Potential use of Bacillus subtilis in a co-culture with Clostridium butylicum for acetone–butanol–ethanol production from cassava starch

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Abstract

A high amylase producing Bacillus subtilis WD 161 was used in a co-culture with Clostridium butylicum TISTR 1032 to enhance acetone–butanol–ethanol (ABE) production from starch. The mixed culture of C. butylicum TISTR 1032 and B. subtilis WD 161 without anaerobic pretreatment by reducing agent and N2 flushing increased amylase activity 10 folds and enhanced ABE production 5.4 and 6.5 folds from soluble starch and cassava starch, respectively, compared to those of the pure culture of Clostridium itself. The medium optimization for ABE production by the mixed culture without anaerobic pretreatment found that cassava starch concentration of 40 g/L, C/N ratio of 4.35 and the mixed nitrogen sources of 265 mM yeast extract and 100 mM NH4NO3 gave the highest ABE production in terms of final concentration and productivity. The benefits of using this high amylase producing aerobic Bacillus in a co-culture with anaerobic Clostridium were not only increasing substrate utilization and ABE production but there was also no requirement to add any costly reducing agent to the medium or flushing with N2 to ensure anaerobic condition. This thus makes the anaerobic fermentation more economical and cost effective. This co-culture system may contribute greatly to developing industrialized ABE production.

Introduction

The use of energy derived from biological reactions (bio-energy) provides many advantages, perhaps the most important being the reduced dependence on a non-renewable fossil fuel sources. It can also provide good opportunities to convert renewable organic waste materials into energy [1]. The most commonly used metabolically derived liquid bio-energy compounds are ethanol and butanol. Butanol, along with small amounts of acetone and ethanol, is produced biologically from renewable biomass by Clostridium spp. under strictly anaerobic condition. This has been called the “acetone–butanol–ethanol (ABE) fermentation” process [2]. This process has been attractive as all these three products are commonly used solvents in many important industries and especially it also has a high theoretical potential for replacing petro-chemical derived energy [2].

In term of application as a biofuel, butanol is more valuable than ethanol as it possesses many favorable physical properties, including higher energy content, higher boiling points and a reduced need to modify combustion engines. Moreover, butanol also has numerous applications in other fields, for example food, plastics, and other industries [3]. Therefore, optimizing ABE fermentation to enhance butanol production over ethanol appears to be the more commercially attractive option. However, the market for ABE is still tight due to its high production costs. Substrate costs can make up to about 63% of the total cost of ABE production [4]. This is not because of the expense of the substrate itself, but mainly because of the low efficiency of Clostridium to convert substrate into ABE [5]. This means that the yield of ABE is often low, and this together with the formation of by-products leads to the high cost for butanol recovery [3], [4]. In addition, the maintenance of strict anaerobic conditions for Clostridium to grow requires special conditions such as an addition of costly reducing agents into the medium, and flushing with N2 gas. These factors additionally increase the costs of the fermentation process.

There are several possible ways to reduce the costs of producing ABE from fermentation. These include using the low cost fermentation substrate or optimizing the fermentation conditions to improve the efficiency of converting substrate to ABE. Another is genetic engineering which could produce highly effective strains able to utilize a variety of substrates so that the need to pretreat substrates will be reduced [4]. Among the cheap and readily available substrates for ABE production, starch is possibly one of the better choices. However, starch utilization is often low due to the low activity of the amylases produced by Clostridia [5]. The high amylolytic strain could be obtained by the mutation or genetic engineering. But this may require the containment of the genetically modified bacteria to allay fears that there could be problems that raise safety concerns for human health or their effects on the environment. The transformed or modified genes may require special media or conditions to maintain their characteristics [6].

The pre-hydrolysis of starch either by commercial enzymes or by acids with high concentration at high temperature both have negative feedback effects. For example, processes are costly, and difficulties in handling procedures may arise. Furthermore, the treatment of substrate by acids results in a low sugar yield and the formation of harmful by-products. These include formate, furfurol, and melanoids that can seriously inhibit the growth of the butanol producing Clostridia [7]. In order to increase substrate utilization and butanol yield, there have been several reports of processes using Clostridia and other organisms in which the substrate was first hydrolysed by a fungus/clostridial mixture to produce amylase, and then butanol production was achieved separately by adding another Clostridia species [8], [9], [10]. However, all these experiments using mixed cultures were carried out under strictly anaerobic conditions and the cultures were established randomly. There has been one report on butanol production by the simultaneous co-culture of Clostridium with Bacillus which had been randomly isolated from the same soil sample as the Clostridium. However, the role of the Bacillus in the co-culture was not established clearly [11].

In this study, a mixed culture of C. butylicum TISTR 1032 and an aerobic B. subtilis WD 161 with high amylolytic activity was attempted to enhance ABE production from starch. It was thought that aerobic organisms with amylolytic activity would not only assist Clostridium in substrate hydrolysis but it would also consume any available oxygen in the medium and maintain anaerobic conditions for clostridial growth. There would thus be a reduced need to supply any costly reducing agent into the fermentation medium. This could significantly reduce the cost of the fermentation process. Firstly, the growth and amylase production of pure culture of B. subtilis WD 161 under conditions with and without anaerobic pretreatment by addition of a reducing agent and flushing with N2 were compared to those under aerobic condition. Then, the preliminary study of co-culturing B. subtilis WD 161 with C. butylicum TISTR 1032 for ABE production from soluble starch was performed under conditions with and without anaerobic pretreatment. Subsequently, the mixed culture of C. butylicum TISTR 1032 and B. subtilis WD 161 system was applied for ABE production from cassava starch. The medium composition, including the concentration of cassava starch, C/N ratio and ratio of organic and inorganic nitrogen sources, were optimized. The medium optimization is not only required for the enhancement of organic acids during the acidogenic phase and ABE production during the solventogenic phase by Clostridium, but it is also required to enhance secretion of amylase by Bacillus in the mixed culture.

Section snippets

Microorganisms

Clostridium butylicum TISTR 1032 was purchased from the Culture Collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. The stock culture was maintained in the form of a spore suspension in 25% glycerol and frozen at −20 °C.

Bacillus subtilis WD 161 was a generous gift from Asso. Prof. Dr. Poonsuk Prasertsan (Environmental Technology Laboratory, Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University). The stock

Growth and amylase production of B. subtilis WD 161 under aerobic and anaerobic conditions

The possibility of using B. subtilis WD 161 for co-culturing with C. butylicum TISTR 1032 was investigated. B. subtilis WD 161 was cultured under three conditions including the conditions with and without anaerobic pretreatment and aerobic condition. In accordance with references on the anaerobic growth of Bacillus, some Bacillus species grow in the presence of NH4NO3. In such cases NO3 replaces oxygen as an electron acceptor and the NO3 is reduced to N2 gas [17], [18], [19]. Thus, the

Conclusions

This study has shown that the use of high amylase producing B. subtilis WD 161 for co-culturing with C. butylicum TISTR 1032 could enhance ABE production from starch without anaerobic pretreatment. The medium optimization for ABE production by the mixed culture found that the optimum cassava starch concentration was 40 g/L. A low C/N ratio enhanced amylase activity and starch utilization and, consequently, the production of ABE. The use of yeast extract or NH4NO3 alone had a negative effect on

Acknowledgements

The research reported in this paper was financially supported by the graduate school of Prince of Songkla University and Thai Government in the fiscal year of 2009–2010 under Grant AGR5311990030S. The authors would like to thank Asso. Prof. Dr. Poonsuk Prasertsan for providing B. subtilis WD 161, Dr. Sompong O-Thong and Miss Supalak Sattayasmithstid for their general helps.

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