THE EFFECT OF COD CONCENTRATION ON ORGANIC ACIDS PRODUCTION FROM CASSAVA ETHANOL STILLAGE

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1 ISBN THE EFFECT OF COD CONCENTRATION ON ORGANIC ACIDS PRODUCTION FROM CASSAVA ETHANOL STILLAGE Agung Nugroho, Kiki Yustendi, Tjandra Setiadi Department of Chemical Engineering, Faculty of Industrial Technology, Institut Teknologi Bandung, Jl. Ganesa, Bandung 432 ABSTRACT The objective of this research is to evaluate the conversion of cassava ethanol stillage into organic acids in an anaerobic process with the variation of initial COD concentration. The stillage was obtained from BPPT bioethanol plant in Lampung and treated in anaerobic batch reactors. Temperature was maintained at 35 o C and ph was about ± 6. Variations of initial COD concentration used were 5, 2, 25, and 3 g COD/L. Parameters analyzed were COD, TVA (total volatile acids), and concentration of each acid formed. Degradation of COD was occurred in all runs and the highest COD removal was 33.6% at COD of 25 g/l, however the highest degree of TVA production (.233 g-c TVA / g-c COD) was observed at the COD of 2 g/l. The optimum time to produce total volatile acids was about 4 hours (2 days). The TVA production increased from the COD of 5 to 2 g/l, and decreased from the COD of 25 to 3 g/l, it might be due to a high-organic-content inhibition. The acetic acid production was increase with the increasing COD concentration, it indicated that there was no accumulation of H 2 gas in the broth fermentation. Key words : acidogenic, anaerobic, cassava, organic acid, ethanol stillage I. INTRODUCTION Typically, -5 liters of ethanol stillage, as a distillation by product, are produced for every liter of alcohol product. This data shows that a large number of wastes will be produced in the bioethanol processes. In , the government of Indonesia has planned to use ethanol as a gasoline substitute as much as 5% of gasoline (premium) consumption or it will be approximately 2.8 billion liters/year of ethanol production []. Therefore, it can be predicted that about 35 billion liters/year of ethanol stillage will be produced. If the stillage can not be recovered or treated, it will create big environmental problems. One of the solution for these problems is to obtain valuable products from it. The possibility to utilize the waste is to produce volatile organic acids from the stillage. It is reported as well that the main substrate for ethanol production in Indonesia will be from cassava. Volatile organic acids include some short fatty acids such as formic acid, acetic acid, propionic acid, butyric acid, and valeric acid. Each acid has a certain value, if it is further proceeded. Like acetic acid (CH 3 COOH), it is one of the simplest carboxylic acids and has many applications in industries. The volatile organic acid production from ethanol stillage by anaerobic fermentation is the most popular method. In general, anaerobic digestions of ethanol stillage will produce methane as an end product. However, to produce methane gas, there are some intermediate organic acids produced. There are four steps of anaerobic digestion: namely hydrolysis, acidogenic, acetogenic, and methanogenic [2]. The schematic main processes in the anaerobic degradation is shown in Fig.. Process of organic acid formation from ethanol stillage is a partial anaerobic digestion, where the anaerobic digestion cycle is terminated after the acetogenic process. Production of organic acids is started by degradation of ethanol stillage with extra cellular enzymes secreted by mixed cultures. The organic acids will be produced during acidogenic and acetogenic. All those processes are done by groups of bacteria participating in anaerobic digestion, e.g.: acidogenic bacteria, hydrogenogenic bacteria, and homoacetogenic bacteria [3]. It is reported that the product formation by a mixed acidogenic population is a very complex process and is greatly influenced by many factors. These factors include wastewater specificity, reactor configuration, hydraulic retention time (HRT), influent organic concentration, organic loading rate, ph, temperature, oxidation reduction potential, and nutritional requirements [4]. In this study, the effect of COD concentration on the production of volatile organic acids from cassava ethanol stillages would be investigated. Moreover, it would be reported how the COD concentration would affect the type of organic acids produced. Yogyakarta-Indonesia, 4-5 th December 27

