New regulatory role of Znf1 in transcriptional control of pentose phosphate pathway and ATP synthesis for enhanced isobutanol and acid tolerance.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production. Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

Optimization of CO fermentation by Clostridium carboxidivorans in batch reactors: Effects of the medium composition.

OBJECTIVES: The objective of this study was to investigate the effects of medium composition on CO fermentation by Clostridium carboxidivorans. The focus was to reduce the medium cost preserving acceptable levels of solvent production. METHODS: Yeast extract (YE) concentration was set in the range of 0-3 g/L. Different reducing agents were investigated, including cysteine-HCl 0.6 g/L, pure cysteine 0.6 g/L, sodium sulphide (Na2S) 0.6 g/L, cysteine-sodium sulphide 0.6 g/L and cysteine-sodium sulphide 0.72 g/L. The concentration of the metal solution was decreased down to 25 % of the standard value. Fermentation tests were also carried out with and without tungsten or selenium. RESULTS: The results demonstrated that under optimized conditions, namely yeast extract (YE) concentration set at 1 g/L, pure cysteine as the reducing agent and trace metal concentration reduced to 75 % of the standard value, reasonable solvent production was achieved in less than 150 h. Under these operating conditions, the production levels were found to be 1.39 g/L of ethanol and 0.27 g/L of butanol. Furthermore, the study revealed that selenium was not necessary for C. carboxidivorans fermentation, whereas the presence of tungsten played a crucial role in both cell growth and solvent production. CONCLUSIONS: The optimization of the medium composition in CO fermentation by Clostridium carboxidivorans is crucial for cost-effective solvent production. Tuning the yeast extract (YE) concentration, using pure cysteine as the reducing agent and reducing trace metal concentration contribute to reasonable solvent production within a relatively short fermentation period. Tungsten is essential for cell growth and solvent production, while selenium is not required.

The effect of Alnus incana (L.) Moench extracts in ameliorating iron overload-induced hepatotoxicity in male albino rats.

Iron overload causes multiorgan dysfunction and serious damage. Alnus incana from the family Betulaceae, widely distributed in North America, is used for treating diseases. In this study, we investigated the iron chelating, antioxidant, anti-inflammatory, and antiapoptotic activities of the total and butanol extract from Alnus incana in iron-overloaded rats and identified the bioactive components in both extracts using liquid chromatography-mass spectrometry. We induced iron overload in the rats via six intramuscular injections of 12.5 mg iron dextran/100 g body weight for 30 days. The rats were then administered 60 mg ferrous sulfate /kg body weight once daily using a gastric tube. The total and butanol extracts were given orally, and the reference drug (deferoxamine) was administered subcutaneously for another month. After two months, we evaluated the biochemical, histopathological, histochemical, and immunohistochemical parameters. Iron overload significantly increased the serum iron level, liver biomarker activities, hepatic iron content, malondialdehyde, tumor necrosis factor-alpha, and caspase-3 levels. It also substantially (P < 0.05) reduced serum albumin, total protein, and total bilirubin content, and hepatic reduced glutathione levels. It caused severe histopathological alterations compared to the control rats, which were markedly (P < 0.05) ameliorated after treatment. The total extract exhibited significantly higher anti-inflammatory and antiapoptotic activities but lower antioxidant and iron-chelating activities than the butanol extract. Several polyphenolic compounds, including flavonoids and phenolic acids, were detected by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) analysis. Our findings suggest that both extracts might alleviate iron overload-induced hepatoxicity and other pathological conditions characterized by hepatic iron overload, including thalassemia and sickle-cell anemia.

Ameliorative Effects of Thunbergia erecta L. Leaves Against the Initiation of Hepatocarcinogenesis Induced by Diethylnitrosamine in the Rat Model.

Thunbergia erecta L. contains cytotoxic and liver-protective compounds. Thunbergia erecta L. leaves were macerated in 70% aqueous ethanol, then fractionated with ethyl acetate (9.3 g) and butanol (12.7 g), and attenuated Den-induced liver cancer in a Wistar rat experimental model. Ethyl acetate and butanol fractions were chromatographed using column chromatography and solid-phase extraction (SPE); Vicenin-II (1), kaempferol (2), biochanin A, sissotrin 7-O-ß-glucopyranoside (3), gentianose (4), acacetin 7-O-ß-glucopyranoside (5), apigenin 7-O-ß-glucopyranoside (6), and rosmarinic acid (7) were extracted, and their structures were determined using NMR spectroscopy and ESI-mass spectrometry. Sixty rats were divided into six groups (ten each): control group, Den group, doxorubicin/Den-treated group, butanol fraction/Den-treated group, and isolated acacetin 7-O-ß-glucopyranoside/Den-treated group. The liver enzymes and proinflammatory biomarkers were used to estimate the liver function. In addition, liver tissues were collected for analysis of oxidative stress markers, gene expression, and histopathology. There is a significant increase in the levels of liver enzymes, AFP, and TNF-ἁ. This was conveyed by a significant increase of IL-1 and caspase-3, elevation of MDA and reduction of GSH, and suppression of Bcl2 and elevation of Bax expression. All parameters in butanol, ethyl acetate fractions, and isolated acacetin 7-O-ß-glucopyranoside (major constituents) of T. erecta L. were significantly improved to values close to those of the control group.

