INITIAL INVESTIGATION ON ACETIC ACID PRODUCTION AS COMMODITY CHEMICAL

INITIAL INVESTIGATION ON ACETIC ACID PRODUCTION AS COMMODITY CHEMICAL 1,2 Mallika Boonmee, 2 Soothawan Intarapanich 1 Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen, Thailand 40002 2 Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, Thailand 40002 e-mail: 1 mallikab@kku.ac.th Introduction Abstract. Acetic acid, generally used in food industry, has potential uses also in many chemical industries especially when used in its salt forms. As a part of the project that explored alternative uses of ethanol in addition to its use as a blend in gasohol, this research studied the production of acetic acid by fermentation. Acetobacter pasteurianus TISTR 520 was chosen based on results from our previous studies and used in this fermentation study. Increased aeration rate did not result in further loss of ethanol due to aeration as the acetic acid produced was 17.5 g/l at aeration of 4 vvm as compared to 21.3 g/l at 2 vvm. Acetic acid production increased to 39.7 g/l, which was 65% of theoretical yield, when inoculum size was increased by pre-cultivating the cells with 2% ethanol prior to addition of ethanol for acetic production phase. Further investigation using ph control fed-batch cultivations with pulse feedings resulted in the increase of acetic acid production to up to 65.6 g/l at ph 3.5, which accounted for the yield of 0.89 g acid/g ethanol or 68% of theoretical yield. While no acetic acid was produced at ph 3.0, the acid production at ph 4.0 and ph 4.5 were similar in term of concentration but slightly lower than that at ph 3.5. This preliminary observation indicated that the production at ph 3.5 may result in elevated acetic acid tolerance level in the organism such that higher concentration was obtained. Lower acetic acid yield in fermentor cultivation, as compared to flask cultivation, dues mainly to loss of substrate ethanol through aeration. However, the advantage was the increased productivity as cultivation time was reduced. Keywords. Acetic acid, fermentation, fed-batch, aeration, ph Acetic acid is a useful chemical especially in food industries as vinegar. It is also used to produce derivatives of which the importance ones included vinyl acetate, which is a monomer for polyvinyl acetate used in paint and adhesives; acetic anhydride, which is used for the production of cellulose acetate for the production of photographic films and also used as a reagent for aspirin production. Apart from its derivatives, acetic acid itself is an important solvent many processes especially in the production of terephthalic acid (TPA), which is the raw material for polyethylene terephthalate (PET). Acetic acid can be synthesized both via chemical or biological means. The chemical syntheses include methanol carbonylation, ethylene oxidation or acetaldehyde oxidation. For biological production of acetic acid, the most practiced method is through the aerobic cultivation of the bacteria in the genus Acetobacter spp. Using alcohol as substrate. Generally, acetic acid production by Acetobacter spp. is subjected to the inhibition by both ethanol and acetic acid. Acetic acid concentration in the range of 45 73.5 g/l, depending on species, was reported to be inhibitive to the growth of the organism (Park et.al. 1989, Park et.al. 1991a, Krisch & Szajáni 1997). Previous study on A. pasteurianus TISTR 520 showed that ethanol concentration above 6% v/v started to have inhibition effect of the growth and acid production of the strain (Boonmee & Intarapanich 2006a). Attempts to overcome the restriction in acid production by ethanol and acetic acid inhibition included the use of various cultivation methods. Higher acetic acid concentration was reported when using repeated batch with or without cellrecycling (Park et.al. 1991b), repeated fed-batch cultivation with 2 fermentors (Ito et.al. 1991) and repeated fedbatch with cell recycling system and intermittently addition of ethanol (Park et.al. 1991a). Another important parameter to the acetic acid production was oxygen as the production is the oxidative process. The oxygen importance is such that a short disruption of oxygen supply to the medium when acid concentration is high could interrupt the acid production by the bacteria due to the damage to bacterial cells (Muraoka et.al. 1982). The amount of dissolved oxygen was also correlated to the growth and hence acetic acid production of the acetic acid bacteria including A. aceti, A. pasteurianus and Gluconobacter oxydans. Higher growth and acid production were observed at higher dissolved oxygen (Drysdale & Fleet 1989). This study investigated the acetic acid production in bench top fermentor using Acetobacter pasteurianus TISTR 520 which was previously tested for its ability in acetic production with the emphasis on the effect of aeration, inoculum size and ph. The study was a part in the project that explored the possible uses of ethanol as a reactant for the production of other compounds. 2081

