Pectinase production by Aspergillus niger LFP-1 using pomelo peels as substrate: An optimization study using shallow tray system

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1 Indian Journal of Biotechnology Vol 14, October 2015, pp Pectinase production by Aspergillus niger LFP-1 using pomelo peels as substrate: An optimization study using shallow tray system I Darah, M M J Taufiq and Sheh-Hong Lim* Industrial Biotechnology Research Laboratory, School of Biological Sciences Universiti Sains Malaysia Penang, Malaysia Received 11 October 2013; revised 8 November 2014; accepted 17 January 2015 A local fungal isolate Aspergillus niger LFP-1 produced high level of pectinase activity when cultivated in a plastic shallow tray ( cm 3 ) system using pomelo peels as a substrate. The highest pectinase production of U/g substrate and 3.36 mg glucosamine/g substrate of fungal growth were obtained on the 6 d of cultivation after using all the improved cultural conditions that consisted of 40 g of pomelo peel or equivalent to 0.6 cm of substrate thickness (particle size of 0.75 mm, moisture content of 1:1 ratio) with the addition of inoculums size of spores/ml, once at every 48 h of mixing frequency and cultivation at room temperature (30±2 C). After improvement of cultural condition, an increment of 51.16% of pectinase production and 8.74% of fungal growth was observed compared to the cultural condition prior to improvement. The results showed that a plastic shallow tray system was suitable for obtaining highest enzyme production in solid state fermentation (SSF), especially in case of the acidic based substrate. Keywords: Aspergillus niger, optimization, pomelo peel, pectinase, solid state fermentation Introduction Solid substrate fermentation (SSF) is a biotechnological process, which is defined as fermentation process involving solids in the absence or near absence of free water 1. It is widely adopted in fermentations used for the production of microbial growth and secondary metabolites. This process is an attractive method for the production of industrially demanded enzymes that employ microorganisms. SSF systems are closer to microbial natural habitats and thus make them usually to produce secondary metabolites more efficiently. Various types of biomass, especially, those rich with lignocellulosic material and pectin are suitable for SSF in pectinase production. The substrate commonly used for pectinase production are of plant origin, such as, agricultural wastes containing large amount of celluloses, hemicelluloses and pectin, that could serve as a carbon and inducer source for the production of enzymes 2. Pectinases are a group of enzymes found in fruits and their functions are to hydrolyze pectin into polygalacturonic acids and finally to galacturonic acid. Pectinases can be divided into pectin lyase (EC ), pectin esterase (EC ), pectate lyase *Author for correspondence: Tel: ; Fax: limshehhong77@gmail.com (EC ) and polygalacturonase (EC ) based on their mode of action of hydrolyzing glycosidic linkages in the pectic polymers 3. Amongst them, polygalacturonase, involved in the hydrolysis of polygalacturonic acid chains, is the most-studied and widely used pectinase 4. Pectinases are extensively used in the feed and drink industries, chiefly in fruit juice clarification because they are capable of reducing the viscosity of liquors 5. Moreover, they also have other application including extraction of vegetable oils 6, curing of coffee and cocoa beans 7, refinement of vegetable fibers and manufacturing of pectin-free starch 8. In the textile industry, pectinases are sometimes used in the treatment of natural fibers, such as, cotton and ramie fibers Therefore, it is important to discover new pectinase producing microbes and to optimize their enzyme production conditions in order to meet increasing industrial demand. In Malaysia, pomelo is widely grown in plantations. The rejected pomelo fruits and peels are discarded or disposed by natural decomposition. Sometimes, the rejected pomelo fruits are burnt in an open and this may cause a severe problem in the community as well as environment. The pomelo peels is reported to contain of 16.9% soluble sugars, 3.75% fibers (9.21% cellulose, 10.5% hemicelluloses, 0.84% lignin and about 42.5% pectin), 3.5% ashes, 1.95% fat

