International Journal of Application or Innovation in Engineering & Management (IJAIEM)

Web Site: www.ijaiem.org Email: editor@ijaiem.org, editorijaiem@gmail.com Volume 3, Issue 1, January 2014 ISSN 2319 - 4847

A study on the effects of environmental factors on fungal -amylase synthesis utilizing cerealprocessing mill residues.
Sibabrata Mukherjee*, Sujata Kulkarni and Milind Kulkarni
Biotech Lab, Chemical division, Thermax Ltd. 97-E, General Block, Bhosari, Pune, Maharashtra. PIN-411026. India

Abstract
Different environmental parameters were monitored and subsequently optimized for the production of fungal -amylase using various cereals processing mill residues viz. wheat, rice and maize barns. The fungal strains were isolated from the natural sources which were screened and selected based on their starch degrading properties. The selected strain of Aspergillus sp. SM 07 was cultivated adopting solid state fermentation (SSF) and wheat bran was found to be the superior carbon source for maximum amylase production. The effects of environmental factors including initial moisture content (IMC), incubation temperature, initial pH, inoculum level and solid to flask volume ratio were studied following one parameter at a time approach during cultivation of Aspergillus sp. SM 07 on wheat bran. The maximum enzyme activity per gram of dry substrate (34220 IU/gds) was attained when the SSF was conducted with 20g of solid substrate in 500 ml Erlenmeyer flask at 300 C for 96h and the IMC, pH and inoculum level were 60%, pH-5 and 4ml respectively.

Keywords: Aspergillus, solid state fermentation, environmental factors, amylase.

1. INTRODUCTION
Among the countries in the Asia-Pacific Region, India has been found to be the second largest producer of agriculture waste and crop residues [1]. Generally the agro-residues belong to two groups: the plant materials left behind in the farm after removal of main crop and agro-industrial residues, the refuses produced by crop processing mills [2]. India generated agro-residues comprising rice hull, rice bran, wheat bran, corn residues etc which had been estimated to be ~890 million tons in 2010, of which ~190 million tons being mill processing residues [2]. Although a small part of such residues are utilized in fish farming, animal feed, etc but larger part of the residues remains as waste [3]. The land disposals of such low bulk density components also require space for treatment time. In recent years there has been increasing interest in the effective utilization of these residues as carbon source for microorganisms to produce added value chemicals, enzymes etc [4]. Solid state fermentation (SSF) technique, has contributed enormously for such utilization [5]. Solid state production of -amylase on agro residues is one example of such value additions [6]. Apart from utilization of agro wastes SSF has been found to be advantageous over submerged fermentation (SmF), the conventional mode of enzyme production, in some other aspects has been reported by scientists [7] & [8]. -amylase (EC3.2.1.1, 1,4--D-glucan-glucanohydrolase) is a starch hydrolyzing enzyme and distributed in all organisms including plants, animals and microorganisms. From the industrial view point the microbial amylase has been received maximum attention [9]. Among the various groups of microorganisms used in SSF, filamentous fungi are the most widely exploited because of their ability to grow on complex solid substrates and production of a wide range of extracellular enzymes [10]. Amylase was first produced industrially from a fungal strain in 1894, for pharmaceutical application [11]. Gradually with time this enzyme found vivid applications in the area of food, feed, fuel, paper, textiles, detergent etc. [12] and consequently the microbial -amylase represents approximately 30% of the world enzyme market. [13]. Many enzyme preparations such as proteases, lipases, xylanases, pullulanases, pentosanases, cellulases, glucose oxidases, lipoxygenases etc. have been alternative to other enzyme substituted but none have -amylases [14]. Due to the increasing demand for these enzymes in various industries, there is enormous interest in developing enzymes with diverse properties, more industrial applications and more economic production processes [15] & [16]. Isolation of new microorganisms suitable for amylase production could provide potential new sources of the enzyme and with novel bio-chemical properties as well. In recent years tremendous research effort is insistently going on in this sector too [17]-[19]. The present R&D work was aimed to isolate potential fungal strain having starch degrading property and evaluation of the effects of environmental factors on the -amylase production during SSF utilizing low-cost cereal processing mills residues.

