Butyrate production enhancement by Clostridium tyrobutyricum using electron mediators and a cathodic electron donor

Electron mediators and electron supply through a cathode were examined to enhance the reducing power for butyrate production by an acidogenic clostridium strain, Clostridium tyrobutyricum BAS 7. Among the tested electron mediators, methyl viologen (MV)‐amended cultures showed an increase of butyrate productivity (1.3 times), final concentration (1.4 times), and yield (1.3 times). The electron flow altered by MV addition from the ferredoxin pool to the NADH pool was shown by one electron model, implying that more available NADH increased butyrate production. In the cathode compartment poised at ?400 mV versus the Ag/AgCl electrode, the neutral red (NR)‐amended cultures of Clostridium tyrobutyricum BAS 7 increased butyrate concentration (from 5 to 8.8 g/L) and yield (from 0.33 up to 0.44 g/g) with no acetate production at all. Given that electrically reduced NR (NR_red, yellow) by the cathode was re‐oxidized (NR_ox, red) in the cells on the basis of color change, electron flow from NR_red to NAD⁺ (i.e., NADH generation) induced an increase in butyrate production. This is the first report to show the increase of butyric acid production by electrically driven acidogenesis. These results show that the electron flow altered NADH formation by electron mediators and by the cathodic electron donor, increasing the yield and selectivity of reduced end‐products like butyrate. Biotechnol. Bioeng. 2012; 109: 2494–2502. © 2012 Wiley Periodicals, Inc.     

Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.     

Ex situ product recovery for enhanced butanol production by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In situ butanol recovery fermentation has been intensively studied as an effective alternative to conventional butanol production, which is limited due to the cellular toxicity of butanol. However, the low biocompatibility of adsorbents often leads to failure of in situ recovery fermentations. In this study, Clostridium beijerinckii NCIMB 8052 was cultured in flasks without shaking and in situ recovery fermentation was performed by using an adsorbent L493. The amounts of acetone, butanol, and ethanol (ABE) increased by 34.4 % in the presence of the adsorbent. In contrast, cell growth and production of organic acids and ABE were retarded in the 7-L batch fermentations with in situ butanol recovery. Cell damage occurred in the fermentor upon agitation in the presence of the adsorbent, unlike in static flask cultures with in situ recovery. Ex situ recovery fermentation using circulation of fermentation broth after mid-exponential phase of cell growth was developed to avoid adsorbent-cell incompatibility. No apparent cell damage was observed and 25.7 g/L of ABE was produced from 86.2 g/L glucose in the fed-batch mode using 7 L fermentors. Thus, ex situ recovery fermentation with C. beijerinckii is effective for enhancing butanol fermentation.     

Production of butanol and isopropanol with an immobilized <Emphasis Type="Italic">Clostridium</Emphasis>

Clostridium beijerinckii optinoii is a Clostridium species that produces butanol, isopropanol and small amounts of ethanol. This study compared the performances of batch and continuous immobilized cell fermentations, investigating how media flow rates and nutritional modification affected solvent yields and productivity. In 96-h batch cultures, with 80 % of the 30 g L^?1 glucose consumed in synthetic media, solvent concentration was 9.45 g L^?1 with 66.0 % as butanol. In a continuous fermentation using immobilized C. beijerinckii optinoii cells, also with 80 % of 30 g L^?1 glucose utilization, solvent productivity increased to 1.03 g L^?1 h^?1. Solvent concentration reached 12.14 g L^?1 with 63.0 % as butanol. Adjusting the dilution rate from 0.085 to 0.050 h^?1 to allow extended residence time in column was required when glucose concentration in fresh media was increased from 30 to 50 g L^?1. When acetate was used to improve the buffer capacity in media, the solvent concentration reached 12.70 on 50 g L^?1 glucose. This continuous fermentation using immobilized cells showed technical feasibility for solvent production.     

CaCO<Subscript>3</Subscript> supplementation alleviates the inhibition of formic acid on acetone/butanol/ethanol fermentation by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis>

Objective

To investigate the inhibiting effect of formic acid on acetone/butanol/ethanol (ABE) fermentation and explain the mechanism of the alleviation in the inhibiting effect under CaCO₃ supplementation condition.

Results

From the medium containing 50 g sugars l^?1 and 0.5 g formic acid l^?1, only 0.75 g ABE l^?1 was produced when pH was adjusted by KOH and fermentation ended prematurely before the transformation from acidogenesis to solventogenesis. In contrast, 11.4 g ABE l^?1 was produced when pH was adjusted by 4 g CaCO₃ l^?1. The beneficial effect can be ascribed to the buffering capacity of CaCO₃. Comparative analysis results showed that the undissociated formic acid concentration and acid production coupled with ATP and NADH was affected by the pH buffering capacity of CaCO₃. Four millimole undissociated formic acid was the threshold at which the transformation to solventogenesis occurred.

Conclusion

The inhibiting effect of formic acid on ABE fermentation can be alleviated by CaCO₃ supplementation due to its buffering capacity.