2 DARAH et al.: POMELO PEELS AS SUBSTRATE FOR PECTINASE PRODUCTION 553 and 6.5% protein 12. Since pomelo peels contains an appreciable amount of pectin and, therefore, it can be used as a substrate for the production of pectinase by microorganisms under SSF system. In the present communication, we are reporting the pectinase production by a local fungal isolate Aspergillus niger LFP-1 using pomelo peels as a substrate in shallow trays. This included the improvement of cultural conditions (physical parameter) that influenced the highest enzyme production. Materials and Methods Fungal Culture, Maintenance and Inoculum Preparation Aspergillus niger LFP-1, which was isolated from a rotten orange, was supplied by the Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia. The fungal culture was maintained on potato dextrose agar slants supplemented with 1.0% citrus pectin (w/v) at 30 C for 5 d aerobically (until sporulation) and then stored at 4 C until further use. The stored cultures were sub-cultured regularly at every month to maintain viability. The fungal spores were extracted from agar slant by transferring 5.0 ml of sterile distilled water into them and then shaking the slant tubes vigorously. The spore concentration was adjusted to a desired concentration (spores/ml) using a haemocytometer slide chamber (Neubauer, Germany) and used further as inoculum. SSF and Initial Profiling of Pectinase Production Fresh pomelo peels used in the present study were obtained from a stall at Changlun, Kedah, Malaysia. The pomelo peels were chopped into small pieces of uniform size and dried under sunlight until constant weight. They were then ground to powder form (specified particle size) prior of its use in SSF. Because of acidic condition of the substrate (ph 4.3), shallow plastic trays with the size of cm 3 were used throughout the study. Before the start of the study, the bed height of the substrate in a tray was standardized. The effect of different bed height or substrate thickness layer was studied to evaluate the optimum substrate thickness for the enzyme production. The substrate thickness used were 0.3, 0.6, 0.9 and 1.2 cm, which were equivalent to 20.0, 40.0, 60.0 and 80.0 g of substrate (0.75 mm substrate size), respectively. All the beds were inoculated with spores/ml of inoculums size and 1.20% (w/v) of ammonium nitrate as a nitrogen source. The moisture content (sterile distilled water) of the bed was maintained at 1:1 ratio and cultivated at room temperature (30±2 C). The substrate mixture was mixed together to ensure the uniformity before spreading it on the tray. The cultivation was carried out for 10 d and the pectinase activity as well as fungal growth was determined at an interval of 24 h. All experiments were performed in triplicates and the results were presented as means of 3 experiments. Improvement of Different Cultural Conditions on Pectinase Production and Fungal Growth The improvement of SSF system for production pectinase and fungal growth was carried out for 7 d based on the modification of parameters of cultural condition, such as, cultivation temperature (25±2, 30±2, 35±2 & 40±2 C), moisture content (in ratios of 1:0.5, 1:1.0, 1:1.5, 1:2.0 & 1:2.5), inoculum size (1 10 5, , , & spores/ml) and mixing frequency (static or once at every 12, 24, 48 & 72 h). All experiments were performed in triplicates and the results were presented as means of 3 experiments. Extraction of Enzyme Crude enzyme of pectinase was extracted by using modified method of Darah et al 13. The sterile distilled water (50 ml) containing 0.1% (v/v) of Tween 80 was added to fermented pomelo peels. The mixture was then mixed using a rotary shaker for 30 min at 30 C and agitated at 150 rpm. The solid residue was separated from enzymatic solution by filtration through Whatman filter paper no. 1. The cell free filtrate was used as the source of pectinase enzyme. Enzyme Assay Pectinase assay was carried out using modified DNS method 14. The reaction mixture (1.0 ml) containing equal amounts of pectin (0.5%) prepared in citrate buffer (0.05 M, ph 4.5) and suitable diluted enzyme was incubated at 50 C for 30 min in a water bath. After incubation, 3.0 ml DNS solution was added to stop the reaction and the tubes were kept in boiling water for another 10 min. On cooling, the developed colour was read at 575 nm using UV-visible spectrophotometer (Spectronic Unicam). The amount of released reducing sugar was quantified using D-galacturonic acid as a standard. The enzyme activity was defined as the amount of enzyme required to release one micromole equivalent of D-galacturonic acid per min under assay condition.