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Web Site: www.ijaiem.org Email: editor@ijaiem.org, editorijaiem@gmail.com Volume 3, Issue 1, January 2014 ISSN 2319 - 4847 2. METHODOLOGY
2.1 Isolation and screening of amylase producing fungi Eleven fungal strains were isolated from the soli and spoiled food samples. One gram of each sample of fungal source were dissolved in 100 ml of sterile normal saline and mixed properly in a rotary shaker for 2h. The stocks were diluted up to 104 dilutions from which 1 ml was pipette out and pour platted in Czapek Dox agar medium. The plates were incubated for 4 days at 300 C. All the strains were subcultured on Czapek Dox agar slants and the pure cultures obtained were stored at 40C for further use. To detect the potential fungal strains for -amylase production, the isolates were spot cultured on the starch agar medium composed of (g/L) of starch soluble, 20; KH2 PO4, 1.0; KCl, 0.5; NaNO3, 2.0; MgSO4.7H2O, 0.5; FeSO4, 0.001; agar, 30 and pH: 4-4.2. The plates were incubated for 7 days at 300C.Starch hydrolysis properties were evaluated by measuring the diameter of the clear-zone developed upon exposure to the iodine solution. The potential isolate was identified on the basis of morphological and microscopic features and stored on Czapek Dox agar slants at 40C. 2.2 Preparation of inoculum Spore suspension was prepared by dislodging the spores from a seven days old culture of the selected strain in the sterile water containing 0.01% Tween-80. The spore suspension of one ml (6*107 spores, estimated by hemocytometer) was used as the inoculum. 2.3 Selection of substrate Different cereal mill residues including soy hull, wheat bran, rice bran and corn bran were used as solid substrate during SSF. All the materials were properly washed with deionized water and dried at 700 C for overnight prior to use. 2.4 SSF process The SSF was carried out in 500 ml Erlenmeyer flak. Ten gram of solid substrate was taken in to the flask and moistened with the mineral salt solution (MSS) composed of (g/L) KH2 PO4, 1.0; KCl, 0.5; NaNO3 , 2.0; MgSO4.7H2 O, 0.5; FeSO4, 0.001; and pH: 4. The mixture was sterilized by autoclaving for 15min. The media was inoculated with 1ml of immoculum and fermentation was carried out up to 144 h at 300C in a humidity controlled chamber with the initial moisture content of 70%. The flask content was aseptically mixed at a regular time interval. 2.5 Extraction of extracellular enzyme The fermented bran was mixed properly and soaked with its five volume of distilled water at 40C for overnight. The liquid was extracted by squeezing the wet mass with cheese cloth and the centrifuged at 10000 rpm at 100C for 20 min for clarification. The mycelia and spore free extract was used as the enzyme source. 2.6 Enzyme assay -amylase activity was estimated adopting the assay method described by Bernfeld (1955) with minor modification [20]. A reaction mixture containing 0.5 ml of 1% soluble starch solution prepared in 0.01 M acetate buffer and 0.5 ml of diluted enzyme solution was incubated at 500C. After 10 min of incubation the reaction was stopped by adding 3 ml of DNS. Reaction mixtures were boiled for 10 min followed by cooling. Absorbance was measured at 540 nm. Appropriate blanks were used. One unit of the enzyme (IU) was defined as the amount of enzyme that produced 1 mol of glucose per minute. 2.7 Evaluation of the effect of different environmental parameters on amylase production The environmental parameters viz. initial moisture content (IMC), inoculum volume, incubation temperature, initial pH, substrate amount to flask volume ratio were monitored at their different levels to evaluate their effect on the enzyme production during SSF. One parameter at time approach study was adopted for this assessment. The IMC (%) was adjusted with the MSS between 40-90% (w/w). The initial pH was varied from pH: 3-7 and incubation temperature was monitored in the range of 25-400C. To analyze the effect of different pH, the pH of the MSS was adjusted to the desired level. The inoculum volume was varied from 1ml-5ml per batch of SSF. The substrate to flask volume ratio was also varied by changing the substrate amount and the range was from 0.01-0.06 (w/v).

3. RESULTS AND DISCUSSION

3.1 Selection of the fungal strain and the substrate for the enzyme production Among the 11 fungal strains isolated from the environmental sources, based on the plate based screening one isolate named (SM 07) was found to be potential (clearing zone dia-2.6mm) and selected for the next stage studies. The fungal strain was identified as Aspergillus sp. on the basis of its morphology (Fig.1) and certain biochemical characterization

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Web Site: www.ijaiem.org Email: editor@ijaiem.org, editorijaiem@gmail.com Volume 3, Issue 1, January 2014 ISSN 2319 - 4847
[21]. The Aspergillus sp. SM 07 was cultivated on different low cost materials by SSF manner. Out of the four substrates wheat bran was found to be superior for the production of amylase (Fig.2). The maximum enzyme activity obtained 253 IU/gds after 96h of fermentation. Similar finding on SSF based amylase production was also reported by Kumar et al., 2013; Sindhu et al., 2009 [22] &[23].