Materials and Methods Microorganism Acetobacter pasteurianus TISTR 520 was procured from MIRCEN, Thailand Institute of Scientific and Technological Research. The bacteria was stored in 30% glycerol at -20 C and was propagated in agar medium consisted of 100 g glucose, 10 g yeast extract, 20 g CaCO 3 and 25 g agar in 1 L medium to obtain single colonies. Inoculum preparation Two loopful of the bacterial single colonies was inoculated to 50 ml of the medium that consisted of 30 g glucose and 10 g yeast extract in 1 L medium and incubated in incubator at 30 C, shaking at 200 rpm for 24 hours. Five milliliters of the first seed was further inoculated to 95 ml of the same medium and incubated at the same conditions for use as the inoculum for the fermentor cultivation. Batch cultivation The cultivations were carried out in the 5-L fermentor (Biostat B, B.Braun, Germany) with 2 L working volume. Prior to the cultivation, the solution containing yeast extract was added to the fermentor, which was autoclaved at 110 C for 40 min. Ethanol and sterile water in the pre-calculated volumes were added to the yeast extract solution to make the final medium composition in the fermentor to consisted of 10 g/l yeast extract and 6% v/v ethanol. The medium was then sparged with sterile air and agitated at 2 vvm/200 rpm or 4 vvm/550 rpm in order to set 100% po 2. The temperature of the cultivation was controlled at 30 C and po 2 at 20% without ph control. Samples were taken at intervals until ethanol was used up which indicated by the increase in po 2. Fed-batch cultivation Similar cultivation procedure to the batch cultivation was applied but the starting cultivation medium consisted of 10 g/l yeast extract and 2% v/v ethanol. The medium was then sparged with sterile air at 4 vvm and agitated 550 rpm in order to set 100% po 2. The temperature of the cultivation was controlled at 30 C and po 2 at 20% with or without ph control. Samples were taken at intervals until ethanol was used up which indicated by the increase in po 2. Extra ethanol was added to make the ethanol concentration in fermentor to 6% v/v. Repeated procedure was carried out until there is no further use of ethanol. Analytical methods Cell growth was monitored by measuring the optical density of cell solution at 600 nm. The value of ph was monitor through the control unit of the fermentor. Ethanol and acetic acid concentrations were determined using gas chromatography (Shimadsu GC 14B, Shimadsu, Japan) fitted with FID detector. The column used was Porapack Q with helium as the carrier gas and propanol as internal standard. The retention times of ethanol and acetic acid were 2.34 and 5.27 minutes. Results and discussion Effect of aeration on acetic acid production in batch cultivation When A. pasteurianus TISTR 520 was cultivated in batch fermentation with 6% v/v ethanol and no ph control, the results in Figure 1 showed that the acetic acid concentrations obtained were similar regardless of amount of air introduced in the fermentor. The concentration when more air (100% po 2 at 4 vvm, 550 rpm) was presented was 17.5 g/l and slightly higher at 21.3 g/l in the case of less air (100% po 2 at 2 vvm, 200 rpm). The results also indicated that the fermentation completed faster when more air was introduced. The overall productivity at 2 vvm aeration was 0.70 g/l.h and 0.82 g/l.h when the aeration was 4 vvm. Although our results from previous study suggested that stirrer speed did not significantly affected the ethanol loss, the aeration rate and initial ethanol concentration did (Boonmee & Intarapanich 2006a). The results in this study implied that the 2 aeration rates used had some effect on the substrate (ethanol) loss. Although the concentrations of the final product (acetic acid) were similar in the 2 cases, the acetic acid yield decreased slightly from 0.57 g/g to 0.43 2082

