Efficient production of polymer-grade L-lactic acid from corn stover hydrolyzate by thermophilic Bacillus sp. strain XZL4
© Xue et al. licensee Springer. 2012
Received: 23 September 2012
Accepted: 23 October 2012
Published: 29 October 2012
Lactic acid has been identified as one of the top 30 potential building-block chemicals from biomass. Therefore, the search for cheap raw materials is an objective to reduce the production costs. Efficient polymer-grade L-lactic acid production was achieved in this report by a thermophilic strain Bacillus sp. XZL4 using corn stover hydrolyzate as sole carbon source. High L-lactic acid concentration (81.0 g L-1) was obtained from 162.5 g L-1 concentrated corn stover hydrolyzate (total reducing sugar of 83.0 g L-1) with a volumetric productivity of 1.86 g L-1 h-1 (0–36 h) and a product yield of 0.98 g g-1 total reducing sugars. This is the highest L-lactic acid concentration and yield reported from corn stover hydrolyzate. And the high optical purity of L-lactic acid obtained in this study also indicated that Bacillus sp. XZL4 is a promising polymer-grade L-lactic-acid producer from cellulosic biomass.
Lactic acid is a valuable chemical and one of its extensive applications is for polymerization of L-lactic acid to poly (L-lactic acid), which is an attractive polymer because it can be produced from renewable resources and is biodegradable. These properties have strengthened interest in developing more efficient production processes for optical purity of L-lactic acid (Wang et al. 2010a). As lactic acid has been identified as one of the top 30 potential building-block chemicals from biomass (http://www.eere.energy.gov/biomass/pdfs/35523.pdf), the search for cheap raw materials is some of the most important objectives to be achieved to reduce the costs. To improve productivity and economy of lactic acid production, some reports have investigated the potential of utilizing low-cost raw materials as carbon sources, such as molasses and cellulosic materials (Patel et al. 2004; Romaní et al. 2008; Wang et al. 2010a). Corn stover, one of the lignocellulosic biomasses, is the agricultural residue left unutilized in harvested. Corn stover is not a food source and has high concentration of mixed sugars, mainly including glucose and xylose, and therefore it is considered as one of the most important global feedstocks for the production of chemicals in future (Georgieva and Ahring 2007). Furthermore, the addition of large amounts of yeast extract in lactic acid fermentation was also economically prohibited for producing such low-value biocommodities (Altaf et al. 2007). Various low-cost raw materials such as tryptic soy (Nancib et al. 2005), soybean hydrolyzate (Kwon et al. 2000), corn steep liquor (Nancib et al. 2001), whey protein hydrolyzate (Fitzpatrick and O’Keeffe 2001) and red lentil and baker’s yeast cells (Altaf et al. 2006) have been investigated to substitute yeast extract for lactic acid production. However, most of the substitutes were not very effective. Therefore, development of an efficient and cost-effective process for lactic acid fermentation from cheap and non-food substrates is highly desired.
Bacillus species are the most widely utilized microorganisms for L-lactic acid production (Budhavaram and Fan 2009; Danner et al. 1998; Patel et al. 2004Qin et al. 2010; Walton et al. 2010; Wang et al. 2010b; Zhao et al. 2010). As potential industrial strains, thermophilic Bacillus species offers several remarkable advantages for lactic acid production, including the reduction of contamination from competing microbes, simple nutrition requirements, and simple maintenance of stock cultures (Patel et al. 2004; Qin et al. 2009). In this study, corn stover hydrolyzate was chosen as sole carbon and different low-cost nitrogen sources (peanut meal, dry corn syrup and soybean meal) were tested as sole nitrogen source, respectively, for polyer-grade L-lactic acid production by a thermophilic Bacillus strain. The aim of this study was to develop an encouraging process for the economical L-lactic acid production based on cheap raw materials. The batch fermentation results of high yield and high optical purity of L-lactic acid from corn stover hydrolyzate indicated that Bacillus sp. strain XZL4 used in this study is a promising L-lactic-acid producer from cellulosic biomass.
Effects of corn stover hydrolyzate concentrations on L-lactic acid production
Effects of nitrogen source on L-lactic acid production by strain XZL4
L-Lactic acid production from corn stover hydrolyzate by batch fermentation
Comparison of lactic acid production from agricultural by-products by lactic acid producing microorganisms
Lactic acid concentration (g/L)
lime-treated wheat straw hydrolyzate
Bacillus coagulans DSM 2314
Continuation of the SSF
Maas et al. 
sugar cane bagasse hemicellulose hydrolyzate
thermotolerant acidophilic Bacillus sp. strain 17C5
Patel et al. 
