Acetic Acid Batch Fermentation Process by Clostridium Thermoaceticum Harumi Veny, Masitah Hasan my_harumi@yahoo.com, masitahhasan@um.edu.my Department of Chemical Engineering Faculty of Engineering University of Malaya ABSTRACT Acetic acid was produced through anaerobic batch fermentation process by using Clostridium thermoaceticum as microorganism. The kinetic parameters (i.e. µ max , Y p/s, Y x/s ) and the relationship between growth and acetic acid product formation were determined for all environmental effects studied in batch fermentation to establish the optimum condition for this system. The optimum condition obtained from this batch fermentation studies was at pH 6 and temperature of 60 o C with the maximum product yield (Y p/s ) of 0.88 g acetic acid /g glucose and maximum cell yield (Y x/s ) of 0.11 g cell/g glucose. The maximum specific growth rate was 0.11 hour -1 achieved at initial glucose concentration of 30 g/L. The acetic acid fermentation profile from these studies showed a mix of growth associated and non-growth associated Keywords: Acetic acid, Clostridium thermoaceticum, Single step anaerobic fermentation. (1) INTRODUCTION Presently, almost all acetic acid in chemical industry products is produced synthetically. About sixty two percent is produced by the carbonylation of methanol and the rest by the liquid phase oxidation on n-butane and direct oxidation of acetaldehyde (Wacker process) [1] The major disadvantages of the synthetic process are the need of high temperature and pressure, a good agitation, the threat of explosion, the high cost of catalyst, and the dependence on non-renewable sources of raw material (crude oil). Hence, acetic acid productions to be carried out by biological (fermentation) mean become more attractive. The production of acetic acid via fermentation by using renewable biomass feedstock has also been studied as alternatives to acetic acid production from petroleum or natural gas feedstock [2]. The fermentation process could reduce the high cost since it uses renewable resource as raw material and low energy utilization. The problem with the conventional fermentation process is that the two step aerobic fermentation could only produce low acetic acid product yield (around 0.67 g acetic acid/g glucose). This process also involved two stages for converting glucose into ethanol followed by oxidation of the ethanol to acetic acid. Hence, it needs two separate fermentors. A possible solution to this problem is a single step anaerobic fermentation which is described in this paper. The single step anaerobic process requires the absence of oxygen; hence the condition during all fermentations should be strictly anaerobic by gassing with CO 2 . The single step anaerobic fermentation process has a higher product yield, could eliminate separate fermentors for the different stages of acetic acid production and reduces the time required. In these experiments, the single step anaerobic fermentation process uses Clostridium thermoaceticum which converts glucose almost quantitatively to the theoretical amount of acetic acid. The reaction for this process is as follows: C 6 H 12 O 6 3 CH 3 COOH The acetic acid product yield from this single step fermentation process from several studies is around 0.80-0.90 gram acetic acid per gram of glucose [4,5,9]. The maximum specific growth rate (µ max ) from previous researchers [4,5,6,8] was 0.1 h -1 to 0.14 h -1 in batch fermentation system. (2) MATERIAL AND METHODS Microorganism Clostridium thermoaceticum bacteria from DSMZ, Germany was used in this experiment. Media and growth condition RCM and a modification of DSMZ recommended medium. RCM medium is used for maintenance and stock cultures in solid and semi solid form, which then is sub cultured for every 2 weeks. The RCM composition is: Yeast extract (3 g/l), Lab–Lemco Powder (10 g/l), Peptone (10 g/l), Glucose ( 5 g/l), Soluble starch (1 g/l), Sodium Chloride (5g/l), Sodium acetate (3 g/l), Cysteine hydrochloride (0.5 g/l), Agar (0.5 g/l) or (15 g/l for solid medium). Medium for cultivation of Clostridium thermoaceticum is a modification of some medium compositions from earlier researches [4, 5,10]. The compositions are: Solution A: Glucose (variable), Water (150) Solution B: Yeast extract Difco(5 g), Tryptone difco(5 g),Sodium acetate (1.55 g) (NH 4 ) 2 SO 4 (1 g), MgSO 4 . 7 H 2 O (0.25 g),Fe(NH 4 ) 2 (SO 4 ) 2 . 6 H 2 O (0.039 g), Co(NO 3 ) 2 . 6 H 2 O (0.03 g), Na 2 WO 4 . 2H 2 O (3.30 mg), Na 2 MoO 4 . 2H 2 O (2.4 mg), ZnCl 2 (1.4 mg),Na 2 SeO 3 . 5H 2 O(0.2 mg), NiCl 2 . 6H 2 O (0.2 mg), Resazurin 0.1% (1 ml), Distilled water (550 ml), Cysteine hydrochloride (0.25 g), Na 2 S.9H 2 O 5%(5 ml) Solution C: NaHCO 3 (16.8 g), K 2 HPO 4 (7 g), KH 2 PO 4 (5.5 g), Distilled water (300 ml). The media was prepared by heating before sterilization, to help driven off dissolved oxygen Growth condition, medium and inoculums preparation, and culture storage condition have been previously described [3]. Analytical methods Acetic acid production was analyzed using Gas chromatograph. Growth of Clostridium thermoaceticum was monitored spectrophotometrically by measuring the optical density at 600 nm. A standard curve relating the optical density to cell dry weight was constructed. The glucose utilization was analyzed by DNS method spectrophotometrically at optical density of 550 nm. Detail analytical methods have been previously described[3]. Batch fermentation studies The batch fermentation studies were carried out in 2.5 L KO biotech fermentor with a working volume of 1.5 L. The inoculums for all batch fermentation runs were incubated for 48 hours at 60 o C. The