g/g when higher aeration was used. These yield values were lower than those in flask cultivations where the yield was in the ranges of 1.00 1.15 g/g (Boonmee & Intarapanich 2006b). This was mainly due to the ethanol loss from aeration in fermentor. This evaporative loss of volatile compounds including substrate ethanol was reported to be the main cause of the reduction in yield. The yield reduction is generally 10 30% of the stoichiometric yield. The fermentation system with gas circulation unit was introduced so that the fermentation occurred in close system. However, the system required high maintenance and operation costs, which limits its use (Tesfaye et.al. 2002). Effect of increased biomass on acetic acid production Due to the ethanol loss by aeration experienced in fermentor cultivation especially towards the beginning of the cultivation when the cells are at the early stage of propagation and ethanol transformation was still low due to small cell numbers, this part of the study investigated the acetic acid production when the initial cell concentration increased. The cultivation started using 2% v/v ethanol as the substrate to produce the cells. After 2% v/v ethanol was used up, extra ethanol for acetic acid production was added to the concentration of 6% v/v in the fermentor. The cell concentration in term of OD 600 after the initial cultivation in 2% v/v of 0.81 was close to 1.00 and 0.92, which were the OD 600 of the prior batch cultivations with 6% v/v initial ethanol (results not shown). Profiles in Figure 2 showed that the acetic acid production phase (after adding 6% v/v ethanol) occurred faster. The ethanol was used up within 10 hours as compared to 22 hours in the batch previously. Acetic acid produced was 39.7 g/l. Further addition of 6% v/v ethanol did not result in further acetic acid production, which could be the result of low ph whose value was approximately 3.2 at the time. The product inhibition was not believed to be the cause as the previous study in flasks showed that the strain was capable to produce acetic acid of 53 g/l (Boonmee & Intarapanich 2006b). The decrease in ethanol after the further ethanol addition was suspected to be the result of aeration. Figure 1. Cultivation profiles for acetic acid production by A. pasteurianus TISTR 520 at 30 C and no ph control: ethanol ph acetic acid; open symbol 100% po 2 set at 2 vvm 200 rpm; closed symbol 100% po 2 set at 4 vvm 550 rpm 2083

Figure 2. Cultivation profiles for acetic acid production by A. pasteurianus TISTR 520 at 30 C and no ph control with initial ethanol of 2% v/v for cell propagation: ethanol ph acetic acid Fed-batch cultivations with pulse feedings Fed batch cultivations were initiated by the biomass production step using 2% v/v prior to 6% v/v addition for acetic acid production phase. The ph value of the cultivations was controlled throughout the cultivation period in order to determine the suitable ph for acetic acid production in addition to the aim to enhance the acetic acid concentration produced from the batch cultivations. At ph 3.0 (Figure 3a), there was no cell growth at all, hence no acetic acid production. The decreases in ethanol concentration were solely from the evaporation by aeration. The highest acetic acid concentration was produced when the ph of the cultivation was controlled at ph 3.5. Figure 2b showed that acetic acid concentration increased up to the value of 65.6 g/l at the end of the cultivation following the 3 ethanol additions. The acetic acid accumulated at high rates corresponded to the decreases in ethanol concentration in the first 2 ethanol additions. However, the accumulation rate was slow after the third addition and acetic acid produced was only approximately 8 g/l. This implied that the cause of ethanol reduction during the last addition period was mainly the evaporation loss. The small production of the acid in the last ethanol addition was due to the production inhibition. The claim agreed with the study in A. aceti M23 that acetic acid has more inhibitive effect than ethanol that the ethanol oxidation was 70% inhibited at acetic acid 60 g/l (Park et. al. 1989). Similar report on A. aceti NCAIM 001379 showed that the organism was more susceptible to inhibition by acetic acid than by ethanol and lethal acetic acid concentration to the cells was 7% v/v or approximately 73.5 g/l (Krisch & Szajáni 1997). When the cultivation was controlled at ph 4.0 and 4.5, the acetic acid production profiles were similar. The maximum acetic acid of 54.5 and 56.2 g/l were obtained at ph 4.0 and 4.5, respectively, following the 2 additions of ethanol. The third ethanol addition did not further increase the acetic acid concentration in both cases. When comparing these results with the cultivation at ph 3.5, the results