**1. Introduction**

The increasing trend in the consumption of healthy food and the growing demand of natural products by consumers coupled with the price margin between the synthetic and natural flavors have motivated the bioconversion of natural flavors like vanillin, acetoin (AC), etc. that is cost-effective and commercialized. Likewise, the new sustainable development goals of the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

united nation, goal 3 and 12 stated the need for the good health and well-being of the consumer and to ensure the sustainable production and consumption patterns of food product. Acetoin (3-hydroxy-2-butanone or acetylmethylcarbinol) is widely used in the food industry as a flavor enhancer, giving a buttery taste [1]. It can also be used as a building block for various chemicals such as alkyl pyrazines, diacetyl, and acetyl butanediol [1, 2]. Currently, most of the commercial acetoin is produced by chemical synthesis from 2,3-butanedione and 2,3-butanediol. However, the use of such chemically derived acetoin is restricted to food and cosmetic industries because of safety concerns [3]. The production of acetoin by microbial fermentation has been reported to gain increasing interest due to its safety and environmental friendliness [3, 4]. Intensive efforts have been taken to enhance microbial production of acetoin, including screening of high-yield acetoin-producing strains [5, 6], metabolic engineering breeding [7–9], and fermentation optimization [9–11]. The price of chemically generated acetoin (racemic) is over \$ 16.2/kg, and biologically generated dietary acetoin can be two times more expensive [12]. One of the chemicals that can use acetoin as a building block is 1,3-butadiene (BD), which has a worldwide market of 11 Mt./year [13]. However, acetoin is only a minor by-product of 2,3-butanediol production, and its accumulation usually needs complex and expensive growth factors. Apart from carbon, which acts as feedstock for most fermentations, nitrogen is another significant nutrition compositional element needed for the active microbial growth for the fermentation of most targeted bioactive compounds [14].

nutritional substitute for expensive complex media like yeast extract, beef extract, and pep-

Statistical Optimization of Acetoin Production Using Corn Steep Liquor as a Low-Cost Nitrogen…

http://dx.doi.org/10.5772/intechopen.79353

87

Studies have shown that CSL can be used as a nutrition supplement and a cheap nitrogen source for cellular growth in the production of some fermentation products [18, 20, 21]. Yang et al. [15] reported that when a high concentration of CSL was used in acetoin production, cell growth improved with a 3.69-fold increase in acetoin and an improved acetoin productivity [15]. Another author reported that a lower concentration of corn steep liquor had negative effects in the screening of medium components for acetoin production using a newly isolated *Paenibacillus polymyxa* CS 107 [2]. The work by Xu et al. [22] also confirms that CSL and yeast extract had a positive influence on acetoin when screened among other nitrogen sources. All these authors report differently on the optimized condition of CSL as nitrogen source and mostly combined its usage with other costly nutrients like yeast extract and beef extract. This necessitated the need for an optimizing tool like response surface methodology (RSM). Response surface methodology (RSM) is an empirical modeling technique used to establish the relationship between a set of controllable experimental factors and the observed results. RSM defines the effect of the independent variables, alone or in combination, in the processes. In addition to analyzing the effects of the independent variables, this methodology also generates a mathematical model [23]. The applicability of RSM to optimization studies has been demonstrated successfully [23–25]. This chapter focuses on the use of corn steep liquor as a cheap replacement for other expensive complex nutrients such that the CSL and other fermentation controlling parameters could be optimized, using RSM technique for acetoin production. The view of this study is to develop a low-cost fermentation medium that precludes redundant nutritional supplements

and minimizes the costs associated with industrial acetoin fermentation.

peptone 10 g/L, yeast extract 10 g/L, and sodium chloride 5 g/L at pH 7.0 [16].

*B. subtilis* CICC 10025 purchased from the China Centre of Industrial Culture Collection was used in this study. It was maintained on agar slants with the following medium (g/L): glucose 10, beef extract 10, peptone 10, sodium chloride 5, and agar 16 at pH 7.0. The seed culture was prepared by growing the bacterium in 50 ml of the following medium in a 250-ml shake flask for 10 h with agitation of 150 rpm and temperature at 37°C: glucose 60 g/L, beef extract 10 g/L,

The fermentation medium used for this work was the significant optimized medium of Xiao et al. [16], which was then modified. The nutrients used comprised the following (g/L): glu-

COONa 0.5 g, NaCl 5 g, and MgSO4

three different nitrogen sources were used in the preliminary experiment (corn steep liquor, yeast extract, and beef extract). The medium was adjusted to pH 7.0 and autoclaved at 121°

.7H<sup>2</sup>

C with an orbital shaker at 150 rpm for 7 days.

O 0.5 g, while 1 g/L of

C

tone in fermentations [17–20].

**2. Materials and methods**

cose 150, K<sup>2</sup>

HP04

**2.1. Microorganism and inoculum preparation**

**2.2. Medium composition for acetoin production**

0.5 g, CH3

for 15 min. The flasks were incubated at 37°

Studies have shown the potential of low-cost media and complex nitrogen sources like urea, peptone, yeast extract, beef extract, soya bean [14–16] for acetoin production but yet to ascertain effectively the optimized condition. Nitrogen source is one of the main contributors in the total material cost of commercial acetoin production. Hence, replacing a costly nitrogen source with less expensive ones for acetoin production may improve the economics of the process. Corn steep liquor (CSL) has been identified as a potential nitrogen source in biochemical industries and a good substitute for other expensive complex media. It is a major by-product found during cornstarch processing. it is a low-cost source of proteins (**Figure 1**), amino acids, minerals, vitamins, and trace elements and can be used as a rich and effective

**Figure 1.** Graphical illustration of complex nitrogen nutrients and price margin.

nutritional substitute for expensive complex media like yeast extract, beef extract, and peptone in fermentations [17–20].

Studies have shown that CSL can be used as a nutrition supplement and a cheap nitrogen source for cellular growth in the production of some fermentation products [18, 20, 21]. Yang et al. [15] reported that when a high concentration of CSL was used in acetoin production, cell growth improved with a 3.69-fold increase in acetoin and an improved acetoin productivity [15]. Another author reported that a lower concentration of corn steep liquor had negative effects in the screening of medium components for acetoin production using a newly isolated *Paenibacillus polymyxa* CS 107 [2]. The work by Xu et al. [22] also confirms that CSL and yeast extract had a positive influence on acetoin when screened among other nitrogen sources. All these authors report differently on the optimized condition of CSL as nitrogen source and mostly combined its usage with other costly nutrients like yeast extract and beef extract. This necessitated the need for an optimizing tool like response surface methodology (RSM). Response surface methodology (RSM) is an empirical modeling technique used to establish the relationship between a set of controllable experimental factors and the observed results. RSM defines the effect of the independent variables, alone or in combination, in the processes. In addition to analyzing the effects of the independent variables, this methodology also generates a mathematical model [23]. The applicability of RSM to optimization studies has been demonstrated successfully [23–25]. This chapter focuses on the use of corn steep liquor as a cheap replacement for other expensive complex nutrients such that the CSL and other fermentation controlling parameters could be optimized, using RSM technique for acetoin production. The view of this study is to develop a low-cost fermentation medium that precludes redundant nutritional supplements and minimizes the costs associated with industrial acetoin fermentation.
