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3rd International Conference on Multidisciplinary Research & Practice P a g e | 293

Comparative Study of Dihydroxyacetone Production Gluconobacter spp and Bacillus spp Isolated from Fruit Source

Pradyna Joshi, Ajay Bhalerao Department of Microbiology, Laxman Devram Sonawane college, Kalyan (W), Maharashtra 421304, India.

Abstract: - Dihydroxyacetone is in demand in various chemical Dihydroxyacetone has several applications such as and pharmaceutical industries. With increasing awareness, for enhancement of photo chemotherapy with PUVA ecofriendly and non-hazardous method, microbial production of (Psoralen+UVA) treatment for stable plaque psoriasis, dihydroxyacetone is more advantageous than chemical method. synthesis of biomaterials with non-toxic degradation products It is also cheaper and easier method. An attempt was made to (diblock polymers) for many applications including drug isolate the organisms from fruit, which can produce dihydroxyacetone. Among isolated organisms, Gluconobacter spp delivery. (Mishra et.al. 2008). and Bacillus spp showed the production of dihydroxyacetone. Bertrand first observed production of Gluconobacter spp showed higher production than that of dihydroxyacetone from through bacterial route in the Bacillus cells. The yield of dihydroxyacetone using immobilized year 1898. He used bacteria called bacillus. Later cells of Gluconobacter spp was more as compared to that of immobilized cells of Bacillus spp. this S. bacillus was found to be identical with the bacterium Bacillus xylinum. Several organisms such as Key words: Dihydroxyacetone, Ecofriendly, Gluconobacter spp, Klebsiella aerogenes, Pichia membranifaciens, Acetomonas Bacillus spp, Immobilized cells. sp., Candida boidinii 2201, Acetobacter xylinum A-9, Gluconobacter melanogenus IFO 3294, Escherichia coli I. INTRODUCTION (strain ECFS) are involved in production of ihydroxyacetone (DHA) is used extensively in many dihydroxyacetone. (Mishra et.al.2008). D industries. It is used in the cosmetic industry as sunless Dihydroxyacetone (DHA) production from glycerol tanning agent to prevent from the harmful effects of UV rays. by Gluconobacter oxydans was studied and developed during It reacts with the amino acids present on the uppermost layer the 1950s and 1960s (Claret C. et al. 1992). Gluconobacter is of skin, i.e., stratum corneum to produce a darkening effect. It also distinguished from the acetobacter by its ability to rapidly is also used for the treatment of , due to its potential carry out limited oxidation of organic compounds and to capacity to cause pigmentation. Dihydroxyacetone is used in quantitatively excrete the oxidation products into the growth the chemical industry as building block for the synthesis of a medium (Claus et.al. 1975). DHA is an oxidation product of various fine chemicals. (Mishra et.al.2008). glycerol produced by microorganisms (Gupta et.al. 2001). When combined with pyruvate, DHA is used as an athletic This study was done to isolate DHA producing bacteria from nutritional supplement which is claimed to be an orally natural source further can be useful for industrial purpose. administered fat-burner. II. MATERIALS AND METHODS It can affect the sensory quality of the wine with sweet/etherish property. Dihydroxyacetone has also been used A. Enrichment Media for Isolation: Small pieces of fully to fi nd the impaired metabolic step, which leads to diabetic ripened apple (1 gm) were added in100ml enrichment media beta cells. It has been found to have an antidotal effect and statically incubated for 5 days. After incubation, one towards cyanide poisoning. It can be of physiological loopful from each enrichment broth was streaked on calcium significance for synthesis of antimalarial drugs. It has carbonate agar plates and incubated at RT. (W.Hanmoungjai antiproliferative effect on Trypanosoma brucei bloodstream et.al 2007) The colonies with clearing zone of calcium forms. carbonate on the agar plates were acetic acid bacteria, as well as other non-acetic acid bacteria were also selected for further Corresponding Author: Dr. (Mrs) Pradyna Joshi --Principal, Laxman Devram Sonawane of College of Arts, & Commerce, Science Kalyan (w) Pin experiments. (Yamada et.al. 1999) Code-421304, Thane, Maharashtra, India. Co-Author:- Mr. Ajay Bhalerao --Head of the Department of Microbiology, B. Characterization and Identification of an Isolated Laxman Devram Sonawane of College of Arts, & Commerce, Science Kalyan Bacteria: - The acetic acid bacteria were then characterized on (w) Pin Code-421304, Thane, Maharashtra, India. the basis of their morphology, physiology and biochemical properties. Selective isolation between Acetobacter and