3 554 INDIAN J BIOTECHNOL, OCTOBER 2015 Determination of Fungal Biomass (Glucosamine) The fungal growth was determined using method previously described by Swift 15. The fermented sample was dried at 80 C until constant weight and the glucosamine content was detected spectrophotometrically at 530 nm. The fungal growth was expressed as mg glucosamine per g of substrate. Glucosamine powder was used as a standard. Statistical Analysis One-way analysis of variance (ANOVA) and Duncan multiple range test (DMRT) with PASW Statistics 18 version were used to analyze the significant difference of the mean of experimental data. A 5% confidence level or α=0.05 was used to test all experimental data. All experiments were carried out in triplicates. Results Profiling of Incubation Period through Different Substrate Thickness Four substrate thicknesses (substrate bed heights), i.e., 0.3, 0.6, 0.9 and 1.2 cm (equivalent to 20.0, 40.0, 60.0 and 80.0 g of substrate of pomelo peels, respectively), were tested for the present tray system (Fig. 1). The bed height of 0.3 cm (equivalent to 20.0 g substrate) produced the highest pectinase (35.73 U/g substrate) and fungal growth (3.03 mg glucosamine/g substrate) on the 6 th d of cultivation (Fig. 1A). However, in case of 0.6 and 0.9 cm substrate thicknesses, the highest pectinase production (52.19 & U/g substrate, respectively) and fungal growth (3.09 & 3.12 mg glucosamine/g substrate, respectively) was obtained on 7 th d of cultivation (Figs 1B-C). Finally, when substrate thickness of 1.2 cm (equivalent to 80.0 g of substrate) was used, the highest pectinase production (55.29 U/g substrate) and fungal growth (3.29 mg glucosamine/g substrate) was achieved on 8 th d (Fig. 1D). Thus the results show that the period of optimized enzyme production get increased with the thickness of substrate and the highest pectinase productions (7.46 U/g/d) was achieved in 0.6 cm (equivalent to 40.0 g) thickness of substrate. However, other substrate thicknesses (0.3, 0.9 & 1.2 cm) achieved lower enzyme production (5.96, 6.97 & 6.91 U/g/d). Improvement of Cultural Conditions for Pectinase Production Based on the initial profiling, the substrate thickness of 0.6 cm (equivalent to 40.0 g) of pomelo peels was selected for further optimization of cultural conditions in a tray system. Effect of Incubation Temperature The effect of five different cultivation temperatures was studied on pectinase production and fungal growth (Fig. 2). It was observed that the production of pectinase (52.28 U/ g substrate) and fungal growth (3.20 mg glucosamine/g substrate) was optimum at room temperature (30±2 C) (Duncan p<0.05). Incubation temperature beyond or lower the optimum level produced lower amount of pectinase. Therefore, room temperature (30±2 C) was used as cultivation temperature for the subsequent studies. Effect of Moisture Content The effect of moisture content on pectinase production was investigated and the results are shown in Fig. 3. Initially, the pectinase production and fungal growth increased when the moisture content increases from 1:0.5 to 1:1 and reached to the highest pectinase production (52.31 U/g substrate) Fig. 1 (A-D) Effect of different bed heights on pectinase production and fungal growth by A. niger LFP-1 in a shallow tray system: (A) Bed height of 0.3 cm (20.0 g substrate); (B) Bed height of 0.6 cm (40.0 g substrate); (C) Bed height of 0.9 cm (60.0 g substrate); & (D) Bed height of 1.2 cm (80.0 g substrate). [The experiment was carried out in triplicate; the different letters means that there are significantly different between means (Duncan, p < 0.05). Error bars indicate the standard deviation values of the triplicates. *mg/g substrate = mg glucosamine/g substrate]

4 DARAH et al.: POMELO PEELS AS SUBSTRATE FOR PECTINASE PRODUCTION 555 and fungal growth (3.13 mg glucosamine/g substrate) (Duncan, p < 0.05). However further increase in moisture content (1:1.5, 1:2 & 1:2.5) abruptly decreased the production of pectinase production (40.05, & 2.15 U/ g substrate) as well as fungal growth (Fig. 3). Thus, the moisture content in the ratio of 1:1 was optimal for the production of pectinase and retained in the next experiment. Effect of Inoculum Size The effect of inoculums size ( spores/ml) of A. niger LFP-1 on the production of pectinase was studied. With the increase in inoculums size ( spores/ml), the production of pectinase and the growth of fungus increased and reached to the optimal (60.97 U/g substrate & 3.43 mg glucosamine/ g substrate, respectively) at inoculum size of spores/ml (Fig. 4). However, further increase in inoculums size ( spores/ml) showed an abrupt decline in enzyme production (31.27 U/g substrate) and fungal growth (3.05 mg glucosamine/g substrate). Thus inoculum size of spores/ml produced the highest pectinase (Duncan p < 0.05) and the same size was used in the subsequent experiments. Effect of Mixing Frequency The frequency of substrate mixing is an important condition for enzyme production in a tray system. An appropriate mixing or agitation can lead to a better production by controlling the heat accumulation during fermentation. The increase in period of substrate mixing from once at every 12 h to 24 h showed an abrupt increase in pectinase production (11.57 to U/g substrate) and fungal growth (2.56 to 3.46 mg glucosamine/g substrate), which further increased to its optimal (73.06 U/g substrate; 3.51 mg glucosamine/g substrate) once at every 48 h of substrate mixing (Fig. 5). However, once at every 72 h of substrate mixing, the pectinase production declined to U/g substrate and Fig. 2 Effect of different cultivation temperature on pectinase production and fungal growth by A. niger LFP-1. [The experiment was carried out in triplicates; the different letters means that there are significantly different between means (Duncan, p < 0.05). Error bars indicate the standard deviation values of the triplicates. rt: room temperature, 30±2 C] Fig. 4 Effect of different inoculum sizes on pectinase production and fungal growth by A. niger LFP-1. [The experiment was carried out in triplicates; the different letters means that there are significantly different between means (Duncan, p < 0.05). Error bars indicate the standard deviation values of the triplicates.] Fig 3 Effect of moisture content on pectinase production and fungal growth by A. niger LFP-1.[The experiment was carried out in triplicates; the different letters means that there are significantly different between means (Duncan, p < 0.05). Error bars indicate the standard deviation values of the triplicates.] Fig. 5 Effect of different mixing frequency on pectinase production and fungal growth by A. niger LFP-1. [The experiment was carried out in triplicates; the different letters means that there are significantly different between means (Duncan, p < 0.05). Error bars indicate the standard deviation values of the triplicates.]

