Evaluation of Potential Activity of Fungi Isolated From ... - IJIRST

IJIRST –International Journal for Innovative Research in Science & Technology| Volume 2 | Issue 11 | April 2016 ISSN (online): 2349-6010

Evaluation of Potential Activity of Fungi Isolated From Bagasse for Gluconic Acid Production Nilesh K. Purane Department of Biotechnology PG Research Center, MGSM’s ASC College, Chopda 425107 Dist. Jalgaon

Prakash S. Lohar Department of Biotechnology PG Research Center, MGSM’s ASC College, Chopda 425107 Dist. Jalgaon

Abstract The prime importance of any viable and successful industrial fermentation process is economics, and to greatest extent depends on selection of material, strain and strategies for fermentation process. The present attempt was to increase gluconic acid production using glucose as sole carbon source with minimum residence time by using isolated strain of A. niger from bagasse. In this experiment, batch fermentation was employed in 50L semiautomatic PLC stirred tank fer-menter, equipped with controller for optimum growth conditions. The high promising gluconic acid (GA) production (85.20 gL-1) was observed with nearly greater than 100% yield over 36 hours. This process provides significant ad-vantages over traditional submerged fermentation strategies, as showed 100% conversion of glucose to GA, made fewer difficulties during product recovery. To reduce analysis time with better accuracy, an effort has been made to use a method for evaluation of parameters like conversion of substrate and production of GA during the fermentation by using High Performance Thin Layer Chromatography (HPTLC) through the quantitative analysis of GA and glucose from microbial fermentation by A. niger. Keywords: Aspergillus niger; Submerged fermentation; Gluconic acid; Glucose; Semiautomatic PLC stirred tank fermenter _______________________________________________________________________________________________________ I.

INTRODUCTION

Organic acids production using microbial submerged fermentation processes through conversion of low cost substrate to high value products is a promising approach for obtaining building-block chemicals from renewable carbon sources. Because of some outstanding properties of GA such as extremely low toxicity, very low corrosiveness, a capability of forming water soluble complexes and the property of plastifying concrete and retarding the setting process, it is regarded as a bulk chemical in the food, beverage, textile, pharmaceutical, and construction industries [1]. Due to low selectivity in chemical synthesis of GA, it is uneconomical for industrial purpose [2]. Hence, the microbial process are employed for conversion of glucose into GA in submerged fermentation using fungal species like A. niger and Penicillium, GliocadiumandGonatabotrysetcand even some bacterial species such as G. oxydans, G. diazotrophicus, Z. mobilis, A. methanolicus, P. florescens, and the species of Morexella, Tetracoccus, pullularia, Micrococcus, EnterobacterandScopulariopsisetc have been tested and reviewed in the past [3, 4, 5, 6]. About 50,000-60,000 tons of GA is produced annually worldwide using glucose as substrate. The most favoured production process is submerged fermentation by A. niger utilizing glucose as a major carbon source, which accompanied product yield of 98% [7]. Among the other, refined glucose & sucrose have also been main substrates for GA production [8]. However, use of GA and its derivatives is currently restricted because of high prices about US$ 1.20–8.50/kg [1]. Its huge market consumption has spurred interest in the development of an effective and economical system for GA production. Now a day, A. niger is an effective process but there is still a certain interest in improving productivity and production yields by trying different production strategies during industrial gluconic acid fermentation approach [1, 9]. So, present study is aimed to evaluate isolated A. niger strain, particularly submerged fermentation conditions may facilitate the strategies of commercial production of gluconic acid. During the fermentation, the various analytical parameters like substrate conversion and product formation are presently carried out by HPLC or enzymaticspectrophotometric methods. This is a time taking and costly affair for industries.Although effort for qualitative analysis of GA and glucose was made by Schleissner et al. (1997)using the traditional TLC technique. HPTLC facilitates automated application and scanning in situ. It offers extreme flexibility for various step of TLC. In present communication efforts were made to quantification of GA and glucose with HPTLC [10].