2 ISBN Figure. Anaerobic process for insoluble carbohydrate [5]. II. MATERIAL AND METHODS A. Operation conditions and experimental set up The anaerobic process was started with seeding and acclimatization. The freshly cow manure was used as seeds, then it was acclimatized to the cassava ethanol stillage obtained from the BPPT ethanol plant in Lampung. After the anaerobic culture was ready, it was used as a main seed for each run. The experiments were carried out in 6 L batch reactors having a working volume of 5 L. Each reactor equipped with a gas pump to circulate the produced gas which has two functions, namely to mix the suspension, and to reduce the hydrogen gas partial pressure. The produced gas was released intermittently. The anaerobic process was maintained at optimal temperature of 35 o C [6]. A water bath was used to maintain temperature during anaerobic digestion, and the initial COD concentration was varied, namely 5, 2, 25 and 3 g/l. Retention time for acidogenesis and acetogenesis process was reported to be 48 hours [7]. However, in this experiment, the process was carried out until 72 hours. This was occurred to explore acids produced after 48 hours of anaerobic digestions. Therefore, the variation retention time was due to the sampling time, i.e., 6, 2, 24, 36 and 72 hours. Acidity in the reactor was controlled at around 6 by adding phosphate buffer every 6 hours. Hydrogen partial pressure in the fermentor was kept in a very low concentration by circulating the nitrogen gas feed into fermentor every 2 hours for 5 min period. Each experiment was conducted in duplicate. B. Analysis Ion Chromatography DIONEX ICS was used to analyze the concentration of volatile organic acids. Parameter analyzed was the concentration of each acids produced for every retention time in each run. Besides that, COD, MLSS, and TVA (total volatile acids were also analyzed according to Standard Methods 992. III. RESULT AND DISCUSSION A. The acclimatization periods The acclimatization process using mixed cultures from freshly cow manure was carried out in the beginning of anaerobic process. It included every day feeding of stillage as a substrate into two different COD concentrations, i.e. 5 and 25 g/l, which the first (I) would be used for the reactor with COD concentration of 5 and 2 g/l, and the second (II) would be used for the reactor with COD concentration of 25 and 3 g/l. After acclimatization process for 2 months, the mixed liquor suspended solid (MLSS) concentration in each reactor was increased from 6 to 2 ppm and from 26 ppm to 28 ppm, respectively. Besides the MLSS concentration, the acclimatization process resulted the distinct color at the end of the process as can be seen in the Fig. 2. Mixed culture for initial COD concentration of 5 and 2 g/l Mixed culture for initial COD concentration of 25 and 3 g/l Figure 2 Mixed cultures produced from the acclimatization process Yogyakarta-Indonesia, 4-5 th December 27

3 ISBN B. COD Removal The anaerobic digestion process for producing TVA resulted in the COD degradation. There were also some variations of the result regarding the variation of the COD concentration used in the anaerobic digestion process. Fig. 3 shows the COD removal in this process depended on the initial COD, it can be seen that the initial COD concentration of 25 g/l gave the best performance for the COD degradation of the stillage, that was 33.6%. The COD degradation increased from initial COD of 5 to 25 g/l, and decreased from 25 to 3 g/l. COD removal (%) Initial [COD] (g/l) Figure 3. Percentage of COD degradation as a function of initial COD concentration. C. Production of Total Volatile Acids (TVA) During the anaerobic digestion, the production of total volatile acids (TVA) from ethanol stillage can be seen in Fig. 4. Fig. 4 (a) shows that the TVA produced was less significant in a retention time of more than 4 hours for every variation of initial COD concentration. The initial COD concentration gave differentiation in the production of TVA. The initial COD concentration of 5 g/l gave the lowest TVA production (.26 g-c) and the initial COD concentration of 3 g/l gave the highest TVA production (2. g-c). Total Volatil Acid (g-c) 3 2,5 2, Time (hour) [COD] = 5 g/l [COD] = 2 g/l [COD] = 25 g/l [COD] = 3 g/l TVA production (g-c/g -C-COD),25,2, Initial [COD] (g/l) (a) Figure 4 (a) The production of TVA at different initial COD concentration (b) The degree of TVA production comparing to the initial COD concentration (g-c) Note: The degree of TVA production in Fig. 4 (b) was calculated from the following equation: Degree of TVA Production = final mass of TVA ( g C) initial mass of TVA ( g C) initial mass of COD ( g C) The degree of TVA production usually stated as the produced TVA (as g-c) divided by the initial COD concentration as g-c [4]. If the data obtained was analyzed and compared to its initial COD in term of g-c, the result was completely different as showed in Fig. 4 (b). It shows that the initial COD of 2 g/l had the highest degree of TVA production (.233 g-c TVA/g-C COD). The degree of TVA production increased from the initial COD of 5 to 2 g/l, and decreased from the initial COD of 25 to 3 g/l. D. Production of Specific Volatile Acids The productions of TVA can be broken down into the production of specific volatile acids such as acetic, butyric, valeric and propionic acids. Fig. 5 (a), (b), (c), and (d) show the results of the production of specific volatile acids during the anaerobic digestion for different initial COD concentrations. (b) Yogyakarta-Indonesia, 4-5 th December 27