anaerobic condition for fermentation process was maintained by continuous supply of sterile CO 2 gas. The pH was maintained by automatic addition of 2N KOH and 2N HCl. The foam formed during the process was reduced by automatic addition of antifoam agent and with the help of foam breaker in the stirrer. The stirrer rate for all batch fermentation was maintained at a low rate of 50 rpm. (3) RESULTS AND DISCUSSIONS The environmental effects on batch fermentations were studied by varying the pH (i.e. pH 5 to 7) and initial glucose concentration (15 to 60 g/L). The environmental effect studies achieved the maximum acetic acid yield of 0.88 g/g and growth yield of 0.1 g/g at pH 6 and 60 o C. This is probably because of the pH optimum conditions for the bacteria growth is near to neutral pH while the pH of acetic acid production was expected to be at acid pH. See Figure 1. According to literature[8], at low pH below pH 6 undissociated acetic acid is responsible for growth inhibition and at high pH (above pH 6) ionized acetate is responsible for growth inhibition . The maximum specific growth rate (µ max ) obtained was 0.11 h -1 at initial glucose concentration of 30 g/L. The specific growth rate increased with increase in initial glucose concentration from 15 g/L to 30 g/L before it tends to decrease up to concentration of 60 g/L. See Figure 2.This may be so as the substrate concentration has exceeded the substrate critical concentration (S>S crit )[7]. The maximum specific growth rates were determined at the exponential phase and through graphical data of plot ln(x/x o ) as a function of time (t) by following the growth rate equation: X d t dx . µ = 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 45678 pH 0 0.02 0.04 0.06 0.08 0.1 0.12 Product Yield Growth yield Fig.1 Acetic acid yield and growth yield at different pH 0 0.02 0.04 0.06 0.08 0.1 0.12 0 20406080 Initial glucose concentration (g/L) Specific growth rate Fig.2 The maximum specific growth rate The maximum kinetic parameters result at the optimum condition are given in Table 1 Table.1 Kinetic parameters Initial glucose concentration Temp pH µ max (h -1 ) Y x/s (g.g -1 ) Y p/s (g.g -1 ) 15 g/L 60 o C 6 0.082 0.108 0.88 30 g/L 60 o C 6 0.11 0.11 0.815 The batch fermentation profile at optimum condition of pH 6 and 60 o C from this study is given in Figure 3. This fermentation profile showed that the acetic acid concentration increased with the increase in biomass/dry cell. However, the acetic acid still increased even when the dry cell had already ceased to grow. This phenomenon illustrates a mixed growths associated which also have been reported previously in some literature [6, 8]. 0 2 4 6 8 10 12 14 16 0 20406080100120140 Time (hours) Acetic acid, Glucose (g/L) 0 2.5 5 7.5 10 Dry weight (g/L) acetic acid glucose dry weight Fig.3 Batch fermentation profile at pH 6 and 60 o C [4] Conclusion The conclusions which could drawn from this acetic acid batch fermentation process from glucose by Clostridium thermoaceticum are: 1. Overall the optimum condition for acetic acid production obtained from these studies was at pH 6 and temperature of 60 o C. 2. The maximum production yield (Y p/s ) achieved was 0.88 g of acetic acid/g glucose which is within the range of earlier reported researches on this process. Y p/s Y x/s 3. The maximum biomass yield (Y x/s ) of 0.11 g biomass/g glucose and maximum specific growth rate (µ max ) of 0.11 hour -1 were obtained at initial glucose concentration of 30 g/L. 4. The relation between the growth and acetic acid production from these studies showed a mix of non growth and growth associated where the acetic acid was still produced even when growth has ceased. [5] References [1]Busche, R.M.(1991). Extractive Fermentation of acetic acid; Economix trade off between yield of Clostridium and concentration of Acetobacter. Appl.Biochem.Biotech.28(29):605-621. [2] Cheryan,M., Parekh,S., Shah,M., Witjitra,K.,(1997). Production of acetic acid by Clostridium thermoaceticum. Adv.Appl.Microbial.V.43.pp.1-31 [3] Harumi.V, Masitah.H,Ramachandran.K.B,.(2003) “Acetic acid production by single step anaerobic fermentation of glucose. In; Proceeding of Technical Postgarduate Symposium 2003 (TECHPOS’03). [4] Ljungdahl,L.G., (1983).Formation of acetate using homoacetate fermenting anaerobic bacteria. In: Organic chemicals from biomass, edited by Wise,D.L., pp.219. The Benjamin/Cummings Publishing Company,Inc. [5]Parekh,S.R., Cheryan,M., (1990). Acetate Production from Glucose by Clostridium thermoaceticum. Process Biochemistry International.117-121. [6] Parekh,S.R., Cheryan.M., (1991). Production of acetate by mutant strain of Clostridium thermoaceticum.Appl.Microbiol.Biotechnol.36:3 84-387 [7] Sugaya,K., Tuse,D., Jones,J.L., 1986. Production of acetic acid by Clostridium thermoaceticum in batch and continous fermentations. Biotech.Bioeng. v.xxviii.pp.678-683. [8] Pirt, S.John, (1975). Principles of microbe and cell cultivation, Halsteed Press Book.UK. [9] Wang,I.C.D., Fleishchaker.J., Wang,Y.G., (1978) A novel route to the production of acetic acid by fermentation. AIChE Symposium series, No.181, vol 74. [10] Witjitra,K., Shah,M.M., Cheryan,M., (1996). Effect of nutrient sources on growth and acetate production by Clostridium thermoaceticum. Enzyme & Microbiol.Technol.19:322-327 . environmental effects studied in batch fermentation to establish the optimum condition for this system. The optimum condition obtained from this batch fermentation. separate fermentors for the different stages of acetic acid production and reduces the time required. In these experiments, the single step anaerobic fermentation