High production of 2,3-butanediol from biodiesel-derived crude glycerol by metabolically engineered Klebsiella oxytoca M1.

Sukhyeong Cho,Taeyeon Kim,Youngsoon Um,Han Min Woo,Yunje Kim,Jinwon Lee

doi:10.1186/s13068-015-0336-6

Abstract

Background2,3-Butanediol (2,3-BDO) is a promising bio-based chemical because of its wide industrial applications. Previous studies on microbial production of 2,3-BDO has focused on sugar fermentation. Alternatively, biodiesel-derived crude glycerol can be used as a cheap resource for 2,3-BDO production; however, a considerable formation of 1,3-propanediol (1,3-PDO) and low concentration, productivity, and yield of 2,3-BDO from glycerol fermentation are limitations.ResultsHere, we report a high production of 2,3-BDO from crude glycerol using the engineered Klebsiella oxytoca M3 in which pduC (encoding glycerol dehydratase large subunit) and ldhA (encoding lactate dehydrogenase) were deleted to reduce the formation of 1,3-PDO and lactic acid. In fed-batch fermentation with the parent strain K. oxytoca M1, crude glycerol was more effective than pure glycerol as a carbon source in 2,3-BDO production (59.4 vs. 73.8 g/L) and by-product reduction (1,3-PDO, 8.9 vs. 3.7 g/L; lactic acid, 18.6 vs. 9.8 g/L). When the double mutant was used in fed-batch fermentation with pure glycerol, cell growth and glycerol consumption were significantly enhanced and 2,3-BDO production was 1.9-fold higher than that of the parent strain (59.4 vs. 115.0 g/L) with 6.9 g/L of 1,3-PDO and a small amount of lactic acid (0.7 g/L). Notably, when crude glycerol was supplied, the double mutant showed 1,3-PDO-free 2,3-BDO production with high concentration (131.5 g/L), productivity (0.84 g/L/h), and yield (0.44 g/g crude glycerol). This result is the highest 2,3-BDO production from glycerol fermentation to date.Conclusions2,3-BDO production from glycerol was dramatically enhanced by disruption of the pduC and ldhA genes in K. oxytoca M1 and 1,3-PDO-free 2,3-BDO production was achieved by using the double mutant and crude glycerol. 2,3-BDO production obtained in this study is comparable to 2,3-BDO production from sugar fermentation, demonstrating the feasibility of economic industrial 2,3-BDO production using crude glycerol.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0336-6) contains supplementary material, which is available to authorized users.

Highlights

In the last few years, considerable effort and progress have been made in the production of bio-based bulk chemicals from renewable resources because of the decrease in fossil fuel availability and increasing concern for global warming [1]. 2,3-Butanediol (2,3-BDO)Previous studies on biological production of 2,3-BDO have focused on sugar fermentation using glucose [5,6,7,8,9,10]Cho et al Biotechnol Biofuels (2015) 8:146 and sucrose [11] as the carbon sources
Flask fermentation of K. oxytoca M1 using pure and crude glycerol To evaluate glycerol utilization and 2,3-BDO production by K. oxytoca M1, flask batch fermentation was performed with pure glycerol and crude glycerol as the sole carbon sources at the initial concentration of 35–40 Glycerol consumption (g/L)
Considering that the final pH of crude glycerol fermentation was lower than the pH value of pure glycerol fermentation, a lower dry cell weight (DCW) in crude glycerol fermentation might be caused by a low pH level as presented

Summary

Introduction

In the last few years, considerable effort and progress have been made in the production of bio-based bulk chemicals from renewable resources because of the decrease in fossil fuel availability and increasing concern for global warming [1]. 2,3-Butanediol (2,3-BDO)Previous studies on biological production of 2,3-BDO have focused on sugar fermentation using glucose [5,6,7,8,9,10]Cho et al Biotechnol Biofuels (2015) 8:146 and sucrose [11] as the carbon sources. Glycerol, which is generated as a by-product from ethanol fermentation, fat saponification, and biodiesel production [17], is an attractive cheap resource for 2,3-BDO production. Because the amount of biodiesel-derived crude glycerol is almost equivalent to 10 % (w/w) of global biodiesel production, there is an increasing surplus of glycerol on the world market [18]. Klebsiella species, such as K. pneumoniae and K. oxytoca, have been found to utilize glycerol as the sole carbon source and produce 1,3-propanediol (1,3-PDO) and 2,3-BDO [18,19,20,21,22]. In addition to the oxidative branch, glycerol is metabolized through the reductive pathway, which results in the generation of 1,3-PDO (Fig. 1). 1,3-PDO is a major by-product generated during the production of 2,3-BDO using glycerol and may serve as an obstacle for obtaining a high purity of 2,3-BDO in downstream processes because of the similar boiling points of 2,3-BDO and 1,3-PDO [3]

Methods

Results

Conclusion