Zhaojuan Zheng

Open fermentative production of l-lactic acid by Bacillus sp. strain NL01 using lignocellulosic hydrolyzates as low-cost raw material

Highly efficient L-lactate production by a thermophilic strain Bacillus sp. NL01 was demonstrated in this study. Lignocellulosic hydrolyzates containing a high content of glucose, which was prepared from corn stover, was used as substrate for L-lactic acid production. The fermentation was carried out under open condition without sterilization and used NaOH as alkaline neutralizing reagent. In batch fermentation, 56.37 g l(-1) L-lactic acid was obtained from lignocellulosic hydrolyzates which contained the solid residues produced in enzymatic saccharification. In fed-batch fermentation, 75.03 g l(-1) L-lactic acid was obtained from lignocellulosic hydrolyzates supernatant. The yield was 74.5% and the average productivity was 1.04 g l(-1) h(-1).

Enzymatic hydrolysis, adsorption, and recycling during hydrolysis of bagasse sulfite pulp

The high costs of enzymatic hydrolysis along with the high enzyme dosage are often considered as the major bottlenecks in lignocellulosic bioconversion. This study investigated the hydrolysis efficiency, cellulase adsorption and enzyme recycling during the hydrolysis of bagasse sulfite pulp (BSP). After 48 h of hydrolysis, more than 70% of the cellulose was hydrolyzed, while the protein concentration and cellulase activity in solution remained 31% and 17% of the initial value, respectively. The cellulase adsorption on the fresh BSP was better fitted by a Sips model, suggesting the occurrence of a multilayer adsorption at low cellulase concentration and monolayer adsorption at high concentration on the BSP surfaces. Desorption profile studies showed that the optimum desorption condition was at pH 4.8 and 40 °C. Moreover, considering the limited ability to desorption, directly empolying the bound enzyme with residual substrate is more effective method to recover cellulase during the hydrolysis of BSP.

Efficient Conversion of Inulin to Inulooligosaccharides through Endoinulinase from Aspergillus niger.

Inulooligosaccharides (IOS) represent an important class of oligosaccharides at industrial scale. An efficient conversion of inulin to IOS through endoinulinase from Aspergillus niger is presented. A 1482 bp codon optimized gene fragment encoding endoinulinase from A. niger DSM 2466 was cloned into pPIC9K vector and was transformed into Pichia pastoris KM71. Maximum activity of the recombinant endoinulinase, 858 U/mL, was obtained at 120 h of the high cell density fermentation process. The optimal conditions for inulin hydrolysis using the recombinant endoinulinase were investigated. IOS were harvested with a high concentration of 365.1 g/L and high yield up to 91.3%. IOS with different degrees of polymerization (DP, mainly DP 3-6) were distributed in the final reaction products.

A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover.

This study established a new more neutral magnesium bisulfate pretreatment (MBSP) using magnesium bisulfate as sulfonating agent for improving the enzymatic hydrolysis efficiency of corn stover. Using the MBSP with 5.21% magnesium bisulfate, 170°C and pH 5.2 for 60 min, about 90% of lignin and 80% of hemicellulose were removed from biomass and more than 90% cellulose conversion of substrate was achieved after 48 h hydrolysis. About 6.19 kg raw corn stover could produce 1 kg ethanol by Saccharomyces cerevisiae. Meanwhile, MBSP also could protect sugars from excessive degradation, prevent fermentation inhibition formation and directly convert the hemicelluloses into xylooligosaccharides as higher-value products. These results suggested that the MBSP method offers an alternative approach to the efficient conversion of nonwoody lignocellulosic biomass to ethanol and had broad space for development.

Cost-effective simultaneous saccharification and fermentation of l-lactic acid from bagasse sulfite pulp by Bacillus coagulans CC17.

The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous β-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol.

Lactic Acid Production from Pretreated Hydrolysates of Corn Stover by a Newly Developed Bacillus coagulans Strain

An inhibitor-tolerance strain, Bacillus coagulans GKN316, was developed through atmospheric and room temperature plasma (ARTP) mutation and evolution experiment in condensed dilute-acid hydrolysate (CDH) of corn stover. The fermentabilities of other hydrolysates with B. coagulans GKN316 and the parental strain B. coagulans NL01 were assessed. When using condensed acid-catalyzed steam-exploded hydrolysate (CASEH), condensed acid-catalyzed liquid hot water hydrolysate (CALH) and condensed acid-catalyzed sulfite hydrolysate (CASH) as substrates, the concentration of lactic acid reached 45.39, 16.83, and 18.71 g/L by B. coagulans GKN316, respectively. But for B. coagulans NL01, only CASEH could be directly fermented to produce 15.47 g/L lactic acid. The individual inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde and p-hydroxybenzaldehyde (pHBal) on xylose utilization by B. coagulans GKN316 was also studied. The strain B. coagulans GKN316 could effectively convert these toxic inhibitors to the less toxic corresponding alcohols in situ. These results suggested that B. coagulans GKN316 was well suited to production of lactic acid from undetoxified lignocellulosic hydrolysates.

Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity

Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

Production of optically pure l-phenyllactic acid by using engineered Escherichia coli coexpressing l-lactate dehydrogenase and formate dehydrogenase

L-Phenyllactic acid (L-PLA) is a novel antiseptic agent with broad and effective antimicrobial activity. In addition, L-PLA has been used for synthesis of poly(phenyllactic acid)s, which exhibits better mechanical properties than poly(lactic acid)s. However, the concentration and optical purity of L-PLA produced by native microbes was rather low. An NAD-dependent L-lactate dehydrogenase (L-nLDH) from Bacillus coagulans NL01 was confirmed to have a good ability to produce L-PLA from phenylpyruvic acid (PPA). In the present study, l-nLDH gene and formate dehydrogenase gene were heterologously coexpressed in Escherichia coli. Through two coupled reactions, 79.6mM l-PLA was produced from 82.8mM PPA in 40min and the enantiomeric excess value of L-PLA was high (>99%). Therefore, this process suggested a promising alternative for the production of chiral l-PLA.

Rational Design of Bacillus coagulans NL01 l-Arabinose Isomerase and Use of Its F279I Variant in d-Tagatose Production

d-Tagatose is a prospective functional sweetener that can be produced by l-arabinose isomerase (AI) from d-galactose. To improve the activity of AI toward d-galactose, the AI of Bacillus coagulans was rationally designed on the basis of molecular modeling and docking. After alanine scanning and site-saturation mutagenesis, variant F279I that exhibited improved activity toward d-galactose was obtained. The optimal temperature and pH of F279I were determined to be 50 °C and 8.0, respectively. This variant possessed 1.4-fold catalytic efficiency compared with the wild-type (WT) enzyme. The recombinant Escherichia coli overexpressing F279I also showed obvious advantages over the WT in biotransformation. Under optimal conditions, 67.5 and 88.4 g L-1 d-tagatose could be produced from 150 and 250 g L-1 d-galactose, respectively, in 15 h. The biocatalyst constructed in this study presents a promising alternative for large-scale d-tagatose production.

Draft Genome Sequence of Bacillus coagulans NL01, a Wonderful l-Lactic Acid Producer

ABSTRACT Here, we report the draft genome sequence of Bacillus coagulans NL01, which could produce high optically pure l-lactic acid using xylose as a sole carbon source. The draft genome is 3,505,081 bp, with 144 contigs. About 3,903 protein-coding genes and 92 rRNAs are predicted from this assembly.