Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum.

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 µM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 µM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 µM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.

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

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.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from sugarcane juice.

Clostridium tyrobutyricum is a promising organism for butyrate and n-butanol production, but cannot grow on sucrose. Three genes (scrA, scrB, and scrK) involved in the sucrose catabolic pathway, along with an aldehyde/alcohol dehydrogenase gene, were cloned from Clostridium acetobutylicum and introduced into C. tyrobutyricum (Δack) with acetate kinase knockout. In batch fermentation, the engineered strain Ct(Δack)-pscrBAK produced 14.8-18.8 g/L butanol, with a high butanol/total solvent ratio of ∼0.94 (w/w), from sucrose and sugarcane juice. Moreover, stable high butanol production with a high butanol yield of 0.25 g/g and productivity of 0.28 g/L∙h was obtained in batch fermentation without using antibiotics for selection pressure, suggesting that Ct(Δack)-pscrBAK is genetically stable. Furthermore, sucrose utilization by Ct(Δack)-pscrBAK was not inhibited by glucose, which would usually cause carbon catabolite repression on solventogenic clostridia. Ct(Δack)-pscrBAK is thus advantageous for use in biobutanol production from sugarcane juice and other sucrose-rich feedstocks.

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 engineering of Clostridium tyrobutyricum for n-butanol production from maltose and soluble starch by overexpressing α-glucosidase.

Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular α-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of α-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Δack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch.

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.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase.

The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (Δack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH 5.0 and 6.0. Compared to C. tyrobutyricum (Δack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22 - 0.26 g/g (vs. 0.10 - 0.13 g/g) and productivity to 0.35 g/l h (vs. 0.13 g/l h) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation.

Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production.

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.

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.

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping.

Acetone-butanol-ethanol (ABE) fermentation with a hyper-butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed-batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid-liquid demixing or phase separation, a final product containing ~610 g/L butanol, ~40 g/L acetone, ~10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed-batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n-butanol production from glucose.

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

Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production.

Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ∼60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.

Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay.

A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.