4 ISBN , , (a) (b), , (c) Figure 5 (a) Volatile acids produced for the initial COD concentration of 5 g/l (b) 2 g/l (c) 25 g/l, (d) 3 g/l Fig. 5 (a) shows the concentration of volatile acids formed during the anaerobic digestion period at the initial COD of 5 g/l. The result shows that there were five volatile acids formed during the fermentation. Those acids were formic, acetic, propionic, butyric, and valeric acids. Each acid was formed in a different rate during the anaerobic digestion. The valeric acid was formed much more than any other acids (.23 g-c) after 2 hours of digestion and reached its peak at 36 hours of digestion (.83 g-c). Fig. 5 (b) shows the result of the initial COD of 2 g/l, there was more likely the similar result as the previous initial COD concentration that there was no formation of formic acid. In this system, the acetic acid did not increased significantly and the propionic acid was formed in a low rate during the process. The different result was detected in the formation of butyric acid that it was produced more than the others and reached its peak (.2 g-c) at 36 hours of digestion. The last two initial COD concentrations gave a completely different volatile acids profile than those of the first two initial COD concentration. In these conditions, acetogens bacteria gave a good performance resulting a high concentration of acetic acid. Fig. 5 (c) shows the result for the initial COD of 25 g/l. The formation of propionic acid reached its peak at 2 hours of digestion and butyric at 24 hours of digestion. After 36 hours of digestion, the formation of acetic acid increased significantly resulting the decrease of propionic and butyric acids. At the end of the digestion, the acetic acid was formed in the mass of.4 g-c, much more than any other acids in the system. Fig. 5 (d) shows the result for the initial COD of 3 g/l. It is noted that the formation of propionic and butyric acid increased after 2 hours of digestion. However, after 36 hours of digestion, the formation of acetic acid incerased significantly and at the same time, propionic and butyric acids decreased. The valeric acid was formed in a very little amount but it was also converted into acetic acid by acetogens bacteria after 36 h of digestion. There was no formation of formic acid in this condition. At the end of the anaerobic digestion process, the concentration of acetic acid was almost six times higher than any other acids in the system. The formation of acetic acid in this condition was the highest among the other COD concentration, it was.97 g-c/g-c COD. E. Discussion In this experiments, the TVA production from cassava ethanol stillage was conducted in batch reactors for 3 days to evaluate acids production in a longer time. The result in the Fig. 4 (a) shows that the optimum time required to produce TVA (acidogenesis) was 4 hours or about 2 days, it was in accordance with the time needed as in reference [7]. The distinct color between two mixed cultures indicated that there was a different group of bacteria existed. These mixed cultures gave different results of TVA produced, like showed in the Fig. 2. The highest acid production was at the COD of 2 g/l. As has been reported [3], that the anaerobic digestion is a complex process involving several different types of microorganisms or mixed cultures that was affected by the initial COD concentration. It was predicted that the initial COD of 2 g/l gave the highest production of TVA because acid-producing bacteria were exist more. It can be proved from the mixed cultures used, that mixed culture used for COD of 5 and 2 g/l was different from the mixed culture used for COD of 25 and 3 g/l, as shown in the Fig. 2. In the COD of 25 and 3 g/l, Yogyakarta-Indonesia, 4-5 th December 27 (d)