Oxidoreduction potential controlling for increasing the fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production.

BACKGROUND: Lignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem. RESULTS: In this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at - 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP+, and NADH/NAD+ were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity. CONCLUSION: ORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid "acid crash". Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved.

Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum.

BACKGROUND: The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. RESULTS: Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. CONCLUSIONS: In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.

Isobutanol production freed from biological limits using synthetic biochemistry.

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

Acetone­butanol­ethanol fermentation from sugarcane bagasse hydrolysates: utilization of C5 and C6 sugars

Background: Fuels and chemicals from renewable feedstocks have a growing demand, and acetone, butanol and ethanol (ABE) are some relevant examples. These molecules can be produced by the bacterial fermentation process using hydrolysates generated from lignocellulosic biomass as sugarcane bagasse, one of the most abundant sources of lignocellulosic biomass in Brazil. It originates as a residue in mills and distilleries in the production of sugar and ethanol. Results: In the present work, two strategies to generate hydrolysates of sugarcane bagasse were adopted. The fermentation of the first hydrolysate by Clostridium acetobutylicum DSM 6228 resulted in final concentrations of butanol, acetone and ethanol of 6.4, 4.5 and 0.6 g/L, respectively. On the other hand, the second hydrolysate presented better results (averages of 9.1, 5.5 and 0.8 g/L, respectively), even without the need for nutrient supplementation, since key elements were already present in the medium. The productivity (QP) and yield (YP/S) of the solvents with second hydrolysate were 0.5 g/L•h-1 and 0.4 g/g, respectively. Conclusions: The results described herein open new perspectives for the production of important molecules from residual lignocellulosic biomass for the fuel and chemical industries within the context of second-generation biorefinery.

Isotope dilution LC/ESI--MS-MS quantitation of urinary 1,4-bis(N-acetyl-S-cysteinyl)-2-butanone in mice and rats as the biomarker of 1-chloro-2-hydroxy-3-butene, an in vitro metabolite of 1,3-butadiene.

1-Chloro-2-hydroxy-3-butene (CHB) is a possible metabolite of 1,3-butadiene, a carcinogenic air pollutant. To demonstrate its formation in vivo, it is desirable to develop a practical biomarker and the corresponding analysis method. CHB can undergo alcohol dehydrogenase- and cytochromes P450 enzymes (P450)-mediated oxidation to yield 1-chloro-3-buten-2-one (CBO), which readily forms glutathione conjugates. We hypothesized that CBO-derived mercapturic acids, which are the expected biotransformed products of CBO-glutathione conjugates, could be used as CHB biomarkers. Thus, in the present study, we investigated the in vivo biotransformation of CHB into CBO-derived mercapturic acids. Because the reaction of CBO with N-acetyl-l-cysteine yields two products, 1,4-bis(N-acetyl-S-cysteinyl)-2-butanone (NC1) and 1-chloro-4-(N-acetyl-S-cysteinyl)-2-butanone (NC2), we first developed an isotope dilution LC/ESI--MS-MS method to quantitate urinary NC1 and NC2, and then determined their concentrations in urine of C57BL/6 mice and Sprague-Dawley rats administered CHB. Since no NC2 was detected in samples, the LC/ESI--MS-MS method was optimized specifically for NC1. NC1 was enriched through solid phase extraction with the recovery being 75-82%. The limits of detection and quantitation were 6.8 and 34 fmol/0.1 mL for mouse urine, and 4.5 and 7.1 fmol/0.1 mL for rat urine, respectively. In urine of animals before CHB administration, no NC1 was detected; in mice administered CHB at 10 and 30 mg/kg, and rats at 5 and 15 mg/kg, NC1 was detected and its concentrations in urine from animals given higher doses were 3-6 fold higher than those given lower doses. Moreover, the NC1 concentrations in urine during 0-8 h were 4-6 fold and 10-11 fold higher than those during 8-24 h for mice and rats, respectively. The results demonstrated that CHB could be in vivo biotransformed into NC1, which could be used as a practical CHB biomarker.