     

Cellulosic Butanol (ABE) Biofuel Production from Sweet Sorghum Bagasse (SSB): Impact of Hot Water Pretreatment and Solid Loadings on Fermentation Employing <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> P260

A novel butanol fermentation process was developed in which sweet sorghum bagasse (SSB) was pretreated using liquid hot water (LHW) pretreatment technique followed by enzymatic hydrolysis and butanol (acetone butanol ethanol (ABE)) fermentation. A pretreatment temperature of 200 °C resulted in the generation of a hydrolyzate that inhibited butanol fermentation. When SSB pretreatment temperature was decreased to 190 °C (0-min holding time), the hydrolyzate was successfully fermented without inhibition and an ABE productivity of 0.51 g L^?1 h^?1 was achieved which is comparable to the 0.49 g L^?1 h^?1 observed in the control fermentation where glucose was used as a feedstock. These results are based on the use of 86 g L^?1 SSB solid loadings in the pretreatment reactors. We were also able to increase SSB solid loadings from 120 to 200 g L^?1 in the pretreatment step (190 °C) followed by hydrolysis and butanol fermentation. As pretreatment solid loadings increased, ABE yield remained in the range of 0.38–0.46. In these studies, a maximum ABE concentration of 16.88 g L^?1 was achieved. Using the LHW pretreatment technique, 88.40–96.00 % of polymeric sugars (cellulose + hemicellulose) were released in the SSB hydrolyzate. The LHW pretreatment technique does not require chemical additions and is environmentally friendly, and the hydrolyzate can be used successfully for butanol fermentation.     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Improvement of butanol production in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> through enhancement of NAD(P)H availability

Clostridium acetobutylicum is a natural producer of butanol, butyrate, acetone and ethanol. The pattern of metabolites reflects the partitioning of redox equivalents between hydrogen and carbon metabolites. Here the exogenous genes of ferredoxin-NAD(P)⁺ oxidoreductase (FdNR) and trans-enoyl-coenzyme reductase (TER) are introduced to three different Clostridium acetobutylicum strains to investigate the distribution of redox equivalents and butanol productivity. The FdNR improves NAD(P)H availability by capturing reducing power from ferredoxin. A butanol production of 9.01 g/L (36.9% higher than the control), and the highest ratios of butanol/acetate (7.02) and C₄/C₂ (3.17) derived metabolites were obtained in the C acetobutylicum buk^- strain expressing FdNR. While the TER functions as an NAD(P)H oxidase, butanol production was decreased in the C. acetobutylicum strains containing TER. The results illustrate that metabolic flux can be significantly changed and directed into butanol or butyrate due to enhancement of NAD(P)H availability by controlling electron flow through the ferredoxin node.     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

Increasing n-butanol production with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by optimizing acetyl-CoA synthesis,NADH levels and trans-2-enoyl-CoA reductase expression

Background

n-Butanol can serve as an excellent gasoline substitute. Naturally, it is produced by some Clostridia species which, however, exhibit only limited suitability for industrial n-butanol production. The yeast Saccharomyces cerevisiae would be an ideal host due to its high robustness in fermentation processes. Nevertheless, n-butanol yields and titers obtained so far with genetically engineered yeast strains are only low.

Results

In our recent work, we showed that n-butanol production via a clostridial acetoacetyl-CoA-derived pathway in engineered yeast was limited by the availability of coenzyme A (CoA) and cytosolic acetyl-CoA. Increasing their levels resulted in a strain producing up to 130 mg/L n-butanol under anaerobic conditions. Here, we show that under aerobic conditions. this strain can even produce up to 235 mg/L n-butanol probably due to a more efficient NADH re-oxidation. Nevertheless, expression of a bacterial water-forming NADH oxidase (nox) significantly reduced n-butanol production although it showed a positive effect on growth and glucose consumption. Screening for an improved version of an acetyl-CoA forming NAD⁺-dependent acetylating acetaldehyde dehydrogenase, adhE^{A267T/E568K/R577S}, and its integration into n-butanol-producing strain further improved n-butanol production. Moreover, deletion of the competing NADP⁺-dependent acetaldehyde dehydrogenase Ald6 had a superior effect on n-butanol formation. To increase the endogenous supply of CoA, amine oxidase Fms1 was overexpressed together with pantothenate kinase coaA from Escherichia coli, and could completely compensate the beneficial effect on n-butanol synthesis of addition of pantothenate to the medium. By overexpression of each of the enzymes of n-butanol pathway in the n-butanol-producing yeast strain, it turned out that trans-2-enoyl-CoA reductase (ter) was limiting n-butanol production. Additional overexpression of ter finally resulted in a yeast strain producing n-butanol up to a titer of 0.86 g/L and a yield of 0.071 g/g glucose.

Conclusions

By further optimizing substrate supply and redox power in the form of coenzyme A, acetyl-CoA and NADH, n-butanol production with engineered yeast cells could be improved to levels never reached before with S. cerevisiae via an acetoacetyl-CoA-derived pathway in synthetic medium. Moreover, our results indicate that the NAD⁺/NADH redox balance and the trans-2-enoyl-CoA reductase reaction seem to be bottlenecks for n-butanol production with yeast.