Figure 1. The clear zone formed due to the starch hydrolysis. (a). Morphology of the fungal isolate (Aspergillus sp SM 07) under light microscope stained with Lacto phenol cotton blue. (b)

Figure 2 Screening of various agro residues for the production of -amylase using Aspergillus sp SM 07 at different incubation period (h) by SSF. Data shown represent the mean standard error of experiments that were conducted in triplicate. 3.2 Effect of IMC on the on enzyme production The enzyme production conducted at different level of IMC for the 96h. It was revealed that the maximum enzyme activity (289 IU/gds) was obtained at 60% IMC (Fig.3). Identical observations were reported earlier by Sindhu et al., 2009 [23]. The subsequent experiments for rest of the parameter investigation were performed at optimum IMC. IMC was known to be required for swelling of the solid matrix like agro residues which in turn facilitated better substrate utilization of the substrate by microorganisms [24]. 3.3 Effect of incubation temperature on the on enzyme production Maximum enzyme activity (297 IU/gds) was observed when the SSF was carried out at 300C (Fig.3). The activity was decreased at lower and higher points from the optimal point. Similar finding was reported by Francis et al. 2003 during SSF production of -amylase using Aspergillus oryzae as source [25].

Figure 3 Effect of IMC (%) and incubation temperature (0C) on the -amylase production by Aspergillus sp SM 07 during SSF on wheat bran medium. Data shown represent the mean standard error of experiments that were conducted in triplicate.

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Web Site: www.ijaiem.org Email: editor@ijaiem.org, editorijaiem@gmail.com Volume 3, Issue 1, January 2014 ISSN 2319 - 4847
3.4 Effect of initial pH on the on enzyme production Total seven sets of batch SSF were carried out covering low, neutral and high range of acidity to investigate the effect of initial pH on the enzyme activity. Although the media pH is generally monitored in SmF, SSF based production had also been reported to be influenced by initial media pH [26]. Maximum enzyme activity (296 IU/gds) was observed at pH: 5.0 (Fig.4). With the increase in pH beyond 5.5 the sharp activity decline was noticed. At lower pH, mycellial growth was poor and enzyme activity was also lesser than that of the optimum point. Further optimization steps were done by conducting the SSF at pH-5.0. 3.5 Effect of inoculum volume on the on enzyme production It was reported that the inoculums level also contributed in productivity of enzymes during SSF [27]. Different volumes of inoculum were tried to point out the optimum inoculum requirement for the enzyme production. There was a significant increase in enzyme activity with the increase in inoculum volume till 4ml from 1ml but beyond this the marginal change in activity was noticed (Fig.4). Four ml of inoculum was used for the subsequent evaluation steps. 3.6 Effect of solid to flask volume ratio on the on enzyme production: Considerable amount of enzyme yield was received when the SSF was conducted at various solid to flask volume ratio. Maximum enzyme activity (32920 IU/gds) was obtained when 20g of wheat bran media was fermented at sequentially optimized conditions.

Figure 4 Effect of initial medium pH and inoculum volume (ml) on the -amylase production by Aspergillus sp SM 07 during SSF on wheat bran medium at 300C and 60% IMC. Data shown represent the mean standard error of experiments that were conducted in triplicate.

4. CONCLUSION
From the present investigation it can be concluded that the cereal processing mills rejects can be utilized for the value added product development, in this case, the enzyme -amylase. The utilization of such low cost material helps to cut down the raw material cost for the development of biotechnological products and reduces the waste handling cost as well. However the environmental factors were also found to be significantly influential on enzyme yield during SSF. One parameter at a time approach to evaluate the effects of the process parameters, viz. IMC, pH, inoculum volume, temperature and solid to flask volume ratio, was also found to be effective. It was revealed from this study that almost 1.5 times (34220/24020 IU/gds) better amylase yield had been achieved following sequentially directed optimization strategy for those five factors. The amylase titre obtained in the present study indicated good potentiality of the fungal isolate, Aspergillus sp. SM 07, to be used for further development on it.

ACKNOWLEDGEMENT
The R&D work was fully funded by Thermax Ltd. India.

References
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Web Site: www.ijaiem.org Email: editor@ijaiem.org, editorijaiem@gmail.com Volume 3, Issue 1, January 2014 ISSN 2319 - 4847
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