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Evaluation of Potential Activity of Fungi Isolated From Bagasse for Gluconic Acid Production (IJIRST/ Volume 2 / Issue 11/ 022)

II. MATERIALS AND METHODS Isolation and Identification of Fungal Strain Sample was taken from solid waste bagasse located in area of the sugarcane farm, Chopda dist. Jalgaon. The sample was added to 10 ml sterile distilled water in test tube. From test tube 0.5 ml volume pipette onto Sabouraud-glucose medium with 50 μg/ml of chloramphenicol or 40 μg/ml of streptomycin plus 40 UI/ml of penicillin. They were incubated at 28 °C for 3-4 days. The isolated fungi were identified through a macromorphological, micromorphological and physiological studies as stated in the literature [11]. Harvesting of A. niger Spores and Inoculum Preparation: The spore of A. nigerfrom slant were harvested with the help of 0.1% pre-sterilized Tween 80. The spores in inoculums were maintained at 3 × 106 and it was inoculated in Erlenmeyer flasks containing spore germination medium with following composition: Glucose 5%, Di-ammonium phosphate 0.2%, MgSO4,0.25%, KH2PO4 0.1% (pH 5.5). The inoculated medium was put on orbital shaking incubator at 280C with 150 rpm for 48 hrs. Fermenter design and Culture Conditions: Batch fermentations were performed in 50L Semiautomatic stirred–tank fermenter (Scigenics India private Ltd. Chennai) equipped with top stirred bearing three 6 blade Ruston turbine type impellers, additionally four removable baffle plates attached to shell wall of fermenter. Agitation speed of stirred was set at 250 rpm. The temperature was constantly set at point 28oC using continuous water flow with thermostat. Fermentation medium with following composition:Glucose 10%, Di-ammonium phosphate 0.09%, MgSO4 0.01%, Urea 0.015%, KH2PO4 0.02%, Soybean oil 0.5% (pH 5.5) sterilized at 121oC for 20 minutes with the help of steam air. The air saturation in fermenter was measured and controlled using polorographic dissolved oxygen probe. Before inoculation, 100% air saturation in sterilized fermenter medium was adjusted using the constant atmospheric air flow rate at 30 LPM. At same time vessel pressure was maintained at 0.5 bars with the help of exhaust diaphragm valve. Sterilized fermentation medium was inoculated with 10 % 48 hrs old inoculums medium by using peristaltic pump. The pH was measured and maintained at set point 5.5 using Gel Filled, glass type pH probe, by automatically adding 15% pre-sterilized CaCO3 slurry. The sampling was carried out at regular interval from bottom fitted diaphragm valve. Determination of Biomass: Culture fluid was filtered through Whatman No 1 paper. The filtered mycelia were washed with acidified (pH 2.5 with 4M HCl) distilled water to convert the insoluble CaCO3 to soluble CaCl2. The separated mycelia were washed several times with deionized water. Then mycelia were dried (75oC) to a constant weight. And its dry weight was determined by subtracting the average predetermined dry weight of Whatman No 1 paper from the combined weight of Whatman No 1 paper along with mycelium [12]. Determination of Glucose and GA: The GA concentrations in samples were determined by measuring calcium in form of calcium gluconate (CG) in fermentation broth. The glucose and GA in fermentation samples were analyzed by HPTLC (CAMAG, Ancrom Enterprises Pvt. Ltd, Mumbai). HPTLC was performed on 10cm X 20cm pre coated silica gel G F254 aluminum TLC plates (1.5554.0007, Merck KGaA, Germany). Plate was pre washed with methanol: water with 4:1 ratio and activated by heating on CAMAG TLC plate heater on 1000C for one hour. Fermentation broth samples and reference (calcium gluconate-Acros Organics Germany; glucoseMerk) were applied to the plated by means of Linomat V TLC applicator (CAMAG Switzerland). The plate were developed at 300C ± 20C to a distance of 8.5 cm, with 20 mL of butanol : acetic acid : water with ratio 6:2:1 as mobile phase, in CAMAG twin trough chamber. After removal from the chamber, plate was dried on CAMG TLC plate heater III for heating for one and half hours at 120oC. Plate was scanned and quantified at 310 nm with the help of CAMAG TLC Scanner – 3 with WinCATS 1.4.4 software (Figure 2). The Rfvalues of CG and glucose were 0.18 and 0.42 respectively (Figure 1). The fermentation samples determination (Biomass, concentration of glucose and GA) were carried out in triplicate for reproducibility of results and it represented in form of mean. Statistical analysis was performed using ANOVA test software.


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Fig. 1: HPTLC chromatogram of GA fermentation samples at UV 366nm. Track no 1 = 25µg std. glucose (Merk), Track no 2 = 180µg std. calcium gluconate (Acros Organics, Germany), Track no 3 to 6 = 24, 28, 32 & 36 hours fermentation samples

Fig. 2: Densitogram curves of GA fermentation samples (24- 36 hours) at 310nm.

III. RESULTS & DISCUSSIONS Identification of Fungal Stain: Based on microscopic structural and growth characterization, it was concluded that the fungus isAspergillusniger. The ability of this strain was further utilized for GA fermentation(Figure 3).