Enterococcus mundtii QU25
Abdel-Rahman et al. 
reed hemicellulose liquor
Lactococcus lactis IO-1 JCM 7638/ Lactobacillus pentosus ATCC 8041
Perttunen et al. 
wheat straw hydrolyzate
fungus Rhizopus oryzae CBS 112.07
Maas et al. 
hot water-extracted Siberian larch
moderate thermophile Bacillus coagulans MXL-9
Walton et al. 
Bacillus coagulan strains 36D1
defatted rice bran
Lactobacillus delbrueckii IFO 3202
Tanaka et al. 
Enterococcus faecalis RKY1
Wee et al. 
corn fiber hydrolyzate
moderate thermophile Bacillus coagulans MXL-9
Bischoff et al. 
corn stover hydrolyzate
Lactobacillus pentosus ATCC 8041
Zhu et al. [2007a]
corn stover hydrolyzate
Bacillus sp. strain XZL4
Additionally, cellulose- and hemicellulose-derived carbohydrate feedstocks contained a variety of mixed sugars, mainly glucose and xylose. In order to maximize lactic acid yield and production, complete utilization of mixed sugars is essential. Carbon catabolite repression (CCR) is a common phenomenon in bacteria and very few bacteria have been reported which consume different sugars simultaneously (Görke and Stülke 2008). Therefore, for industrialization of lactic acid production from cellulosic materials, it is desirable to use CCR-positive strain for lactic acid production from mixed sugar substrates. Bacillus sp. strain XZL4 could utilize both the two sugars simultaneously, although glucose was utilized a little faster than xylose (Figure 1), proving its feasibility for L-lactic acid production from low-cost raw materials.
High L-lactic acid concentration with a volumetric productivity of 1.86 g L-1 h-1 (0–36 h) and a product yield of 0.98 g g-1 total reducing sugars was obtained in batch fermentation by Bacillus sp. strain XZL4. The mechanisms that strain XZL4 produced L-lactic acid so effectively could be explained by the analysis results from genome sequencing data (Su et al. 2011). The genome size of Bacillus sp. strain XZL4 is only 2.8 Mb. The small genome size with less genomic redundancy was thought to improve the productivity of platform chemicals or other products (Zhu et al. 2007b; Morimoto et al. 2008). The pathway of EMP is well known for its high efficiency to utilize hexose. Compared to the hexose, the pathways of utilization of pentose are more flexible. In theory, the transketolase/transaldolase pathway has higher carbon efficiency than the phosphoketolase pathway. Based on carbohydrate metabolism analysis, the key enzymes (xylose/arabinose isomerase, ribulokinase, and ribulose-5-phosphate 4-epimerase) involved in the pentose metabolite were found in the genome. The transketolase/transaldolase pathway, instead of phosphoketolase, was in the genome, implying that strain XZL4 could utilize pentose more efficiently. Furthermore, few pyruvate-dissipating enzymes were found in strain XZL4 (Su et al. 2011). Therefore, simple and efficient carbohydrate metabolism systems, especially the absence of pyruvate decarboxylase and the existed transketolase/transaldolase pathway in thermophilic Bacillus sp. strain XZL4, should be responsible for the high-yield lactic acid production from corn stover hydrolyzate. Additionally, the absence of D-lactate dehydrogenase genes in the genome of Bacillus sp. strain XZL4 also resulted in producing such high-optical purity L-lactic acid.
In conclusion, the highest lactic acid concentration (81.0 g L-1) and yield (0.98 g g-1 total reducing sugars) was obtained from corn stover hydrolyzate in this study. Corn stover hydrolyzate can provide an economic L-lactic acid production process with cheap and renewable biomass by Bacillus sp. strain XZL4. Although the relatively higher concentrations of lactic acid were previously reported by using paper sludge (Budhavaram and Fan 2009) and wood hydrolysate (Wee et al. 2004) (Table 1), expensive yeast extract used as nitrogen source reduced their industrial applicability. And more, the absence of pyruvate decarboxylase and D-lactate hydrogenase genes in the genome demonstrated that Bacillus sp. strain XZL4 is a high-efficient polymer-grade L-lactic-acid producer from cellulosic biomass.