Optimisation of Simultaneous Saccharification and Fermentation (SSF) for Biobutanol Production Using Pretreated Oil Palm Empty Fruit Bunch.

This study was conducted in order to optimise simultaneous saccharification and fermentation (SSF) for biobutanol production from a pretreated oil palm empty fruit bunch (OPEFB) by Clostridium acetobutylicum ATCC 824. Temperature, initial pH, cellulase loading and substrate concentration were screened using one factor at a time (OFAT) and further statistically optimised by central composite design (CCD) using the response surface methodology (RSM) approach. Approximately 2.47 g/L of biobutanol concentration and 0.10 g/g of biobutanol yield were obtained after being screened through OFAT with 29.55% increment (1.42 fold). The optimised conditions for SSF after CCD were: temperature of 35 °C, initial pH of 5.5, cellulase loading of 15 FPU/g-substrate and substrate concentration of 5% (w/v). This optimisation study resulted in 55.95% increment (2.14 fold) of biobutanol concentration equivalent to 3.97 g/L and biobutanol yield of 0.16 g/g. The model and optimisation design obtained from this study are important for further improvement of biobutanol production, especially in consolidated bioprocessing technology.

Mechanistic simulation of batch acetone-butanol-ethanol (ABE) fermentation with in situ gas stripping using Aspen Plus™.

Process simulations of batch fermentations with in situ product separation traditionally decouple these interdependent steps by simulating a separate "steady state" continuous fermentation and separation units. In this study, an integrated batch fermentation and separation process was simulated for a model system of acetone-butanol-ethanol (ABE) fermentation with in situ gas stripping, such that the fermentation kinetics are linked in real-time to the gas stripping process. A time-dependent cell growth, substrate utilization, and product production is translated to an Aspen Plus batch reactor. This approach capitalizes on the phase equilibria calculations of Aspen Plus to predict the effect of stripping on the ABE fermentation kinetics. The product profiles of the integrated fermentation and separation are shown to be sensitive to gas flow rate, unlike separate steady state fermentation and separation simulations. This study demonstrates the importance of coupled fermentation and separation simulation approaches for the systematic analyses of unsteady state processes.

Detoxification of Organosolv-Pretreated Pine Prehydrolysates with Anion Resin and Cysteine for Butanol Fermentation.

Bioconversion of lignocellulose to biofuels suffers from the degradation compounds formed during pretreatment and acid hydrolysis. In order to achieve an efficient biomass to biofuel conversion, detoxification is often required before enzymatic hydrolysis and microbial fermentation. Prehydrolysates from ethanol organosolv-pretreated pine wood were used as substrates in butanol fermentation in this study. Six detoxification approaches were studied and compared, including overliming, anion exchange resin, nonionic resin, laccase, activated carbon, and cysteine. It was observed that detoxification by anion exchange resin was the most effective method. The final butanol yield after anion exchange resin treatment was comparable to the control group, but the fermentation was delayed for 72 h. The addition of Ca(OH)2 was found to alleviate this delay and improve the fermentation efficiency. The combination of Ca(OH)2 and anion exchange resin resulted in completion of fermentation within 72 h and acetone-butanol-ethanol (ABE) production of 11.11 g/L, corresponding to a yield of 0.21 g/g sugar. The cysteine detoxification also resulted in good detoxification performance, but promoted fermentation towards acid production (8.90 g/L). The effect of salt on ABE fermentation was assessed and the possible role of Ca(OH)2 was to remove the salts in the prehydrolysates by precipitation.

Simultaneous acetone-butanol-ethanol fermentation, gas stripping, and full-cell-catalyzed esterification for effective production of butyl oleate.

In this study, aiming to improve the economic feasibility of acetone-butanol-ethanol (ABE) fermentation process, generate valuable products and extend the product chain, esterification catalyzed by Candida sp. 99-125 cells was hybrid with the ABE fermentation-gas-stripping integration system. The gas-stripping condensate that contained concentrated ABE products was directly used for esterification without the participation of toxic organic solvents. Full-cell catalysis temperature and the cell dosage rate on oleate production were evaluated and optimized in the esterification process. Under the optimized conditions (35 °C, 8% of cells), ~ 68% of butyl oleate and ~ 12% of ethyl oleate were obtained after 4 h of esterification. The Candida sp. 99-125 cells were able to be reused for at least four cycles. The novel cascade process showed environmental benefits, which also showed promising in improving the economic feasibility of the conventional ABE fermentation process.