5 ISBN it was predicted that acid-producing bacteria were suppressed or inhibited due to the high of initial COD concentration. The differences of specific acids produced at different initial COD concentration were observed (Fig. 5 (a) to (d)). The results also showed that the rate of production decreases for some acids as the digestion progresses. The higher intermediates had been converted to lower acids as the digestion progresses, like propionic and butyric acids were converted into acetic acid [8]. Following the pathway of anaerobic digestion as showed in Fig., all the volatile acids formed during hydrolysis and also acidogenesis phase would be converted into acetic acid in the acetogenesis phase. It was assumed that during the anaerobic process, production of acetic acid would be inhibited by H 2 gas produced according to the following reactions: Propionic + 3 H 2 O Acetic + HCO H 2 + H + Butyric + 2 H 2 O 2 Acetic + 2 H 2 Valeric + 2 H 2 O + H H 2 Acetic + Propionic The reactions show that H 2 gas will be produced as a byproduct in the acetic acid production from the propionic and butyric acids.the higher of initial COD concentration, then the content of valeric, butyric, and propionic acids would be higher too. In a high initial COD concentration, the acetic acid and H 2 gas formation would be generated, but the H 2 gas would be formed in amount more than that of acetic acid (see the coefficient of reactions). Therefore, in the higher of initial COD concentration, the acetic acid production should be lower. However, in this experiment, the results obtained was quite different from the assumption. The results gave that the acetic acid production was higher along with the increasing of initial COD concentration. It can be seen from Table, that at the initial COD of 25 and 3 g/l, the acetic acid percentage at the end of process was higher than those of at the initial COD of 5 and 2 g/l. Table Percentage of specific acids at the end of the process Component [COD] = 5 g/l [COD] = 2 g/l [COD] = 25 g/l [COD] = 3 g/l Formic acid (%) Acetic acid (%) 2,88 49,57 76,22 Propionic acid (%) 8,48 26,7 7,56 9,3 Butyric acid (%) 24,5 46,92 34,8 3,9 Valeric acid (%) 67,47 4,5 8,6,47 These results might be related to its experiment design. The experiment was designed to minimize the accumulation of H 2 gas in the broth fermentation by circulating the fermentation gas, and sparging with N 2 gas was conducted also. So, it was predicted that the accumulation of H 2 gas in the broth fermentation was not occurred. It was also found that hydrolysis, acidogenesis, and acetogenesis occurred consecutively, as has been reported in many literatures. It can be seen that acetic acid formation occurred after propionic and butyric acid formation, that was after 36 hours of digestion (Fig. 5 (a) and (b)). This phenomena took place due to the increasing of acetogenic bacteria activities after 36 hours of digestion. Before that time, acidogenic bacteria gave high productivity resulting an increase of butyric and propionic acid. With the increase of volatile acids, then at the certain stage, they would suppress the activity of acidogenic bacteria and gave a very good environment for acetogenic bacteria to convert those acids into acetic acid. It was observed that production of butyric acid was more than that of propionic acid in all cases and more than that of acetic acid in the first two cases. In the case of consumption of different TVA, propionic acid was consumed more, followed by butyric acid. The propionic acid consumption can be related to the formation of acetic acid and the consumption of acetic acid can be related to the methane formation. IV. CONCLUSION From this study, it can be concluded as follows:. Degradation of COD was occurred in all runs and the highest COD removal was 33.6% at COD of 25 g/l. 2. The optimum time to produce total volatile acids was about 4 hours (2 days). 3. The initial COD of 2 g/l gave the highest degree of TVA production (.233 g-c TVA / g-c COD ). The TVA production increased from the initial COD of 5 to 2 g/l, and decreased from the initial COD of 25 to 3 g/l, it might be due to a high-organic-content inhibition. Yogyakarta-Indonesia, 4-5 th December 27

6 ISBN The acetic acid production was increase with the increasing COD concentration, it indicated that there was no accumulation of H 2 gas in the broth fermentation. ACKNOWLEDGMENT - This study was funded by LPPM ITB through Riset KK-ITB 27 with contract No. 82c/K.9/PL/27. - The cassava ethanol stillage was obtained from the BPPT ethanol plant in Lampung. - Thank to Dr. Udin Hasanudin from UNILA, for examining the specific acids in the samples using the Ion Chromatography DIONEX ICS. REFERENCES [] Anon., Produksi Bioetanol di Indonesia, Kompas, March 6 th 26. [2] de Mes. T.Z.D., Stams JH., Reith JH. and Zeeman G., 23, Methane production by anaerobic digestion of wastewater and solid wastes, Dutch Biological Hydrogen Foundation pp [3] Lyberatos, G., 999, Modeling of Anaerobic Digestion A Review, Global Nest: the Int. J. Vol, No 2, pp [4] Speece, RE., 996, Anaerobic Biotechnology for Industrial Wastewaters, Archae Press, Tennessee. [5] Angelidaki, I. dan Ahring, B.K., 992, Effects of free long-chain fatty acids on thermophilic anaerobic digestion, Appl. Microbiol. Biotech., 37, [6] Hwang, S., Yongse L., dan Keunyoung Y., 2, Maximization of Production in Partial Acidogenesis of Swine Wastewater, Biotechnology and Bioengineering Vol.75 No. 5. [7] Yeoh BG., 997, Two Phase Anaerobic Treatment of Cane-molasses Alcohol Stillage, Wat. Sci. Tech. Vol. 36 No. 6-7, pp [8] Lata, K., Rajeshwari, KV., Pant DC., and Kishore VVN., 22, Volatile Fatty Acid Production during Anaerobic Mesophilic Digestion of Tea and Vegetable Market Wastes, World Journal of Microbiology & Biotechnology 8: , Kluwer Academic Publishers. Printed in the Netherlands. Yogyakarta-Indonesia, 4-5 th December 27

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