Fig. 3:Microscopic view of isolated A. niger for Gluconic acid production.

The Establishment of GA Fermentation System: Submerged process is significantly affected by various factors. Among these, selection of suitable strain, substrate and process parameters are crucial. For significant GA production CaCO3 used as buffering agent during fermentation [4].Rogalski et al. (1988) concluded that calcium carbonate appeared to be important in preventing the acidification of the culture broth during


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cultivation [13]. Petruccioli et al. (1995) stated that the addition of calcium carbonate to the growth medium in shake flasks and fermenters prevented pH drop during cultivation [14]. This in turn is necessary for optimal glucose oxidase production. However, CaCO3limits the possibility of obtaining high concentrations of product due to the limited solubility of the resulting calcium gluconate (4% at 30oC) that precipitates over the mycelium thus inhibiting oxygen uptake [15]. To overcome this problem glucose concentration was maintained up to 8-10%. Aeration of growing aerobic cultures fulfillthe requirements of oxygen supply and also removes gaseous waste products. Agitation increases the efficiency of aeration by forcing the supplied air bubbles to disintegrate into smaller bubbles resulting in an increased interface between the gas and the liquid [16]. Because foaming affect on oxygen transfer rate from air and creating several problem in microbial respiration in aerobic fermentation, 0.5% soybean oil was used an antifoaming agent during fermentation [17]. In the present study A. niger grown in air phase by providing continuous filtered air with 100% dissolved oxygen and maintained 0.5 bar vessel pressure. This showed increased bioconversion of glucose to GA with less fermentation time. Time Course of GA Production: In present study, the glucose appeared to be a potential substrate resulting into high level of gluconicacidproduction at batch fermentation level (Figure 4and Figure 5). When initial reducing sugar level was used as 83.30 gL -1, the biosynthetic activity of A. niger initially increased proportionally up to 24 hours with 83.16% selectivity, 1.49 gL -1h-1 volumetric production rate, 51.68% sugar conversion and produced 35.80 gL-1 GA. An increase in level of GA production was observed after 28 hours with 99.59% selectivity, 2.08 gL-1h-1 productivity and 58.29 gL-1 GA production. It also showed significant biomass development (31.44 gL-1). This was followed by maximum production of GA (85.20 gL -1) over 36 hours.

Fig. 3: Time course changes of glucose, GA concentration and dry biomass weight during GA batch fermentation with A. niger.

Fig. 4: Time course changes of productivity, selectivity and yield during GA batch fermentation with A. niger.

In addition, the proper fermentation conditions or well optimized nutrient supplementation are needed to improve product formation during microbial fermentation. The kinetic analysis of gluconic acid production by mutant ORS-4.410 indicated that fermentation of glucose results in 94.5% yield after 144 hours of incubation [18].Glucose at a concentration of 10–15% has long been proposed as the main carbon source for GA production, with a 90–95% yield [8, 19, 20 21]. However, this attempt was made to utilized glucoseas substrate; the high promising GA production (85.20gL-1) with nearly greater than 100% yield was obtained within 36 hours.


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IV. CONCLUSION Batch processes exhibit some advantages in comparison with solid state fermentation e.g. easy control of microbial contamination and maintained proper aeration and finally increased product concentration. The industrial fermentative production of GA using A. niger is considered a mature process but there is still a certain interest in improving production yields by trying different glucose feeding strategies or more appropriate fermentation conditions. In these studies, we suggest a careful analysis of diversity of fungal potential, particularly submerged fermenting conditions may facilitate the strategies for commercial production of GA. The comparative investigation of GA production activity of A. niger species (isolated from bagasse) against the glucose as substrate will be used in developing peak height performing GA production in bioreactor. For quantitative evaluation of fermentation products HPTLC could be a strong tool for qualitative and quantitative determination with it’s off line facility high flexibility, reliability and cost efficiency. It also facilitates repeated detection (scanning) of the chromatogram with same or different parameter. We made effort to used HPTLC for more sophisticated and reliable analysis of fermentation samples. It has main advantage to analyse both residual sugar and GA concentration of many samples (nearly 15 samples) in single step and reduces the analysis time. Although further several experimentation is still needed, the technical feasibility of these fermentation strategies along with bagasse isolated A. niger strain appears realistic. ACKNOWLEDGEMENTS We take this opportunity to express our sincere thanks and gratitude to Dr. A.K. Agnihotri sir for their valuable guidance and help. Finally we acknowledge with deep appreciation, the indispensable help, encouragement and moral support received our departmental colleagues. The authors declare that they have no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

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