may imply some combined effect of ph and acetic acid concentration to the production of acetic acid such that higher acetic acid tolerance may be induced at lower ph but not too low to inhibit the intracellular cell functions. A possible explanation may be drawn from the result of the study on evolution of acetate resistant A. aceti that cytoplasmatic anion accumulation is an important component of acetate toxicity and the intracellular acetate concentrations were significantly lower in evolved A. aceti (Steiner & Sauer 2003). As the pk a of acetic acid is 4.76, the acetic acid was presented more in the undissociated form at lower ph and hence less free anion (acetate) to promote the acetate toxicity to the cell. Therefore, it was possible that cells cultivated at ph 3.5 were more tolerant to acetate toxicity than those cultivated at higher ph. However, more detailed studies would be needed to confirm this hypothesis. a b 2084

c d Figure 3. Profiles of fed-batch cultivations with pulse feedings for acetic acid production by A. pasteurianus TISTR 520 at 30 C and various ph controls (a) ph 3.0 (b) ph 3.5 (c) ph 4.0 and (d) ph 4.5: ethanol and acetic acid The acetic acid yields and total productivities, calculated up to the point where ethanol was no longer utilized, showed in Table 1. The values had been accounted for ethanol losses due to aeration and sampling. Similar acetic acid yields and productivities were obtained from the fed-batch cultivations at ph 4.0 and 4.5. However, higher yield was observed at ph 3.5 due to higher amount of acetic acid produced. Lower productivity at ph 3.5 was the resulted from prolonged cultivation after the last ethanol addition. Table 1. Summary of parameters in acetic acid production by A. pasteurianus TISTR 520 in fed-batch cultivations with pulse feedings Cultivation ph 3.0 3.5 4.0 4.5 Cultivation time (h) 77.3 98.2 43.5 45.9 Ethanol used (g) 0 137.2 150.4 138.6 Acetic acid produced (g) 0 121.9 108.5 112.1 Y p/s 0 0.89 0.72 0.81 Q p (g/h) 0 1.242 2.494 2.441 Acknowledgements The authors would like to give their gratitude to the Office of the Cane and Sugar Board for the financial support on this project. References Boonmee M. and Intarapanich S. (2006) Significance of Substrate Loss during Fermentation on Product Yield Calculation: a Case Study of Acetic Acid Production, In: The 16th Annual Conference of Thailand s Chemical Engineering and Applied Chemistry Society, 26-27 October 2006. Bangkok, Thailand. Boonmee M. and Intarapanich S. (2006) Evaluation of 6 Acetobacter spp. for Acetic Acid Production Capabilities. In: The Proceedings of 18th Annual Meeting of the Thai Society for Biotechnology. 2 3 November 2006 The Montien Riverside Hotel, Bangkok, Thailand. Drysdale, G. S. and Fleet, G. H. (1989). The growth and survival of acetic acid bacteria in wines at different concentrations of oxygen. American Journal of Enology and Viticulture, 40(2): 99-105. Ito, T., Sota, H., Honda, H., Shimizu, K. and Kobayashi, T. (1991). Efficient Acetic Acid Production by Repeated Fed- Batch Fermentation using Two Fermentors. Applied Microbiology & Biotechnology, 36(3): 295-299. Krisch J. and Szajáni B. (1997) Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti. Biotechnology Letters, 19(6): 525 528. Muraoka, H., Watabe, Y. and Ogasawara, N. (1982). Effect of Oxygen Deficiency on Acid Production and Morphology of Bacterial Cells in Submerged Acetic Fermentation by Acetobacter aceti. Journal of Fermentation Technology, 60(3): 171-180. Park Y.S., Ohtake H., Fukaya M., Okumura H., Kawamura Y. and Toda K. (1989) Effects of Dissolved Oxygen and Acetic Acid Concentrations on Acetic Acid Production in Continuous Culture of Acetobacter aceti. Journal of Fermentation and Bioengineering, 68(2): 96 101. Park, Y. S., Fukaya, M., Okumura, H., Kawamura, Y. and Toda, K. (1991). Production of Acetic Acid by a Repeated Batch Culture with Cell Recycle of Acetobacter aceti. Biotechnology Letters, 13(4): 271-276. 2085

Park, Y. S., Toda, K., Fukaya, M., Okumura, H. and Kawamura, Y. (1991). Production of a High Concentration Acetic Acid by Acetobacter aceti using a Repeated Fed-Batch Culture with Cell Recycling. Applied Microbiology & Biotechnology. 35(2): 149-153. Steiner P. and Sauer U. (2003) Long-Term Continuous Evolution of Acetate Resistant Acetobacter aceti. Biotechnology and Bioengineering, 84(1): 40 44. Tesfaye W., Morales M.L., García-Parrilla M.C. and Troncoso A.M. (2002) Wine vinegar: technology, authenticity and quality evaluation. Trends in Food Science & Technology, 13: 12 21. 2086