 20.24 U/g DM equivalent to 10.83 U/ml [34].

Olive oil: Venkat et al. [40] showed that oil content significantly affects the lipase yield. It has been demonstrated that 2% of olive oil as an enhancer showed a maximum lipase activity of about 30.30 U/g initial dry weight after 24 h of cultivation, whereas with peptone showed a activity of 27.80 U/g of initial dry weight [40]. Earlier investigations showed that an optimum growth of the fungal Rhizopus oligosporous GCBR-3 had maximum activity of 30 ± 2.1 U/g and use of Rhizopus strains also enhanced activity [44,45]. These fungal strains also showed greater activity with other cultures for lipase production [31,46].

Substrates with supplements/inducers: Aspergillums niger NCIM1207 illustrated higher levels of extracellular enzyme at 45°C. The maximum lipase activity of 630 IU/gdss was observed with wheat bran as substrate and the enzyme extraction was done with NaCl by supplementing Triton X-100 [35]. However, in the case of Rhizopus, Rhodotorula and Aspergillus, the lipase production seems to be independent of substrate type [47-49]. Extracellular lipase production from different microorganisms using lipids, carbohydrates and also a mixture of both has been reported on enzyme production [48-51]. The use of surfactants like Triton X-100 during SSF helps to increase enzyme production, with up to three fold increase in enzyme production both in SmF and SSF. Wheat bran has sufficient nutrients and remains loose even in moist conditions, thereby providing a large surface area for the inoculums to give maximum activity [52].

In another report, lipase production by mutant Aspergillus niger 11T53A14 using wheat bran with the fermentation medium containing 100 g of wheat bran grain (size ≤ 5 mm), humidified with a solution containing nitrogen source (ammonium sulfate) and inducer (sunflower soap stock) in a concentration of 3% (w/w) is demonstrated. The activity was tested for different nutrient compositions at pH 7.0, temperature 32°C, and 78 h and the maximum enzyme activity was observed to be 153.2 U/gdm at 0.6 % (w/w) nitrogen concentration [53].

Olive oil cake and Sugar cane bagasse: Cordova et al. [31] reported lipase production with an equal combination of olive oil cake and sugarcane bagasse as substrates. The lipase production was carried out using thermo stable fungal cultures of Rhizomucor pusillus and Rhizopus rhizopodiform is subjected to solid state fermentation. A mixture of 50% each substrate has shown an enzyme activity of about 43.01 U/ml. It was observed that under standard conditions, bagasse has low protein, less fat content and high level of cell wall component whereas olive oil cake possess high protein, fat and higher amount of lignin content [31].

Oily substrates: Out of 34 fungal species isolated from different oil substrates, nine exhibited lipolytic activities [54-56]. Maximum lipase production of 1152 μ/g was obtained after 96 h of incubation by Aspergillus species [38], whereas 8 days of incubation using A.niger at 80% moisture content attained a high enzyme titer of 1216.0 U/g [57]. Increase in the moisture level is believed to reduce the porosity of the wheat rava, thus limiting oxygen transfer [52]. At the same time low moisture content causes reduction in the solubility of nutrients of the substrates and low degree of swelling [58]. Different optimized factors, including wheat rawa with olive oil 1%, corn steep liquor 1% and 80% moisture content increase the solubility of the nutrient with the productivity of lipase in SSF 1.94 times higher than that in SmF [38].

Regiane et al. [59] used mutant strain of Aspergillus niger 11T53A14 for fractional factorial design to optimize lipase production. The soap stocks which are produced during the processing of canola, sun flower and corn has rich amount of lipid by products as inducers. The maximum lipase activity of 201.5 U/gds was observed for sun flower soap stock of 0.5% nitrogen and 3% inducers, when compared with canola and corn respectively [59].

Comparison of lipase activity with substrates

Comparison studies using fungal stains of Penicillium chrysogenum, Trichoderma harzianum and Aspergillus flavus was carried out through solid state fermentation using agro-industrial residues as substrates for optimum extracellular lipase production. For all three strains, the growth temperature was 29 ± 1°C, and 65% w (g/gds) moisture content. The effect of fermentation conditions on production are; initial pH (6.0 and 7.0), time of fermentation (72 h, 96 h and 120 h), and type of mixed substrate (wheat bran-olive oil, and wheat bran-castor oil cake). The Aspergillus flavus showed maximum lipase production of 121.35 U/gds which is five and nine times more than Trichoderma harzianum and Penicillium chrysogenum at pH-7.0 for 30.3°C having 96 h fermentation time [60].