5 556 INDIAN J BIOTECHNOL, OCTOBER 2015 fungal growth to 3.18 mg glucosamine/g substrate. The figures for pectinase production (62.58 U/g substrate) and fungal growth (3.48 mg glucosamine/g substrate) in static state or no substrate mixing condition were comparable to the figures for once at 24 h of substrate mixing. Thus, the mixing frequency once at every 48 h gave the highest enzyme production as well as fungal growth (Duncan, p < 0.05), and used for the next experiment. Profile Post-improvement of Cultural Conditions in a Tray System for Pectinase Production A time course profile after the improvement of cultural conditions (40.0 g of pomelo peel with 0.75 mm of substrate size, 1:1 ratio of moisture content, spores/ml inculum size & once at every 48 h mixing frequency of substrate) was studied for 10 d. The results show a gradual increase in the productivity of enzyme and fungal growth initially, which reached to its maximum productivity (78.89 U/g substrate; 3.36 mg glucosamine/g substrate) on the 6 th d of cultivation time (Fig. 6). However, further increase in cultivation time beyond 6 th d showed a gradual decrease in both parameters and, on 10 th d, it reached to U/g substrate for the productivity of enzyme with 2.28 mg/g substrate fungal growth. Thus the results of present study show that optimization had substantial effect on the production of pectinase. This study further shows that the relationship between fungal growth and enzyme production was linearly related to enzyme production (Duncan p < 0.05). Fig. 6 Time course profile of pectinase production and fungal growth under SSF in a shallow tray system after the improvement of physical parameters. [The experiment was carried out in triplicates; the different letters means that there are significantly different between means (Duncan, p < 0.05). Error bars indicate the standard deviation values of the triplicates.] Discussion In SSF, substrate thickness is an important factor. The fungal growth on the surface of substrate is almost similar, but growth within the substrate varies depending on the substrate thickness, also known as the bed height. Pandey 16 reported that tray bioreactor was limited because of mass transfer and heat. It could develop large internal temperature gradient and gas concentration with substrate thickness above 40 mm. Higher bed thickness would cause in decreased oxygen availability at the middle and bottom area of substrate, finally affecting the fungal growth as well as the enzyme activity. In the present study, it was observed that 40 g of pomelo peels or equivalent to 0.6 mm of substrate thickness produced optimum enzyme yield. Substrate size is also an important parameter in SSF to produce high yield of enzymes because smaller particle size may lead to clumping of substrate and result in reduced accessibility to nutrients and insufficient oxygen supply that finally lower the enzyme production. According to Membrillo et al 17, the size of substrate (sugar cane bagasse fibers) has strong influence in SSF. In the present study, production of pectinase was poor at moisture level lower or higher than 1:1 ratio. The low level of moisture content leads to the reduction of substrate swelling, solubility of solid substances, nutrient diffusion and also prevents the nutrient absorption from the substrate. These factors result in insufficient nutrient supply to microorganism, which in turn causes the reduction in microbial growth and enzyme production 18, and sometimes causes higher water retention. Although a rise in moisture up to optimum level enhances enzyme production, too high level of initial moisture content has unfavorable effect on enzyme production 19. SSF is an exothermic process where temperature directly influences the growth of microorganisms and product formation. In the present study, we used room temperature (30±2 C) as this temperature was found optimal for the production of pectinase in SSF. Darah et al 20 and Syarifah et al 21 have also reported that temperature higher to optimum level (30 C, room temperature) led to the reduction of mannanase yield. Temperature of 30 C has been proven to be the best temperature in enzymatic synthesis. In our case, 30±2 C was the room temperature and by using this temperature we can save on the energy cost.

