Bioconversion of whey to 2,3-butanediol using Klebsiella oxytoca NRRL

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1 Indian Journal of Biotechnology Vol 13, April 2014, pp Bioconversion of whey to 2,3-butanediol using Klebsiella oxytoca NRRL Sadhana Vishwakarma* Department of Engineering Chemistry, TIT Group of Institutions, Bhopal , India Received 6 November 2012; revised 28 January 2013; accepted 23 April 2013 Biotechnological production of 2,3-butanediol (BD), a value added chemical, from wastes and with excessive biomass is a promising and attractive alternative to traditional chemical synthesis. The production of BD by Klebsiella oxytoca NRRL from deproteinated whey was studied in batch fermentations at ph 6.5. Lactose present in the deproteinated whey was found to be effectively utilized by K. oxytoca, producing BD g/g of lactose utilized after an incubation period of 96 h. Moreover, addition of acetate at a concentration of 50 mm was found to increase the yield by 1.5-fold, resulting in BD g/g lactose utilized. During the process, COD (chemical oxygen demand) reduction of 87% was observed in addition to 91% BOD (biological oxygen demand) reduction. Thus, the present process of utilization of whey by K. oxytoca clearly shows that it not only helps in the production of value added chemical BD from the waste but also helps in reducing the environmental problems faced with the disposal of untreated or unfermented whey directly into the river. Keywords: Biotechnological conversion, 2,3-butanediol, butylenes glycol, Klebsiella oxytoca, whey, whey permeate Introduction Whey is generated at a rate of 9 kg/kg of cheese or 6 kg/kg of cottage cheese manufactured and 100 kg of whey is equivalent to the sewage produced by 5 people 1. In many countries including India, most of the whey is discarded as waste creating severe environmental pollution problems because of its high chemical oxygen (COD) and biological oxygen demands (BOD) 2. The BOD is mainly due to the lactose, which is present at a concentration between 4-5% 3. The cost-effective disposal or utilization has become increasingly important for the modern dairy industries as they contribute high organic pollution load. In recent years, this necessity has been heightened by several reasons, such as, the increased volume of whey being produced, the increased centralization of diary manufacturing at fewer processing sites and more stringent legislative requirements for effluent quality. The most recent approach for whey utilization is ultra-filtration to separate proteins from permeate. The protein fraction is used as food but the permeate or deproteinated whey (DPW) still exerts a BOD greater than 30 kg/m 3 and is normally disposed of without any treatment 4. A number of options have been proposed to convert permeate to value added *Author for correspondence: Mobile: vish_sadhana@rediffmail.com products and other more profitable alternatives. These alternatives include alcoholic beverages 5, gasohol 6, lactic acid 7, acetic acid, propionic acid 2, acetone and butanol 8. Utilization of whey or whey permeate as a raw material for fermentation industries is not only a techno-economically feasible waste utilization alternative, but is also a mean of reducing problems associated with the use of lignocellulosic materials. 2,3-Butanediol (BD) is an interesting chemical because of its diverse potential uses. It can be converted to butadiene, acetoin, diacetyl or methyl ethyl ketone, all having industrial applications 9. It can also be used in the manufacture of antifreeze 10. A more promising future for BD is in plastic industry 11. It is also used in nonaqueous foam, drugs, cosmetics, ointments and antiperspirants 12. The advantage of BD fermentation over that of ethanol and butanol is that it is less toxic to the microorganisms those produce it and thereby allow higher concentration to be maintained in the fermentation broth 13. A number of bacteria 14,15 can biotechnologically convert carbonaceous substrates to BD The present research work was undertaken to investigate the utilization of whey by Klebsiella oxytoca NRRL as an alternate renewable resource for the production of 2,3-butanediol. Materials and Methods Chemicals and Microbial Culture All the solvents and salts used in the study were of analytical grade and used directly without any further