4-(4-Hydroxyphenyl)-2-butanol (rhododendrol)-induced melanocyte cytotoxicity is enhanced by UVB exposure through generation of oxidative stress.

4-(4-Hydroxyphenyl)-2-butanol (rhododendrol, RD), a skin-whitening agent, was reported to cause skin depigmentation in some users, which is attributed to its cytotoxicity to melanocyte. It was reported that cytotoxicity to melanocyte is possibly mediated by oxidative stress in a tyrosinase activity-dependent manner. We examined the effect of UV radiation (UVR) on RD-induced melanocyte cytotoxicity as an additional aggravating factor. UVR enhanced RD-induced cytotoxicity in normal human epidermal melanocytes (NHEMs) via the induction of endoplasmic reticulum (ER) stress. Increased generation of intracellular reactive oxygen species (ROS) was detected. Pretreatment with N-acetyl cysteine (NAC), antioxidant and precursor of glutathione significantly attenuated ER stress-induced cytotoxicity in NHEMs treated with RD and UVR. Increase in cysteinyl-RD-catechol and RD-pheomelanin in NHEMs treated with RD and UVR suggested that, after UVR excitation, RD or RD metabolites are potent ROS-generating substances and that the tendency to produce RD-pheomelanin during melanogenesis amplifies ROS generation in melanocytes. Our results help to elucidate the development mechanisms of RD-induced leukoderma and provide information for innovation of safe skin-whitening compounds.

Bioactivation of 1-chloro-2-hydroxy-3-butene, an in vitro metabolite of 1,3-butadiene, by rat liver microsomes.

1-Chloro-2-hydroxy-3-butene (CHB) is an in vitro metabolite of 1,3-butadiene, a rodent/human carcinogen. To search for an approach detecting CHB in vivo, it is vital to obtain a full understanding of CHB metabolism. Previously, we demonstrated that CHB was bioactivated to 1-chloro-3-buten-2-one (CBO) by alcohol dehydrogenase. However, CHB metabolism by cytochrome P450s has not been reported. Thus, in the present study, CHB metabolism by rat liver microsomes was investigated. The results showed that CHB was converted to 1-chloro-3,4-epoxy-2-butanol (CEB) and CBO. 4-Methylpyrazole, a cytochrome P450 2E1-specific inhibitor, inhibited the formation of both CEB and CBO, while 1-benzylimidazole, a generic cytochrome P450 inhibitor, completely abolished the formation of CEB and CBO, suggesting that CHB metabolism was mediated by cytochrome P450s. Because the molecules have two chiral centers, CEB was detected as two stereoisomers, which were designated D-CEB and M-CEB, and were characterized as (2S,3R)-/(2R,3S)-CEB and (2R,3R)-/(2S,3S)-CEB, respectively. The amounts of M-CEB were more than those of D-CEB by 50-80%. The amounts of CEB and CBO increased linearly over time from 10 (or 20 min for CBO) to 50 min. CHB metabolism followed Michaelis-Menten kinetics; the Km and Vmax values were determined to be 6.4 ±â€¯0.7 mM and 0.10 ±â€¯0.01 nmol/min/mg protein for D-CEB, 4.2 ±â€¯0.5 mM and 0.16 ±â€¯0.01 nmol/min/mg protein for M-CEB, and 4.0 ±â€¯0.5 mM and 4.6 ±â€¯0.5 nmol/min/mg protein for CBO, respectively. Thus, CBO was the dominant product of CHB metabolism. Moreover, CEB was genotoxic at ≥ 50 µM as evaluated by the comet assay. Collectively, the data showed that CHB could be bioactivated to CEB and CBO by cytochrome P450s with CBO being the predominant product. Thus, the formation of CEB and CBO can be used as evidence of CHB production. The products may also play a role in toxicity of CHB.

The Draft Genome Sequence of Clostridium beijerinckii NJP7, a Unique Bacterium Capable of Producing Isopropanol-Butanol from Hemicellulose Through Consolidated Bioprocessing.