Aspergillus niger J-1 has a maximum enzyme activity of 9.14 IU/g of dry solid substrate equivalent to 4.8 IU/ml of lipase activity using the solid state fermentation techniques with wheat bran as substrate having 0.75% ammonium sulphate, 0.34% urea and nitrogen source as the medium composition at pH-6.0 for 40°C within 24 h [61]. In another report, rice bran as substrate for Aspergillus niger has a maximum lipase production of 121.53 U/gdss at 30.3°C. These activities can further be improved by optimizing process variables such as oil concentration, glucose concentration and humidity ratio [62]. It has also been reported that endophytic fungal strain Colletotrichum gloeosporioides possess more potential for producing extracellular lipolytic enzymes in SSF with sunflower oil as substrate with a production of 2560 U/g DM at 25.0°C [63]. Extracellular lipase production using Yarrowia lipolytica NCIM 3589 with palm kernal cake as substrate alone has a maximum lipase activity of 18.58 U/gds at 96 h [64].

Scale up studies for the production of lipase using Aspergillus niger MTCC 2594 was reported with a maximum activity of 745.7 U/ gds with tri-fermented substrates and suggested that these techniques have a wide potential in cost-effective biofuel production [65]. It has been reported that fungal stain Fusarium oxysporum produced serine peptidase and lipase in alkaline condition with higher enzyme stability. Wherein, wheat bran alone showed a high activity of 228.88 U/ml for proteolytic activity and 111.48 U/ml for lipolytic activity. Study of enzyme production by varying temperature and pH suggested that these enzymes are used for application in the detergent industry [66].

Stability of lipase for different substrates

In Cordova et al. [31] study, finely ground sugar cane bagasse sample was screened to 1mm particle size and assayed for components like minerals, fat, total nitrogen, cell wall components, total fibers, lingo cellulose and lignin, fat and ash whose presence improves the enzyme production [31,52,67,68]. They observed that Rhizomucor pusillus and Rhizopus rhizopodiformis showed maximum lipase production of 1.73 U/ml and 0.97 U/ml at 16 h and 13 h respectively, whereas bagasse and olive oil cake showed a maximum production of 10.83 U/ml and 43.04 U/ml respectively. They showed that olive oil cake has more protein content than bagasse. Kamini et al. (1998) [32] showed that Aspergillus niger MTCC 2594 with gingelly oil cake had a good stability of enzyme at pH of 4.0-10.0 and temperature of 4-50°C. The results were compared with submerged fermentation, where lipase showed low thermal stability and specific activity of 57% at 60°C and 69% activity at 60°C which makes the enzyme stable in all detergents [32]. Similar results have been reported for lipase production from C.cylindracea, A.species and Rhizopus species [69,70].

Gombert et al. [33] investigated three enzymes i.e. lipase, glucoamylase and protease and observed that of three enzymes, maximum activity was obtained with media of C/N ratio [33]. A cake with 1% peptone lipase showed an activity of 4.3 times in the basal medium, whereas protease and gluco amylase showed 1.2 and 1.5 times the activity, respectively. High nitrogen concentration in culture media is effective in enhancing the production of lipase by micro-organisms [37,50,71]. Different nitrogen sources for lipase production by Penicillium strain in laboratory-scale fermentor have also been explored [71,72]. The basal medium rich in protein content as co-factors, amino acids which match P.restrictum physiological characteristics is beneficial for lipase biosynthesis. Previous investigations involving SmF showed that P. restrictum lipase is not stable at alkaline pH levels with a decrease in the lipase activity but a serine protease inhibitor (PMSF) when added at later fermentation stages showed improved activity [72,73]. The accumulation of proteases in SSF affecting the stability of lipases was also observed with fungus and with different species of Penicillium [46].