2 VISHWAKARMA: PRODUCTION OF 2,3-BUTANEDIOL FROM WHEY THROUGH BIOCONVERSION 237 purification. BD was obtained from Merk India. Distilled water was used throughout the experimental study. K. oxytoca NRRL was obtained from the Biochemical Engineering Research Centre, Indian Institute of Technology, New Delhi. It was maintained on nutrient agar slants (ph 6.5) at 4 o C. Medium for Culture The medium used for the culture of K. oxytoca was described by Pirt and Callow and known as PC medium 19. The medium contains (g/l): MgSO 4.7H 2 O, 0.3; CaCl 2.6H 2 O, 0.09; FeSO 4.7H 2 O, ; ZnSO 4.7H 2 O, ; EDTA, 50.51; (NH 4 ) 2 SO 4, 7.2; (NH 4 ) 2 HPO 4, 6.0; and KOH, The ph of the medium was adjusted to 6.5 using phosphoric acid. Glucose or lactose was used as the carbon source. The medium was prepared and autoclaved in three separate solutions, viz., i) solution of the nutrients, ii) solution of ammonium salts and iii) sugar solution Preparation of K. oxytoca Inoculum Glucose or lactose solution (0.1%) in 100 ml portion were autoclaved at 0.7 kg/cm 2 pressure for 30 min. Ammonium salt solution and nutrient medium, which were previously sterilized, were added to the flask followed by inoculation from a 24-h-old slant culture of K. oxytoca and incubated on a shaker (60 rpm) at 30 o C for 24 h. This culture was used as inoculum (1% v/v). Whey and Content Estimation Whey was obtained from a local dairy. The ph of whey was adjusted to 7.0 by using 1 N NaOH and then steamed for 30 min to precipitate protein. It was then cooled, kept at 4 o C and filtered through ordinary filter paper. The filtrate known as deproteinated whey (DPW) was used for further studies. Various constituents of DPW were estimated. Reducing sugar was estimated by dinitrosalicylic acid method 20 ; lactic acid by titrimatric method 21 ; protein according to the modified method of Lowery et al 22 ; *nitrogen by macro Kjeldahl method; calcium and magnesium by complexometric titration method; and sulphates by turbidity method 23. Total dissolved solids, ash content, COD and BOD were estimated by standard methods 23. Production of BD Glucose or lactose solutions (3%) in 100 ml portion were autoclaved in 250 ml flasks. PC minerals were added and then inoculated with K. oxytoca. When DPW was used for fermentation, PC minerals were not added. Autoclaved whey was directly inoculated with K.oxytoca inoculum. Agitation of 60 rpm was provided throughout the experimentation. Samples were withdrawn after every 24 h and then incubated, centrifuged and analyzed for residual sugar, BD, acetoin, acetic acid and ethanol. Effect of Acetate Sodium acetate stock solution (5 M) was prepared and 0.2, 0.5, 1.0, 1.5, and 2.0 ml of it were added to 100 ml DPW, so as to get final concentration of 10, 25, 50, 75, and 100 mm of acetate. The ph of all the solutions was adjusted to 6.5. Then they were autoclaved, inoculated and samples were withdrawn at 24 h interval to analyze the content of BD, acetoin, acetic acid and ethanol. BD, acetoin, acetic acid and ethanol in the fermentation broth were determined by Gas chromatography. The fermentation broth was first centrifuged and the supernatant after proper dilution was injected into GC (Perkin Elmer Sigma 3B) equipped with flame ionization detector. The column used was a stainless steel column with dimension of 2.0 m (length) x 0.3 cm (outer diameter) packed with chromosorb 101, 80/100 mesh, coated with 3% FFAP. The injector, detector and oven temperatures were maintained at 210, 230 and 200 o C, respectively. Lactose was estimated by HPLC using waters Microbondapack carbohydrate analysis column 14 (30 cm x 3.9 mm internal diameter) with differential refractometer detector. Acetonitril:water (80:20) mixture was used as the mobile phase at a flow rate of 1.5 ml/min. Micro elements in DPW were estimated by atomic absorption spectrophotometer using GBC 904 Atomic Absorption Spectrophotometer (dual beam) with graphite furnace. Sodium and potassium were estimated by flame emission photometer clinical model M 4 10 (Ciba Corning UL made) using NaCl and KCl as standards 23. Results and Discussion Characterization of DPW The gross composition, macro elements and trace element composition for DPW obtained in the present study as well as of whey permeate obtained by other researchers are given in Tables 1 and 2. The values obtained for all the minerals were found to be lower than reported by Hobman 9 and Hargrove et al 24. HPLC determination of carbohydrates in DPW showed that only lactose was present in whey,