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3rd International Conference on Multidisciplinary Research & Practice P a g e | 294

Gluconobacter spp was also done using acetate and lactate Yield Gluconobacter spp Bacillus spp oxidation by modified Leifson’s method (W. Hanmoungjai g/liter 85.28 72.42 et.al 2007). Biochemical tests for identification were Percentage (%) out of Glycerol added 77.52% 65.83% performed as per Bergey’s Manual of Determinative Bacteriology. Non-Acetic acid bacterial isolates were also Table 2. Average yield of Dihydroxyacetone obtained after identified microscopically (Al-Allaf: 2011). 30 hr of incubation of Calcium Alginate Immobilized cells of Gluconobacter and Bacillus spp. C. Production of Dihydroxyacetone: - Dihydroxyacetone production was done using biphasic batch cultivation Yield Gluconobacter spp Bacillus spp Gluconobacter and Bacillus. The organism was permitted to g/liter 95.28 84.47 grow on the surface of nutrient medium of pH-5.5 at about 25- Percentage (%) out of Glycerol added 86.01% 76.79% 300C for about 3 days statically. Then were added to the D. Production of Dihydroxyacetone (DHA) by Intact Free and medium and pH -5.5 and incubated at 28-300C for 48 hrs on Immobilized cells of Gluconobacter and Bacillus spp: - shaker at 280 rpm (W.Charney et.al 1978). DHA production by Gluconobacter spp. and Bacillus spp. was also done using The yield of dihydroxyacetone obtained by using immobilized cells (Patrick et al.1985). free, intact cells of Gluconobacter (77.52%) was similar in D. Estimation of Dihydroxyacetone: - DHA was range (75-90%) of yield shown by William et.al. 1978. It is assayed by Seliwanoff’s test for (Batzing et.al, 1971, confirmed that Gluconobacter spp were suitable for R.Asnis, et.al 1952). dihydroxyacetone production than of Bacillus spp. The DHA production after 30hrs of incubation; Gluconobacter spp show E. Identification of DHA: - Detection of DHA as a reaction higher production as compared to Bacillus. spp. Similar product is done by paper chromatography using n-butanol- production was reported with other strains of Acetomonas, pyridine- (10:3:3) solvent system (Flickinger et.al. Candida and microorganism from soil, Pichia 1977). membranifaciens by Mishra et al.2008.

III. RESULTS AND DISCUSSION Immobilized Gluconobacter cells show increase in dihydroxyacetone production about 0.02g/ml after 6 hrs of A. Screening, Characterization and Identification of incubation as compared to intact cells, where free, intact cells Gluconobacter and Bacillus spp: - of Gluconobacter spp after 6 hrs show dihydroxyacetone From apple source, acetic acid as well as non acetic production about 0.01428 g/ml. (Fig. 1.) Immobilized Bacillus acid bacteria were able to be isolated and shown the cells show slight increase in dihydroxyacetone production production of DHA. In modified Leifson’s method, colour of about 0.0152g/ml after 6 hrs of incubation as compared to medium changed from blue to yellow. The isolated spp also intact cells, where free, intact cells of Bacillus spp after 6 hrs showed heavy growth on dextrose-sorbitol-mannitol medium. show dihydroxyacetone production about 0.0125 g/ml. (Fig. The isolated spp show positive growth at 300C, but not at 2). 370C and produced acid from . It was Gram variable, Catalase positive, Oxidase negative and show growth in peptone. It was motile and non-endospore forming. Thus, the organism was confirmed as Gluconobacter spp. Non-Acetic acid bacterial isolates were Gram positive, Endospore Forming Rod Shaped Bacteria, found to be positive in citrate utilization, oxidase, urease, gelatin hydrolysis, catalase, motility, nitrate reduction, Voges- Proskauer and hydrolysis as well as they were grew well in 7 % NaCl. All Sugars were fermented by isolated spp. Thus, by Bergey’s Manual of Determinative, it was confirmed that isolated spp was Bacillus spp (Al-Allaf: 2011).

B. Identification of Dihydroxyacetone as Reaction Product: By paper chromatography, it was confirmed that reaction product was dihydroxyacetone. (Rf = 0.68). C. Production of Dihydroxyacetone: -

Table 1. Average yield of DHA obtained after 30 hr of Fig. 1. DHA production by Free, Intact cells of Gluconobacter and Bacillus incubation of free, intact cells of spp. spp.

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immobilized cells is higher than free cells suggest that the use of immobilized cells is recommended in industrial production.