Ikram ul-Haq et al. [34] studied parameters like temperature, inoculum size etc., and showed that either lower or higher temperature result in increased energy requirements and hence, decrease the product yield [34]. Several investigations have been carried out at different cultivation temperatures and higher values as units per liter are reported [74-78]. Increase in mycelia mass, production of enzyme was shown to decline due to exhaustion of nutrients in the fermentation mash. Enzyme extraction was found to be maximum using phosphate buffer, because of permeability of membrane. Rate of enzyme synthesis using R. oligosporous showed maximum results at 48h with maximum enzyme synthesis at 2.16 u/g substrate per hour, whereas by using optimum concentration of substrate was found to be 0.0245-0.65 U/g per hour, 1.95 U/g per hour, 0.91 U/g per hour and 5.05 U/g per hour [46,79].

It is reported that the enzyme requires an energy of 114.25 and 116 KJ at lower and higher temperatures respectively for conversion of 1 mole substrate with the maximum activity at 45°C and pH 7.0- 8.5 [34]. Moisture content is a crucial factor in determining the process and physical properties of the solid substrate [52,80]. Whereas higher moisture content lead to decreased porosity and thus lowering oxygen transfer, lower moisture decrease the solid solubility, degree of swelling and produces a higher water tension. Maximum lipase production was obtained with wheat bran and synthetic oil based medium (SOB) medium in the ratio of 1:2.5 [35]. A. niger strains are known to be active at pH 4-7 and at temperatures of 40°C and 55°C. Lipase from A. niger NICM 1207 was found to be active at extreme acidic pH of 1.5-2.0 and over a broad pH range of 2.5-9.0 for up to 24 h at room temperature.

The optimum temperature of lipase production from A. niger NCIM 1207 is at 50°C and is stable at 60°C for 5 h. Addition of water to A. niger NCIM 1207 reduces the synthesis of esters shifting the equilibrium of the reaction towards hydrolysis, producing fatty acids. Thus, transesterification becomes dominant only when the availability of water is restricted. This enzyme also showed similar optimum pH in organic solvent. This phenomenon was well explained as pH memory and also reported in several literatures [81]. Characterization and partial purification of iso-lipases (Lip A, Lip B, Lip C) for biomedical application in pharmaceutical industry through SSF is well explained in detail by other investigators [82].

Assays for Determining Lipase Activity

Various techniques have been used to determine the lipase enzyme activity. The commonly used techniques are turbidimetry, interfacial tensiometry, atomic force microscopy, , titrimetry, colorimetry, flourimetry, chromatography, electron microscopy and immunodetection. Colorimetry is one of the known methods used for determining the enzyme activity. In this technique the substrates used are lipids, olive oil emulsions and para-nitrophenyl esters. The principle involved in lipid water interface oil emulsion is absorbance. Safranin absorbance change is due to the net negative charge at the lipid water interface and the product analyzed with this principle is safranin. The olive oil emulsion with copper reagent as substrate involves the estimation of copper complex at 440 nm. The complex develops a pink colour absorbance at 513 nm and the product analysed is rhodamine G-FFA complex. Another method of identifying the lipase activity is using the yellow coloured product which can be measured at 410 nm with para-nitrophenyl esters as substrate to analyze the product [83-86].

Titrimetry is the easiest way of identifying the activity of the enzyme. To identify the lipase enzyme activity involves use of stirred emulsion of TAG, tributyrin, olive oil emulsified with gum Arabic as substrates and the principle involved is pH stat-method-Neutralization of released FFAs using titrated NaOH. This is the most common procedure and is sensitive to within 1 μmol fatty acid released per min [87-88].

Turbidimetry assay is a method used for determining the concentration of a substance in a solution by the degree of cloudiness or by the degree of clarification [89]. The principle used in this technique is- increase in optical density of the sample at 500 nm due to precipitation in the form of calcium salts and the substrate used is tween20 in the presence of CaCl₂, product analysed being is free fatty acid. Electron microscopy applies the principle of detecting fatty acids by using lipids as substrates [90]. This is simple but, initial cost of the equipment is high for this assay technique. Atomic force microscopy uses lipid bilayer supported on mica as a substrate and involves the principle of lipid dissolution which forms holes on the bilayer with time and it monitored by real time images [91]. Its main advantage is that it is applicable even for the micro as well as nano-size particles. Similarly, other assay techniques like interfacial tensiometry, , flourimetry, chromatography, immuno detection are practiced based on the substrate availability the product is analyzed. Interfacial tensiometry, infrared spectroscopy, atomic force microscopy techniques are highly expensive and sophisticated in practice. The different methods for estimating the lipase activities by quantitatively and qualitatively are given in Table 2.