Development of an industrial medium for economical 2,3-butanediol production through co-fermentation of glucose and xylose by Klebsiella oxytoca

Abstract

An industrial medium containing urea as a sole nitrogen source, low levels of corn steep liquor and mineral salts as nutrition factors to retain high 2,3-butanediol production through co-fermentation of glucose and xylose (2:1, wt/wt) by Klebsiella oxytoca was developed. Urea and corn steep liquor were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. Under the optimal medium, the yield of 2,3-butanediol plus acetoin relative to glucose and xylose was up to 0.428 g/g, which was 85.6% of theoretical value. The cheap nitrogen source and nutrition factors combining the co-fermentation process using lignocellulose derived glucose and xylose as the carbon source in the developed medium would be a potential solution to improve the economics of microbial 2,3-butanediol production.

Introduction

Development of bio-refineries has recently attracted increasing attention as a means to provide sustainable alternative solutions to depleting petroleum resources and environmental pollution. Many chemicals, which could only be produced by chemical processes in the past, could potentially be generated biologically from annually renewable resources (Ragauskas et al., 2006). Microbial production of 2,3-butanediol (2,3-BD) is one of the example. Interest in this bioprocess has been increasing recently due to that 2,3-BD has large number of industrial applications and this course would alleviate the dependence on oil supply for the production of platform chemicals. The dehydration of 2,3-BD yields the industrial solvent methyl ethyl ketone (Tran and Chambers, 1987). Further dehydration produces 1,3-butadiene, which is the building block of synthetic rubber (van Haveren et al., 2007). And the high octane rating of 2,3-BD makes it a potential aviation fuel (Magee and Kosaric, 1987, Wu et al., 2008). Besides, 2,3-BD has potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives, plasticizers, foods and pharmaceuticals (Syu, 2001).

However, the raw material costs still hinder the large-scale application of this process. In the precious study, some cheap substrates such as food industry residues (starch hydrolysate, molasses and whey permeate) and lignocellulosic biomass (wood and corn cob hydrolysate) were used for 2,3-BD fermentation (Afschar et al., 1993, Cao et al., 1997, Grover et al., 1990, Martinez and Speckman, 1988, Perego et al., 2000). Among them, lignocellulosic materials represent the largest reservoir of potentially fermentable carbohydrates. These carbohydrates obtained either via an acid-promoted hydrolysis or by an enzymatic hydrolysis, are intrinsically a mixture of pentose (e.g. xylose and arabinose) and hexose (e.g. glucose and mannose, etc.) (Stephanopoulos, 2007). For example, complete hydrolysis of corn stover will ideally generate a solution primarily containing glucose and xylose at a ratio around 2:1 (wt/wt) (Yan et al., 2009). However, most of the present 2,3-BD fermentation processes either considered only the cellulose fraction while ignoring the hemicellulose (Cao et al., 1997), or just used the hemicellulose derived pentose (Saddler et al., 1983). If such a process of 2,3-BD production from lignocellulosic materials is to be economically attractive, effective co-fermentation of mixed sugar must be achieved.

In addition, as for the nitrogen sources in 2,3-BD fermentation, most of the previous works have used yeast extract or ammonium salts (Laube et al., 1984, Qin et al., 2006). The use of these nitrogen sources for the industrial production of 2,3-BD may not be feasible because of their relatively high cost. Encouragingly, the commonly used 2,3-BD producing Klebsiella species have the native ability to use urea as a nitrogen source (Brisse et al., 2006). On an equivalent nitrogen basis, urea is much cheaper than yeast extract and is typically half the cost of ammonium salt. Also, using urea as nitrogen source has additional benefits. Unlike ammonium salts, the metabolism of urea does not contribute to the acidification of the medium (Teixeira de Mattos and Neijssel, 1997), thus reduces the amount of base required for pH control. Corn steep liquor (CSL) which is a by-product of the wet milling of corn for starch is extensively used as a component of medium for the culture of microorganisms in industrial fermentation. It provides a rich source of nutrients, vitamins and minerals, so it can be used as a cheap source of the required nutrients for the substitution of laboratory used yeast extract.

In this paper, the Plackett–Burman design, the steepest ascent method, and the response surface analysis, which were useful statistical methodologies, were adopted to develop an inexpensive industrial medium that contained urea as a sole nitrogen source, low levels of CSL and mineral salts as nutrition factors to retain high 2,3-BD production through co-fermentation of glucose and xylose by Klebsiella oxytoca.