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[1]. Arun Gupta, Vinay K. Singh, G.N. Qazi and Anil Kumar: (2001):- Gluconobacter oxydans: Its Biotechnological Applications. J. Mol. Microbiol. Biotechnol. 3(3): 445-456. [2]. B. L. Batzing and G. W. Claus: Oct. 1971: Biphasic Growth of Acetobacter suboxydans on a glycerol-limiting medium. Journal of Bacteriology, vol. 108, No. I, p. 592-595. [3]. Bergey’s Manual of Determinative Bacteriology – 9th edition. [4]. C. Flickinger and D. Perman: (Mar.1977): Application of - enriched aeration in the conversion of glycerol to dihydroxyacetone by Gluconobacter melanogenus IFO 3293. Applied and environmental microbiology.vol. 33, No. 3. p.706-712. [5]. Cirigliano, M. C. (1982): A selective medium for the isolation and differentiation of Gluconobacter and Acetobacter. J. Food Sci., 47, 1038–1039. [6]. Claret C, Bories A, Soucaille P: (1992): Glycerol inhibition of growth and dihydroxyacetone production by Gluconobacter oxydans. Current Microbiology vol. 25, pp. 149-155. [7]. G. W. Claus, Barry L. Batzing, C. A. Baker, and E. M. Goebel: Fig. 2. DHA production by Immobilized cells of Gluconobacter and Bacillus Sept. 1975: Intracytoplasmic Membrane formation and increased spp oxidation of glycerol during growth of Gluconobacter oxydans. Journal of Bacteriology, vol. 123, No. 3, p. 1169-1183,. Calcium alginate immobilized Gluconobacter and [8]. J. De Ley and J. Frateur: January 1970: The status of generic name Bacillus cells maintained consistent activity oxidation of Gluconobacter. International journal of systemic bacteriology, vol. glycerol to dihydroxyacetone, whereas in the case of free 20, no. 1 pp. 83-95. intact cells the activity was decreased in case of free, intact [9]. J.De.Ley: (1961): Comparative metabolism and a proposal for a phylogenetic relationship of the acetic acid bacteria cells of Gluconobacter spp and Bacillus spp. Similar Results J. gen. Microbiol. 24, 31-50. were reported by (Patrick et al.1985), which indicates that for [10]. May A. A. Al-Allaf: 2011: Isolation of Bacillus spp. from some batch fermentation such process can be adopted to increase sources and study of its proteolytic Activity. Tikrit Journal of Pure the yield. Science.16 (4). [11]. Patrick Adlercreutz, Olle Holst and Bo mattiasson: (1985): Characterization of Gluconobacter oxydans immobilized in calcium alginate. Appl. Microbiol. Biotechnol. 22:1-7. [12]. Robert E. Asnis and Arnold F. Brodie: December 31, 1952: A from Escherichia coli. (From the Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania). [13]. Ruchi Mishra, Seema Rani Jain, and Ashok Kumar: (2008): Research review paper: Microbial production of dihydroxyacetone. Biotechnology Advances 26 293–303. [14]. T.T.Kadere, T.Miyamoto, R. K. Oniagng`o, P.M.Kutima and S.M. Njoroge: 18 August, 2008: Isolation and identification of the genera Acetobacter and Gluconobacter in coconut toddy (mnazi). African Journal of Biotechnology Vol.7 (16), pp 2963-2971. [15]. Watcharee Hanmoungjai, Ekachai Chukeatirote, Wasu Pathorn- aree, Yuzo Yamada and Saisamorn Lumyoung: 2007: Identification of acidotolerant acetic acid bacteria isolated from Thailand sources. Research Journal of Microbiology 2(2): 194-197. [16]. William Charney, Montclair, N.J.: Feb.28, 1978:- Process for production of dihydroxyacetone. U.S.patent. [17]. Yuzo Yamada, Reiko Hosono, Puspita Lisdyanti, Yantyati Widyastuti, Susono Saono, Tai Uchimura and Kazuo Komagata: (1999): Identification of acetic acid bacteria isolated from Indonesian sources, especially of isolates classified in the genus Gluconobacter. J. Gen. Appl. Microbiol, 45, 23–28. Fig. 3. Dihydroxyacetone Production in Percentage. [18]. Websites:- www.wikipedia.com IV. CONCLUSION http://www.cosmeticsinfo.org/ingredient_detail www.Gluconobacteroxydans.Microwiki.html Gluconobacter species from ripen apple is a good source of www.dihydroxyacetoneproduct.info.com dihydroxyacetone production which can be used as ecofriendly, cheaper microbial method. The yield of DHA by

Volume IV Issue I IJRSI ISSN 2321-2705