I-Ching Tang

Metabolic engineering of Clostridium tyrobutyricum for n‐butanol production through co‐utilization of glucose and xylose

The glucose‐mediated carbon catabolite repression (CCR) in Clostridium tyrobutyricum impedes efficient utilization of xylose present in lignocellulosic biomass hydrolysates. In order to relieve the CCR and enhance xylose utilization, three genes (xylT, xylA, and xylB) encoding a xylose proton‐symporter, a xylose isomerase and a xylulokinase, respectively, from Clostridium acetobutylicum ATCC 824 were co‐overexpressed with aldehyde/alcohol dehydrogenase (adhE2) in C. tyrobutyricum (Δack). Compared to the strain Ct(Δack)‐pM2 expressing only adhE2, the mutant Ct(Δack)‐pTBA had a higher xylose uptake rate and was able to simultaneously consume glucose and xylose at comparable rates for butanol production. Ct(Δack)‐pTBA produced more butanol (12.0 vs. 3.2 g/L) with a higher butanol yield (0.12 vs. 0.07 g/g) and productivity (0.17 vs. 0.07 g/L · h) from both glucose and xylose, while Ct(Δack)‐pM2 consumed little xylose in the fermentation. The results confirmed that the CCR in C. tyrobutyricum could be overcome through overexpressing xylT, xylA, and xylB. The mutant was also able to co‐utilize glucose and xylose present in soybean hull hydrolysate (SHH) for butanol production, achieving a high butanol titer of 15.7 g/L, butanol yield of 0.24 g/g, and productivity of 0.29 g/L · h. This study demonstrated the potential application of Ct(Δack)‐pTBA for industrial biobutanol production from lignocellulosic biomass. Biotechnol. Bioeng. 2015;112: 2134–2141.

A novel fermentation process for calcium magnesium acetate (CMA) production from cheese whey

A novel, anaerobic fermentation process is developed to produce calcium magnesium acetate (CMA) from cheese whey. CMA can be used as a noncorrosive road deicer. It poses no environmental threats and has many advantages over road salt. A coculture consisting of homolactic and homoacetic bacteria was used to convert whey lactose to lactate and then to acetate in a continuous, immobilized cell bioreactor. The acetate yield from lactose was ~95% (wt/wt) in this homofermentative process. The acetic acid produced from fermentation was recovered in a concentrated CMA solution by using an energyefficient extraction process. The development of a novel, extractive fermentation process to reduce product inhibition and to further increase reactor productivity is also discussed.

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

SummaryAcetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na+ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.

Butanol production in acetone-butanol-ethanol fermentation with in situ product recovery by adsorption

Activated carbon Norit ROW 0.8, zeolite CBV901, and polymeric resins Dowex Optipore L-493 and SD-2 with high specific loadings and partition coefficients were studied for n-butanol adsorption. Adsorption isotherms were found to follow Langmuir model, which can be used to estimate the amount of butanol adsorbed in acetone-butanol-ethanol (ABE) fermentation. In serum-bottle fermentation with in situ adsorption, activated carbon showed the best performance with 21.9g/L of butanol production. When operated in a fermentor, free- and immobilized-cell fermentations with adsorption produced 31.6g/L and 54.6g/L butanol with productivities of 0.30g/L·h and 0.45g/L·h, respectively. Thermal desorption produced a condensate containing ∼167g/L butanol, which resulted in a highly concentrated butanol solution of ∼640g/L after spontaneous phase separation. This in situ product recovery process with activated carbon is energy efficient and can be easily integrated with ABE fermentation for n-butanol production.

Metabolic process engineering of Clostridium tyrobutyricum Δack–adhE2 for enhanced n‐butanol production from glucose: Effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics

Butanol biosynthesis through aldehyde/alcohol dehydrogenase (adhE2) is usually limited by NADH availability, resulting in low butanol titer, yield, and productivity. To alleviate this limitation and improve n‐butanol production by Clostridium tyrobutyricum Δack–adhE2 overexpressing adhE2, the NADH availability was increased by using methyl viologen (MV) as an artificial electron carrier to divert electrons from ferredoxin normally used for H2 production. In the batch fermentation with the addition of 500 μM MV, H2, acetate, and butyrate production was reduced by more than 80–90%, while butanol production increased more than 40% to 14.5 g/L. Metabolic flux analysis revealed that butanol production increased in the fermentation with MV because of increased NADH availability as a result of reduced H2 production. Furthermore, continuous butanol production of ∼55 g/L with a high yield of ∼0.33 g/g glucose and extremely low ethanol, acetate, and butyrate production was obtained in fed‐batch fermentation with gas stripping for in situ butanol recovery. This study demonstrated a stable and reliable process for high‐yield and high‐titer n‐butanol production by metabolically engineered C. tyrobutyricum by applying MV as an electron carrier to increase butanol biosynthesis. Biotechnol. Bioeng. 2015;112: 705–715.

Calcium magnesium acetate (CMA) production from whey permeate: process and economic analysis

Abstract About 28 billion lbs of liquid whey produced from cheese manufacture are being wasted in the US. An anaerobic fermentation process is developed to produce calcium magnesium acetate (CMA) from whey permeate. CMA can be used to replace salt as a noncorrosive road deicer. A co-culture consisting of homolactic and homoacetic bacteria was used to convert whey lactose to lactate and then to acetate in continuous, immobilized cell bioreactors. The acetate yield from lactose was ∼ 95% (w/w), and the final concentration of acetic acid obtained from this homofermentative process was 4%. The acetic acid produced from fermentation can be readily recovered by solvent extraction with a tertiary amine and reacted with dolomitic lime (CA/MgO) to form a concentrated (>25%) CMA solution. This CMA solution can then be dried to form the granular CMA deicer. About 40 tons CMA can be produced from a plant processing 1.5 million lbs whey permeate per day, at a cost of

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing xylose catabolism genes for glucose and xylose co-utilization.

215/ton. The total capital investment is estimated at ∼ 7 million dollars, with a return rate of less than 1.5 years at the current market price of

Surface Modification for Enhancing Antibody Binding on Polymer-Based Microfluidic Device for Enzyme-Linked Immunosorbent Assay

600/ton.

Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates.