Clostridium aceticum—A potential organism in catalyzing carbon monoxide to acetic acid: Application of response surface methodology

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Abstract

Carbon monoxide is a poisonous gas that results from the automobile emission, gasification of biomass and sewage sludge. The conversion of carbon monoxide as the gaseous substrate to acetic acid by Clostridium aceticum is an alternative usage route to curb the CO problem at relatively low cost. Mixed levels of full factorial design have been conducted to study the effects of CO partial pressure ranging from 1.40 to 2.02 atm and the fermentation time (0–120 h) over the cell density, CO residue and acetic acid concentration in the batch system. The applied mixed gas composition were 4% H2: 18% Argon: 78% CO. The response surface plot computed from experimental design was employed to optimize the process operating conditions for maximum cell density, minimum CO residue and maximum acetic acid productivity. The results from the surface plot shows that acetic acid fermentation is best operated at 1.40 atm CO partial pressure and at 48 h fermentation times in order to achieve process optimization.

Introduction

Acetic acid or more commonly known as vinegar in our daily life is an important industrial feedstock where it is primarily produced from mineral oil or natural gas. Acetic acid is an important industrial feedstock for the manufacture of vinyl acetate monomer and some commonly used esters such as ethyl acetate, butyl acetate and cellulose acetate [1], [2]. In 2003, the worldwide demand for acetic acid was estimated over 15 billions of pounds [1]. Therefore, renewable resources should be used to produce acetic acid since oil as the important acetic acid feedstock is depleting.

In the past decade, renewable resources such as synthesis gas which acts as the simple and cheap feedstock for organic chemical acetic acid has drawn considerable attention. Synthesis gas consisting mainly of CO, CO2 and H2 resulted from the gasification of the sewage sludge, biomass and municipal solid waste which are abundantly available in agricultural country like Malaysia [3]. Malaysia was reported to generate more than 70 million tonnes of biomass from agricultural waste annually. Therefore, it is advantageous and essential to develop an alternative route for acetic acid production from non-renewable process feedstock (petroleum) to renewable feedstock (biomass).

At present, acetic acid is mainly produced by three chemical processes: (i) n-butane oxidation, (ii) methanol carbonylation and (iii) acetaldehyde oxidation which employ mineral oil or natural gas as feedstock [4]. Biological acetic acid production has several advantages over chemical processes in terms of lower energy costs required by the system, lower major input costs into the process, allows a higher specificity, bacteria possesses greater resistance to poisoning and lastly permit a minimum pollutants emissions [5].

Oxidative fermentation has been used to produce acetic acid by utilizing genus acetobacter as biocatalyst under the stream of oxygen. The theoretical maximum yield of the aerobic acetic acid fermentation from glucose is 0.67 g acetic acid per gram of glucose due to the loss of one carbon as CO2 in the glucose during ethanol fermentation prior to acetic acid fermentation. In comparison, fermentation with Clostridium thermoaceticum (acetogenic bacteria) offers significant advantage in terms of acetic acid yield with 3 g acetic acid per gram of glucose being achieved theoretically [6]. Acetogenic bacteria are anaerobic bacteria and capable of fermenting variety of substrates including one-carbon compounds almost stoichiometrically to acetic acid. The acetogens includes C. thermoaceticum [7], Eubacterium limosum [8] and Peptostreptococcus productus [9]. Most of the acetogens can also synthesize acetic acid from reduced one-carbon compounds such as carbon monoxide, formate and methanol [10]. Lynd and Zeikus [11] first observed the ability of acetogens to grow on CO as an energy source. Butyribacterium methylotrophicum can be adapted to grow rapidly on 100% CO in the culture headspace while Acetobacterium woodii exhibited slow growth under 55% CO atmosphere.

Clostridium aceticum was the first acetogenic bacterium isolated by Wieringa [12]. C. aceticum have been proven to grow chemoorganotrophically on variable organic sources and chemolithotrophically only with H2/CO2 at different ratio as shown in reaction 1:2CO2 + 4H2  CH3COOH + 2H2OC. aceticum was discovered as the potential acetate producer in early 1936, but no efforts have been done to explore the natural capability of C. aceticum to grow autotrophically on CO. Therefore, our interest is to grow C. aceticum autotrophically under mixed gas (4% H2: 18% Argon: 78% CO) while producing valuable organic chemical, acetic acid. In this paper, strain C. aceticum isolated by Wieringa (DSMZ 1496) were employed in batch fermentation under a series of different CO partial pressure. The CO tolerances for C. aceticum that grows under different concentrations of gaseous substrates were determined through its cell concentration, the moles of CO consumed and acetic acid production. A statistical experimental design with mixed levels was implemented in order to analyze the corresponding responses in a statistical manner thus resulting in a valid and objective conclusion [13]. Mixed levels full factorial design is an experimental matrix that comprises the combination of different studied levels for each dependent variable. About 5 and 11 levels have been selected for variable CO partial pressure and fermentation time, respectively, and attributed to a total of 55 runs. Such factorial design have been selected due to a considerably long fermentation period (approximately 5 days) is essential for acetic acid fermentation with 12 h interval time of sample taken. This is to ensure a complete and thorough monitoring for the biocatalyst's activity. During statistical analysis, the response surface methodology quantifies the relationships between three measurable responses with the two vital factors and thus determines the optimum operating conditions in a process.

Section snippets

Microorganism

Bacteria strain of C. ceticum (DSMZ 1496) which was obtained from Braunschweig, Germany's culture collection (DSMZ) was used throughout the experiment.

Media and cultivation

The C. aceticum was grown in the liquid medium with the compositions as described in Table 1. The prepared medium was boiled for a few minutes while continuously degassed with N2. A 50 ml of medium were equally distributed into glass serum bottles while being continuously degassed under N2 for a few minutes. The bottles were then fitted with gas

Results and discussions

Cell density, CO residue inside the batch system together with the acetic acid produced are the three responses considered to be important in evaluating the most operability region for acetic acid fermentation. In batch fermentation, different CO partial pressure means different gaseous substrate concentrations and carbon source concentrations, which directly affect the cell density and acetic acid production. Fermentation time is another important factor that indicates the rate of the gaseous

Conclusion

Statistical experimental design provides the useful information on the effect of each individual factor to the concerned responses. CO concentration is not an inhibitory factor to the C. aceticum growth and also to the acetic acid production by the bacteria when operated at 1.40–2.02 atm CO partial pressures. Based on the developed empirical model, fermentation time was discovered to be a great factor influencing the amount of total cell generated and the total acetic acid that can be produced

Acknowledgements

The present research was made possible through an IRPA grant project (01-02-05-32230EA011) sponsored by Ministry of Science, Technology and Innovations (MOSTI), Malaysia and Universiti Sains Malaysia (USM).

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