3 238 INDIAN J BIOTECHNOL, APRIL 2014 Characteristics DPW (present study) whereas glucose was absent. Lactose was comprised of approx 75% of the dissolved solids, but overall concentration of lactose was only %, which is less than the values of % reported by other authors 9,24. The ph of the DPW was found to be , whereas the optimum ph for the production of BD was reported to be 6.5 in other studies 25. This indicates the necessity of ph adjustment before fermentation. The elemental composition shows that all the elements or trace elements required for the growth of the organism K. oxytoca are present in the DPW and hence addition of minerals to whey was not considered. DPW was, therefore, used directly after adjustment of ph to 6.5. Production of BD As major carbohydrate component in whey is lactose, an experiment was carried out to study the efficacy of K. oxytoca for production of BD in PC medium with 3% lactose and the production was compared with 3% glucose. The results are depicted in Fig 1. Results indicate that the utilization of lactose by K. oxytoca was very slow as compared to that of glucose. BD concentration of 2.1 g/l was achieved Table 1 Composition of DPW and whey permeate Lactic casein whey permeate (Hobmann) 8 Ceddar cheese whey permeate (Hargrove) 24 Cottage whey permeate (Hargrove) 24 ph Moisture, % Total dissolved solid (TSS), % Protein, % Phosphate, mg/l Sulphate, m/l Total nitrogen, mg/l Lactic acid, % Lactose, % Ash, % Microelements Potassium Calcium Magnesium Table 2 Trace elemental composition of DPW and permeate powder Trace elements DPW (ppm) (present study) Permeate powder (Hargrove) 35 Copper Lead Maganese Iron Nickel Cadmium Zinc Chromium with lactose after 168 h incubation period. No BD production was observed with glucose at 48 h, whereas 6.8 g/l BD was obtained after 72 h of incubation. Similar observations were made by Champluvier et al 26. The lower rate of lactose consumption could be due to difference in the uptake mechanism, which is energy demanding for lactose but not for glucose 27. Production of BD from DPW is depicted in Fig 2. A BD concentration of 6.1 g/l was achieved form 2.35% lactose utilized after 96 h of incubation period. This corresponds to BD production of g/g lactose utilized. The present results were found to be higher compared to those reported by Speckman and Collins 27, who used Bacillus polymyxa for the production BD from whey containing 4.9% lactose. The yield of BD achieved by them was 0.06 and 0.15 g/g lactose utilized after 72 and 168 h, respectively. Similarly, a BD yield of g/g was obtained by Kadathur et al 28 using K. oxytoca, which is comparatively lower than the present study. Ethanol 0.43%, acetic acid 0.09%, and acetoin 0.02% were also produced as byproducts during fermentation. Further it was observed that K. oxytoca produced a higher concentration of BD from DPW as compared to PC medium with 3% lactose. Influence of Acetate It has been reported that acetate induces the production of acetoin and BD by Aerobacter aerogenes 29. In the present study, it was found that addition of acetate increased the production of BD by K. oxytoca and the results are shown in Fig 3. Acetate at a concentration of 50 mm was found to be the optimum and produced 8.4 g/l of BD from 2.3%

4 VISHWAKARMA: PRODUCTION OF 2,3-BUTANEDIOL FROM WHEY THROUGH BIOCONVERSION 239 Fig. 1 Production of BD from glucose and lactose. Fig. 3 Effect of acetate on BD production from DPW. Table 3 COD and BOD of DPW before and after fermentation Time (h) COD (kg/m 3 ) %COD reduction BOD (kg/m 3 ) % BOD reduction Fig. 2 Production of BD from DPW. lactose. It corresponds to a BD yield of g/g lactose utilized, which amounts to a 1.5-fold increase in the production of BD without appreciable increase in other byproducts. The concentrations above 50 mm acetate resulted in the production of decreased BD. Stromer 29 noted the key role of acetate in the pathway of BD production. Acetate induces 3 enzymes, viz., acetoacetate forming enzyme, acetolactate decarboxlylase and dicetyl reductase, which are involved in the conversion of pyruvate to BD. It also activates the acetoacetate forming enzyme and regulates the balance between acetoin and BD. It was reported that acetate increased the production of BD by increasing the rate of carbohydrate utilization. Effect on COD and BOD COD and BOD of DPW were determined before and after the fermentation and the results are shown in Table 3. About 65 and 80% reduction was observed in COD and BOD, respectively with in 24 h of fermentation. However, further reduction in COD and BOD showed slow down of pace with the passage of time, which might be due to inhibition of lactose utilization by the organism at higher product concentration. Finally, 88 and 92% reduction in COD and BOD, respectively were achieved in 120 h of fermentation (Table 3). Conclusion Characterization of DPW shows that it can be used as a substrate for the production of BD. Lactose present in the DPW was found to be effectively utilized by K. oxytoca, producing BD g/g of lactose utilized. Addition of acetate at concentration of 50 mm was found to increase BD yield by 1.5-fold, resulting in BD g/g lactose utilized. Further, 88 and 92% reduction in COD and BOD, respectively clearly indicates that this process of utilization of whey not only helps in the production of value added chemical BD from the waste, but it also helps in