A wild type solventogenic Clostridium beijerinckii NJP7 capable of converting polysaccharides, such as hemicellulose, into butanol and isopropanol via a unique acetone-isopropanol-butanol (AIB) pathway was isolated and characterized. This represents the first wild type isopropanol-butanol generating bacterium which could achieve butanol production directly from lignocellulose through consolidated bioprocessing (CBP). Strain NJP7 was isolated from decomposite soil from Laoshan Nature Park, China, and its genome shows 98.6% identical to 89.5% of the Clostridium diolis submitted genome sequence. The assembled draft genome contains 5.76 Mb and 5101 predicted encoding proteins with a GC content of 29.73%. Among these annotated proteins, hemicellulase and the secondary alcohol dehydrogenase play key roles in achievement of AIB production from hemicellulose through CBP.

Selection of Schizochytrium limacinum mutants based on butanol tolerance

Background: Mutation breeding is one of the most important routes to achieving high docosahexaenoic acid (DHA) productivity using Schizochytrium. However, few selection strategies have been reported that aim to generate a high DHA content in Schizochytrium lipids. Results: First, culture temperature altered the butanol tolerance of Schizochytrium limacinum B4D1. Second, S. limacinum E8 was obtained by selecting mutants with high butanol tolerance. This mutant exhibited a 17.97% lower proportion of DHA than the parent strain S. limacinum B4D1. Third, a negative selection strategy was designed in which S. limacinum F6, a mutant with poor butanol tolerance, was obtained. The proportion of DHA in S. limacinum F6 was 11.22% higher than that of parent strain S. limacinum B4D1. Finally, the performances of S. limacinum B4D1, E8 and F6 were compared. These three strains had different fatty acid profiles, but there was no statistical difference in their biomasses and lipid yields. Conclusion: It was feasible to identified the relative DHA content of S. limacinum mutants based on their butanol tolerance.

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria.

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)3 as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L-1). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)3 as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L-1. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L-1 (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L-1 for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

High-efficiency acetone-butanol-ethanol production and recovery in non-strict anaerobic gas-stripping fed-batch fermentation.

Conventional acetone-butanol-ethanol (ABE) fermentation coupled with gas stripping is conducted under strict anaerobic conditions. In this work, a fed-batch ABE fermentation integrated with gas stripping (FAFIGS) system using a non-strict anaerobic butanol-producing symbiotic system, TSH06, was investigated for the efficient production of butanol. To save energy and keep a high gas-stripping efficiency, the integrated fermentation was conducted by adjusting the butanol recovery rate. The gas-stripping efficiency increased when the butanol concentration increased from 6 to 12 g/L. However, in consideration of the butanol toxicity to TSH06, 8 g/L butanol was the optimal concentration for this FAFIGS process. A model for describing the relationship between the butanol recovery rate and the gas flow rate was developed, and the model was subsequently applied to adjust the butanol recovery rate during the FAFIGS process. In the integrated system under non-strict anaerobic condition, relatively stable butanol concentrations of 7 to 9 g/L were achieved by controlling the gas flow rate which varied between 1.6 and 3.5 vvm based on the changing butanol productivity. 185.65 g/L of butanol (267.15 g/L of ABE) was produced in 288 h with a butanol recovery ratio of 97.36%. The overall yield and productivity of butanol were 0.23 g/g and 0.64 g/L/h, respectively. This study demonstrated the feasibility of using FAFIGS under non-strict anaerobic conditions with TSH06. This work is helpful in characterizing the butanol anabolism performance of TSH06 and provides a simple and efficient scheme for butanol production.

A dynamic metabolic flux analysis of ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum ATCC 824, with riboflavin as a by-product.

The present study reveals that supplementing sodium acetate (NaAc) strongly stimulates riboflavin production in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 with xylose as carbon source. Riboflavin production increased from undetectable concentrations to ∼0.2 g L-1 (0.53 mM) when supplementing 60 mM NaAc. Of interest, solvents production and biomass yield were also promoted with fivefold acetone, 2.6-fold butanol, and 2.4-fold biomass adding NaAc. A kinetic metabolic model, developed to simulate ABE biosystem, with riboflavin production, revealed from a dynamic metabolic flux analysis (dMFA) simultaneous increase of riboflavin (ribA) and GTP (precursor of riboflavin) (PurM) synthesis flux rates under NaAc supplementation. The model includes 23 fluxes, 24 metabolites, and 72 kinetic parameters. It also suggested that NaAc condition has first stimulated the accumulation of intracellular metabolite intermediates during the acidogenic phase, which have then fed the solventogenic phase leading to increased ABE production. In addition, NaAc resulted in higher intracellular levels of NADH during the whole culture. Moreover, lower GTP-to-adenosine phosphates (ATP, ADP, AMP) ratio under NaAc supplemented condition suggests that GTP may have a minor role in the cell energetic metabolism compared to its contribution to riboflavin synthesis.