6 DARAH et al.: POMELO PEELS AS SUBSTRATE FOR PECTINASE PRODUCTION 557 Fungi endure a contact between hyphae and substrate via its hyphal growth. Therefore, increment or decrement of spore on a substrate surface will affect the availability of nutrients from the medium. Low inoculum size influences the time extension for cell proliferation to utilize substrate and produces the desired product 22. In the present study, this problem could be observed at inoculums size of spores/ml where the lowest pectinase enzyme was produced. On the other hand, higher inoculums size induces the cell production, biomass synthesis and shortens the lag phase during fungal growth. Thus, an inoculam size of spores/ml was found to be optimal for the production of pectinase, while enzyme production declined with further increase in inoculam size. Kumar et al 23 have also suggested that higher inoculum than the optimum may produce too much biomass and may deplete the nutrient that is necessary for microbial metabolite production. Microbial activity of anaerobic cultures is markedly affected by the air supply to the system. There are varying functional aspects of aeration in SSF processes including maintenance of oxygen supply and removing carbon dioxide from the system, heat transfer and the control of moisture level. The results of the present study showed that aeration rate by mixing frequency improved the production of pectinase. The favourable effect of air flow on the enzyme production could be attributed to the enhancement of product formation by microorganism under-forced aeration. The optimum mixing frequency obtained from the present study was once at every 48 h. The optimum level of mixing frequency could depend on the selected microorganism, the particular amount of oxygen for product synthesis, the level of heat evolution to be removed, the quantity of carbon dioxide and other volatile metabolites which would be dissipated, the thickness of substrate bed height and also the volume of pore space in the substrate. As the major parameters affecting the fermentation process for enzyme production were studied and optimal levels were identified. It was noted that the 6 th d of cultivation time produced optimum yield of pectinase in the present study. Thus, optimum cultivation conditions of the present study are cost-effective and such technologies are needed for economical production of pectinase using pomelo peels as substrate in SSF. Conclusion The present work suggests that pomelo peel could be employed as a promising substrate for the production of pectinase by A. niger LFP-1 in a shallow plastic tray system. The optimum cultivation conditions significantly affected the pectinase production in SSF. Under optimized conditions, pectinase yield was U/g substrate, which corresponded to 51.16% of increment compared to the yield before the improvement of cultivation conditions. Acknowledgement The authors would like to thank Universiti Sains Malaysia for awarding Prototype Research Grant Scheme (PRGS) grant to MMJT to complete his M Sc dissertation. References 1 Krishna C, Solid-state fermentation systems An overview, Crit Rev Biotechnol, 25 (2005) Patil S R & Dayanand A, Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions, Bioresour Technol, 97 (2006) Kuhad R C, Kapoor M, & Rustagi R, Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation, World J Microbiol Biotechnol, 20 (2004) Kashyap D R, Vohra P K, Chopra S & Tewari R, Application of pectinases in the commercial sector: A review, Bioresour Technol, 77 (2001) Singh S & Gupta R, Apple juice clarification using fungal pectinolytic enzymes and gelatin, Indian J Biotechnol, 3 (2004) Demir H, Gogus N, Tari C, Heerd D & Lahore M F, Optimization of process parameters for the utilization of orange peel to produce polygalacturonase by solid-state fermentation from an Aspergillus sojae mutant strain, Turk J Biol, 36 (2012) Quattara H G, Koffi B L, Karou G T, Sangare A, Niamke S L et al, Implication of Bacillus sp. in the production of pectinolytic enzymes during cocoa fermentation, World J Microb Biotechnol, 24 (2008) Pedrolli D B, Monteiro A C, Gomes E & Carmona E C, Pectin and pectinases: Production, characterization and industrial application of microbial pectinolytic enzymes, Open Biotechnol J, 3 (2009) Tzanov T, Calafell M, Guebitz G M & Cavaco-Paulo A, Bio-preparation of cotton fabric, Enzyme Microb Technol, 29 (2001) Basu S, Saha M N, Chattopadhyay D & Chakrabarti K, Large scale degumming of ramie fiber using a newly isolated Bacillus pumilus DKS1 with high pectate lyase activity, J Ind Microbiol Biotechnol, 36 (2009) Sharma D C & Satyanarayana T, Biotechnological potential of agro residues for economical production of thermoalkalistable pectinase by Bacillus pumilus dcsr 1 by solid-state

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