5 240 INDIAN J BIOTECHNOL, APRIL 2014 reducing the environmental problems faced due to disposal of untreated or unfermented whey directly into river. References 1 Jelen P, Industrial whey processing technology: An overview, J Agric Food Chem, 27 (1979) Tyagi R D & Kluepfel D, Bioconversion of cheese whey to organic acids, in Bioconversion of waste materials to industrial products, edited by A M Martin (Springer Science plus Business Media, New York) 1998, Modler H W, Muller P G, Elliot J T & Emmons D B, Economics and technical aspects or feeding whey to livestock, J Dairy Sci, 63 (1980) Mawson A J, Bioconversions for whey utilization and waste abatement, Bioresour Technol, 47 (1994) Kosikowski F V & Wzorek W, Whey wines from concentrates of reconstituted acid whey powder, J Dairy Sci, 60 (1977) Kosikowski F V, Whey utilization and whey production, J Dairy Sci, 62 (1979) Kellar A K & Gerhardt P, Continuous lactic acid fermentation of whey to produce a ruminant feed supplement high in crude protein, Biotechnol Bioeng, 17 (1975) Hobman P G, Review of processes and products of utilization of lactose in deproteinated milk serum, J Dairy Sci, 67 (1984) Syu M J, Biological production of 2,3-butanediol, Appl Microbiol Biotechnol, 55 (2001) Clendenning K A & Wright D E, Production and properties of 2,3-butanediol: XVII. Antifreeze properties of ternary aqueous solutions containing levo-2,3-butanediol as a major component, Can J Res, 24F (1946) Fedor W S, Plasticizers: The competitive struggle grows keener, Chem Eng News, 39 (1961) Sanders P, Nonaqueous foams enter aerosols: Glycols used in place of water give stable foams with widely varying properties, Chem Eng News, 38 (1960) Sablayrolles J M & Goama G, Butanediol production by Aerobacter aerogenes NRRL B199: Effect of initial substrate concentration and aeration agitation, Biotechnol Bioeng, 26 (1984) Ji X J, Huang H, Du J, Zhu J G, Ren L J et al, Development of an industrial medium for economical 2,3-butanediol production through co-fermentation of glucose and xylose by Klebsiella oxytoca, Bioresour Technol, 100 (2009) Biswas R, Yamaoka M, Nakayama H, Kondo T, Yoshida K et al, Enhanced production of 2,3-butanediol by engineered Bacillus subtilis, Appl Microbiol Beiotechnol, 94 (2012) Wang A, Wang Y, Jiang T, Li L, Ma C et al, Production of 2,3-butanediol from corncob molasses, a waste by-product in xylitol production, Appl Microbiol Biotechnol, 87 (2010) Motwani M, Seth R, Daginawala H & Khanna P, Microbial production of 2,3-butanediol from water hyacinth, Bioresour Technol, 44 (1993) Gadgil N J, The biotechnological production of chemicals from organic wastes. PhD Thesis, Nagpur University, Nagpur, Pert S J & Callow D S, Extracellular product formation by microorganisms in continuous culture, J Appl Bacteriol, 21 (1958) Miller G L, Use of dinitrosalicylic reagent for the determination of reducing sugars, Anal Chem, 31 (1959) Eckles C H, Combs W B & Macy H, Milk and milk products, 4 th edn (Tata Mcgraw Hill Publishing Co. Ltd., New Delhi) 1993, pp Lowry O, Rosebrough N J, Farr A L & Randall R J, Protein measurement with the Folin-phenol reagent, J Biol Chem, 193 (1951) Standard methods for the examination of water and wastewater, 18 th edn (American Public Health Association-American Water Works Association-Water Environment Federation) Hargrove R E, McDonough F E, LaCroix D E & Alfrid J A, Production and properties of deproteinized whey powders, J Diary Sci, 59 (1976) Zeng A-P, Biebl H & Deckwer W-D, Effect of ph and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture, Appl Microbiol Biotechnol, 33 (1990) Champluvier B, Decallonne J & Rouxhet P G, Influence of sugar source (lactose, glucose galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199, Arch Microbiol, 152 (1989) Speckman R A & Collins E B, Microbial production of 2,3-butylene gycol from cheese whey, Appl Environ Microbiol, 43 (1982) Ramachandran K B, Hashim M A & Fernandez A A, Kinetic study of 2,3-butanediol production by Klebsiella oxytoca, J Ferment Bioeng, 70 (1990) Stormer F C, Evidence for regulation of Aerobacter aerogenes ph 6 acetolactate forming enzymes by acetate ion, Biochem Biophys Res Commun, 74 (1977)