The corn stover hydrolyzate, kindly provided by Energy Research Institute of Shandong Academy of Sciences (China), was prepared by following the procedures: 1) Pretreatment: The corn straw was grinded, then the powder was sieved and the particle sizes ≤2 mm were collected; 2) HCl treatment: The collected powder was treated with 6% HCl at 90°C for 1 h, then washed by water and adjusted to pH 4.8-5.0; 3) Cellulase hydrolysation: The powder of corn straw was hydrolyzed with cellulase (20 FPIU/g dry mass, solid–liquid ratio was 1:50–1:10) at 50°C for 48 h, and the corn straw hydrolysate was concentrated and used in this study. The contents of concentrated corn stover hydrolyzate was glucose 555.3 (g L-1), xylose 174.2 (g L-1), arabinose 19.9 (g L-1), acetic acid 7.1 (g L-1), 2-furfural 1.9 (g L-1) and 5-hydroxymethyl-2-furaldehyde 0.7 (g L-1). All other chemicals were of analytical grade and commercially available.
Strain and culture conditions
Bacillus sp. strain XZL4 used in this study is a homofermentative L-lactic acid producer (Su et al. 2011). The strain has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM №23183). The slant was inoculated at 50°C after 24 h of incubation and stored at 4°C. Strain XZL4 was inoculated in the culture medium containing (per liter) 50 g glucose, 10 g yeast extract (YE), 30 g calcium carbonate (Wang et al. 2010b). The seed culture was prepared as follows: a loop of cells from the fully grown LB agar slant was inoculated into 30 mL of the above sterile medium in 100-mL conical flasks and incubated at 50°C for 24 h without agitation. All experiments were 10% (v/v) inoculum volume and carried out in triplicate.
Effects of corn stover hydrolyzate concentrations on L-lactic acid production
The medium used to study the effects of corn stover hydrolyze concentrations on L-lactic acid production contained 41–133 g L-1 reducing sugars and 10 g L-1 YE. Calcium carbonate was added as 60% (w/w) of the reducing sugars to the medium (Wang et al. 2010a). The well mixed samples were taken periodically and the concentrations of total residual reducing sugar, glucose, xylose and the L-lactic acid production were determined.
Effects of different nitrogen sources on L-lactic acid production
The variables used in the study were peanut meal and soybean meal (with 0.3 g L-1 neutral proteinase, respectively, to release the nitrogen element), peptone, yeast extract, dry corn syrup, (NH4)2SO4 and (NH4)2HPO4. The 162.5 g L-1 corn stover hydrolyzate (containing 83.0 g L-1 reducing sugar) and 49.5 g L-1 CaCO3 were added. The quantities of nitrogen in all medium were controlled at 4.5 g L-1. The well mixed samples were taken periodically and the concentrations of total residual reducing sugar and the L-lactic acid production were determined.
Effects of peanut meal concentrations on L-lactic acid production
The fermentation medium for studying nitrogen concentration utilization contained 1.3-13.2 g L-1 peanut meal and 162.5 g L-1 corn stover hydrolyzate, calcium carbonate was added as 60% (w/w) of reducing sugars to the medium. Fermentations were carried out at 50°C under static conditions in 100-mL Erlenmeyer flasks each containing 30 mL medium. The well mixed samples were taken periodically and the concentrations of total residual reducing sugar and the L-lactic acid production were determined.
Batch fermentation was conducted in a 3-L Erlenmeyer flask containing 1 L fresh medium at 50°C under static conditions. The corn stover hydrolyzate (162.5 g L-1) containing 83.0 g L-1 of total reducing sugars was used. The medium contained 9.9 g L-1 peanut meal and 0.3 g L-1 neutral proteinase, and the culture pH was maintained at 5.1-6.3 by calcium carbonate present in the medium. The well mixed samples were taken periodically and the concentrations of total residual reducing sugar and L-lactic acid production were determined.
The glucose and L-lactate concentration were measured by SBA-40D biosensor analyzer (Institute of Biology, Shandong Academy of Sciences, China). The total concentration of reducing sugars was measured by SGD-IV automatic analyzer of reducing sugar (Institute of Biology, Shandong Academy of Sciences, China). The xylose concentration was determined by xylose assay kit (Nanjing Jiancheng Technology Company Ltd, China). For quantification of formatic acid and acetatic acid, an Aminex HPX-87H column (Bio-Rad, Hercules, CA) was used. The column was maintained at 65°C and eluted with 5 mM H2SO4 at a flow rate of 0.6 mL/min. Peaks were detected by Refractive Index Detector and quantified by comparison to retention times of authentic standards. The optical purity of L-lactic acid was determined by HPLC equipped with a chiral column (MCI GEL CRS10W, Japan) at 254 nm. The mobile phase was 2 mM CuSO4 at a flow rate of 0.5 ml/min (25°C).
This work was supported by grants from the Chinese National Programs for High Technology Research and Development (2011AA02A202), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-2-3) and the National Natural Science Foundation of China (30900022).
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