Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing Class I heat shock protein GroESL

Highlights

• Phenolic compounds and furan derivatives inhibited the growth of C. tyrobutyricum.

• Overexpression of groESL in C. tyrobutyricum enhanced its tolerance to inhibitors.

• 30 g/L butyrate at a productivity of 0.31 g/L·h and yield of 0.37 g/g was obtained.

• This provides an effective strategy for enhanced biomass hydrolysates utilization.

Abstract

Lignocellulosic biomass is the most abundant and renewable substrate for biological fermentation, but the inhibitors present in the lignocellulosic hydrolysates could severely inhibit the cell growth and productivity of industrial strains. This study confirmed that overexpressing of native groESL in Clostridium tyrobutyricum could significantly improve its tolerance to lignocellulosic hydrolysate-derived inhibitors, especially for phenolic compounds. Consequently, ATCC 25755/groESL showed a better performance in butyric acid fermentation with hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw, respectively. When corn straw and rice straw hydrolysates, which showed strong toxicity to C. tyrobutyricum, were used as the substrates, 29.6 g/L and 30.1 g/L butyric acid were obtained in batch fermentation, increased by 26.5% and 19.4% as compared with the wild-type strain, respectively. And more importantly, the butyric acid productivity reached 0.31 g/L·h (vs. 0.20–0.21 g/L·h for the wild-type strain) due to the shortened lag phase.

Introduction

Butyric acid is an important industrial chemical that has been widely used in food, drug, feed and cosmetics industries, and is mainly produced by chemical synthesis at present (Dwidar et al., 2012, Yang et al., 2013, Zhang et al., 2009). Recently, bio-fermentation of butyric acid has regained attention because of the reduction of fossil resources and serious pollution caused by fossil fuel (Baumann and Westermann, 2016, Rephaeli et al., 2000, Wang et al., 2016). However, butyric acid from biological fermentation was hampered by its low yield and high cost of substrate and downstream process; therefore the cost of bio-based butyric acid was much higher than that of chemical synthesis method (Dwidar et al., 2012). In order to promote the industrialization of butyric acid fermentation, it is crucial to reduce the cost of substrate, which accounts for half of the total costs. Clostridium tyrobutyricum is an excellent strain for butyric acid fermentation (Jiang et al., 2010, Zhu and Yang, 2003), and has been extensively studied in various substrates including glucose, xylose, sucrose (Jiang et al., 2009, Sjoblom et al., 2015), and starch-based biomass (Huang et al., 2011). In addition, C. tyrobutyricum is also a potential strain for bio-butanol fermentation (Yu et al., 2015, Yu et al., 2011).

Currently, the focus of research on the substrates of butyric acid fermentation has turned to using lignocellulosic biomass, which is considered as the most widespread and abundant renewable carbon source for the production of bio-based chemicals (Baroi et al., 2015a, Fu et al., 2017a, Huang et al., 2016a, Liu et al., 2013, Sjoblom et al., 2015). However, during the conversion of lignocellulosic biomass to monosaccharides (mainly glucose and xylose), several kinds of inhibitors such as phenolic compounds, furan derivatives and dilute acids could be generated (Jönsson et al., 2013). These inhibitors generally cause DNA damage, affect products formation, inhibit the growth of cells and activity of key enzymes (Allen et al., 2010, Mills et al., 2009). For instance, the lag phase of C. tyrobutyricum was up to 72 h when un-detoxified soybean hull or sugarcane bagasse hydrolysates (>90 g/L total sugar) was used as carbon source (Fu et al., 2017a). Hence, it is essential to examine the inhibitory effects of the representative inhibitors on butyric acid fermentation and improve the resistance of C. tyrobutyricum to various lignocellulosic hydrolysates.

Chaperonins GroESL is a widely-studied heat shock protein, and has been shown to play an important role in enhancing cell's adaptability to the external environment. Numerous studies have been confirmed that intracellular GroESL levels were significantly upregulated under various environmental stresses, which could prevent protein aggregation and assist in protein folding and refolding (Chapman et al., 2006, Kerner et al., 2005). Therefore, overexpression of native groESL has been shown to significantly improve the tolerance of bacteria to temperature, acid, solvent, osmotic pressure and drying (Chaurasia and Apte, 2009, Corcoran et al., 2006, Suo et al., 2017, Tomas et al., 2003). In addition, GroESL protein also performed key functions in resistance to lignocellulosic inhibitory compounds. For instance, the groESL expression levels of C. beijerinckii were consistently upregulated under the stress of furfural (Zhang and Ezeji, 2013). And, overexpression of groESL in C. beijerinckii NCIMB 8052 led to improved tolerance to ferulic acid (Lee et al., 2015a).

The objective of this study was to evaluate the feasibility of enhancing the resistance of C. tyrobutyricum to lignocellulosic inhibitory compounds by overexpression of groESL. At first, butyric acid fermentations were performed in serum bottles with glucose/xylose mixture and hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw. Then, in order to better explain the change of fermentation characteristics between the C. tyrobutyricum wild-type and ATCC 25755/groESL in various hydrolysates, the growth rate of both strains was detected under the stress of representative inhibitors (ferulic acid, syringaldehyde, vanillin, p-coumaric acid, formic acid, levulinic acid, furfural and 5-hydroxymethyl-furfural). Finally, butyric acid fermentations with corn straw and rice straw hydrolysates as carbon source were further studied in bioreactor.