     

Characterization of alcohol dehydrogenase from <Emphasis Type="Italic">Kangiella koreensis</Emphasis> and its application to production of all-<Emphasis Type="Italic">trans</Emphasis>-retinol

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min^?1 mg^?1, a K _m of 1.8 μM, and a k _cat of 1.7 min^?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD ⁺ as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l^?1, and 2,200 mg all-trans-retinal l^?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l^?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l^?1 h^?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.     

Enhancement of NAD(H) pool for formation of oxidized biochemicals in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The NAD⁺/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD⁺-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD⁺ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to l-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final l-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to l-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L l-tagatose, corresponding to 90% conversion and a yield of 0.95 g l-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD⁺-dependent biotransformations.     

Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae

Anaerobic fermentation was relatively difficult to optimize due to lack of monitoring parameters. In this paper, a new method was reported using extracellular oxidoreduction potential (ORP) to monitor 1,3-propanediol (1,3-PD) biosynthesis process by Klebsiella pneumoniae. In batch fermentation, cell growth, 1,3-propanediol production and by-products distribution were studied at four different ORP levels: 10, −140, −190 and −240 mV. From the results, the ORP level of −190 mV was preferable, which resulted in fast cell growth and high 1,3-propanediol concentration. The NAD⁺/NADH ratio was determined at different ORP levels, and a critical NAD⁺/NADH ratio of 4 was defined to divide fermentation environments into two categories: relatively oxidative environment (NAD⁺/NADH>4) and relatively reductive environment (NAD⁺/NADH<4). The former was correlative with high 1,3-propanediol productivity and high specific growth rate. The mechanism of ORP regulation was discussed. It is suggested that ORP regulation of fermentation might be due to its influence on the ratio of NAD⁺/NADH, which determined metabolic flux. Furthermore, a batch fermentation of modulating ORP following a profile in different levels corresponding to different fermentation stage was tested. The 1,3-PD concentration was 22.3% higher than that of constant ORP fermentation at −190 mV. Therefore, ORP is a valuable parameter to monitor and control anaerobic fermentation production.     

An oxidoreduction potential shift control strategy for high purity propionic acid production by Propionibacterium freudenreichii CCTCC M207015 with glycerol as sole carbon source

The effects of oxidoreduction potential (ORP) regulation on the process of propionic acid production by Propionibacterium freudenreichii CCTCC M207015 have been investigated. Potassium ferricyanide and sodium borohydride were determined as ORP control agents through serum bottle experiment. In batch fermentation, cell growth, propionic acid and by-products distribution were changed with ORP levels in the range of 0–160 mV. Based on these analysis results, an ORP-shift control strategy was proposed: at first 156 h, ORP was controlled at 120 mV to obtain higher cell growth rate and propionic acid formation rate, and then it was shifted to 80 mV after 156 h to maintain the higher propionic acid formation rate. By applying this strategy, the optimal parameters were obtained as follows: the propionic acid concentration 45.99 g L^?1, productivity 0.192 g L^?1 h^?1, the proportion of propionic acid to total organic acids 92.26 % (w/w) and glycerol conversion efficiency 76.65 %. The mechanism of ORP regulation was discussed by the ratio of NADH/NAD⁺, ATP levels, and metabolic flux analysis. The results suggest that it is possible to redistribute energy and metabolic fluxes by the ORP-shift control strategy, and the strategy could provide a simple and efficient tool to realize high purity propionic acid production with glycerol as carbon source.     

Production of biobutanol from acid-pretreated corncob using Clostridium beijerinckii TISTR 1461: Process optimization studies

Corncob is a potential feedstock in Thailand that can be used for fermentable sugar production through dilute sulfuric acid pretreatment and enzymatic hydrolysis. To recover high amounts of monomeric sugars from corncob, the sulfuric pretreatment conditions were optimized by using response surface methodology with three independent variables: sulfuric acid concentration, temperature, and time. The highest response of total sugars, 48.84 g/L, was found at 122.78°C, 4.65 min, and 2.82% (v/v) H₂SO₄. With these conditions, total sugars from the confirmation experiment were 46.29 g/L, with 5.51% error from the predicted value. The hydrolysate was used as a substrate for acetone–butanol–ethanol fermentation to evaluate its potential for microbial growth. The simultaneous saccharification and fermentation (SSF) showed that C. beijerinckii TISTR 1461 can generate acetone–butanol–ethanol products at 11.64 g/L (5.29 g/L acetone, 6.26 g/L butanol, and 0.09 g/L ethanol) instantly using sugars from the hydrolysed corncob with Novozymes 50013 cellulase enzyme without an overliming process.     

Cloning and characterization of two distinct water-forming NADH oxidases from <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> for the regeneration of NAD

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K _M of 99.0 μM (LpNox1) and 27.6 μM (LpNox2), and yielding catalytic efficiency k _cat/K _M of 1.0 and 0.2 μM^?1 s^?1, respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD⁺ was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD⁺ regeneration in dehydrogenase-catalyzed oxidations.