Vomifoliol isolated from mangrove plant Ceriops tagal inhibits the NFAT signaling pathway with CN as the target enzyme in vitro.

Vomifoliol, a natural sesquiterpene compound, is a secondary metabolite isolated from the mangrove plant Ceriops tagal. The present study aimed to determine the immunosuppressive effects and underlying mechanisms of vomifoliol on Jurkat cells in vitro. The results show that vomifoliol significantly inhibited calcineurin (CN) at concentrations resulting in relatively low cytotoxicity. Moreover, vomifoliol was found to exert an inhibitory effect on phorbol 12-myristate 13-acetate (PMA)/ ionomycin (Io) -induced Jurkat cells and the dephosphorylation of NFAT1. In addition, it reduced the expression of IL-2. Based on these results, we concluded that vomifoliol may inhibit the immune response of Jurkat cells, and vomifoliol may use CN as the target enzyme to inhibit NFAT signaling pathway. Therefore, vomifoliol may be promising as a low-toxic natural immunosuppressant.

Ionic liquid-based in situ product removal design exemplified for an acetone-butanol-ethanol fermentation.

Selecting an appropriate separation technique is essential for the application of in situ product removal (ISPR) technology in biological processes. In this work, a three-stage systematic design method is proposed as a guide to integrate ionic liquid (IL)-based separation techniques into ISPR. This design method combines the selection of a suitable ISPR processing scheme, the optimal design of an IL-based liquid-liquid extraction (LLE) system followed by process simulation and evaluation. As a proof of concept, results for a conventional acetone-butanol-ethanol fermentation are presented (40,000 ton/year butanol production). In this application, ILs tetradecyl(trihexyl)phosphonium tetracyanoborate ([TDPh][TCB]) and tetraoctylammonium 2-methyl-1-naphthoate ([TOA] [MNaph]) are identified as the optimal solvents from computer-aided IL design (CAILD) method and reported experimental data, respectively. The dynamic simulation results for the fermentation process show that, the productivity of IL-based in situ (fed-batch) process and in situ (batch) process is around 2.7 and 1.8fold that of base case. Additionally, the IL-based in situ (fed-batch) process and in situ (batch) process also have significant energy savings (79.6% and 77.6%) when compared to the base case.

Separation of biobutanol from ABE fermentation broth using lignin as adsorbent: A totally sustainable approach with effective utilization of lignocellulose.

Adsorption is considered to be a promising butanol recovery method for solving the issue of inhibition in the ABE (acetone-butanol-ethanol) fermentation. As a byproduct in the second generation biobutanol industry, lignin was found to be a good adsorbent for the butanol enrichment. It is conducive to the full utilization of renewable lignocellulose biomass resource. Kinetic and equilibrium experiments indicated that lignin had a satisfactory adsorption rate and capacity that are comparable to those of many synthetic materials. Multicomponent adsorption experiments revealed that lignin had higher adsorption selectivity toward butanol than that of ethanol and acetone. The adsorption capacity of lignin for butanol first increased and then gradually decreased with increasing temperature. And maximum adsorption capacity reached 304.66 mg g-1 at 313 K. The inflection point of temperature is close to the ABE fermentation temperature of 310 K. The condensed butanol by desorption was 145 g L-1, with a satisfying regeneration performance. 1H NMR and FT-IR spectra indicated that the aromatic units of lignin formed π-systems with A/B/E. The π-system is particularly significant for butanol due to its longer hydrocarbon chain. These results could contribute to the emerging lignin-based materials for butanol separation.

Bioactivity of volatile organic compounds by Aureobasidium species against gray mold of tomato and table grape.

Aureobasidium strains isolated from diverse unconventional environments belonging to the species A. pullulans, A. melanogenum, and A. subglaciale were evaluated for Volatile Organic Compounds (VOCs) production as a part of their modes of action against Botrytis cinerea of tomato and table grape. By in vitro assay, VOCs generated by the antagonists belonging to the species A. subglaciale showed the highest inhibition percentage of the pathogen mycelial growth (65.4%). In vivo tests were conducted with tomatoes and grapes artificially inoculated with B. cinerea conidial suspension, and exposed to VOCs emitted by the most efficient antagonists of each species (AP1, AM10, AS14) showing that VOCs of AP1 (A. pullulans) reduced the incidence by 67%, partially confirmed by the in vitro results. Conversely, on table grape, VOCs produced by all the strains did not control the fungal incidence but were only reducing the infection severity (< 44.4% by A. pullulans; < 30.5% by A. melanogenum, and A. subglaciale). Solid-phase microextraction (SPME) and subsequent gas chromatography coupled to mass spectrometry identified ethanol, 3-methyl-1-butanol, 2-methyl-1-propanol as the most produced VOCs. However, there were differences in the amounts of produced VOCs as well as in their repertoire. The EC50 values of VOCs for reduction of mycelial growth of B. cinerea uncovered 3-methyl-1-butanol as the most effective compound. The study demonstrated that the production and the efficacy of VOCs by Aureobasidium could be directly related to the specific species and pathosystem and uncovers new possibilities for searching more efficient VOCs producing strains in unconventional habitats other than plants.

Determination of the alcoholic content in whiskeys using micellar electrokinetic chromatography on microchips.

This report describes the development of a methodology based on micellar electrokinetic chromatography for the separation of alcohols on chip-based systems aiming the determination of alcoholic content in whiskey samples. The separation conditions were optimized the best results were achieved using 50 mmolL-1 phosphate buffer containing 30 mmolL-1 sodium dodecyl sulfate. The alcoholic content was determined in 16 seized whiskey samples from 4 different brands as well as in the original samples. The methodology presented herein allowed the correct classification of 75% of the seized samples as adulterated and the data obtained did not statistically differ from those recorded by a reference technique. The proposed analytical approach emerges as a promising tool to provide a rapid screening of the beverages authenticity and it may be useful to be widely explored for the quality control.

Bioactivity-guided isolation of rosmarinic acid as the principle bioactive compound from the butanol extract of Isodon rugosus against the pea aphid, Acyrthosiphon pisum.

Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest control practices involving the excessive use of synthetic insecticides over many years have resulted in aphid resistance to a number of pesticides. In nature, plants produce secondary metabolites during their interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and deterrents towards the insects. In a previous study, we demonstrated that the butanol fraction from a crude methanolic extract of an important plant species, Isodon rugosus showed strong insecticidal activity against the pea aphid, Acyrthosiphon pisum. To further explore this finding, the current study aimed to exploit a bioactivity-guided strategy to isolate and identify the active compound in the butanol fraction of I. rugosus. As such, reversed-phase flash chromatography, acidic extraction and different spectroscopic techniques were used to isolate and identify the new compound, rosmarinic acid, as the bioactive compound in I. rugosus. Insecticidal potential of rosmarinic acid against A. pisum was evaluated using standard protocols and the data obtained was analyzed using qualitative and quantitative statistical approaches. Considering that a very low concentration of this compound (LC90 = 5.4 ppm) causes significant mortality in A. pisum within 24 h, rosmarinic acid could be exploited as a potent insecticide against this important pest insect. Furthermore, I. rugosus is already used for medicinal purposes and rosmarinic acid is known to reduce genotoxic effects induced by chemicals, hence it is expected to be safer compared to the current conventional pesticides. While this study highlights the potential of I. rugosus as a possible biopesticide source against A. pisum, it also provides the basis for further exploration and development of formulations for effective field application.

Crotonols A and B, two rare tigliane diterpenoid derivatives against K562 cells from Croton tiglium.

Crotonols A and B (1 and 2), two tigliane diterpenoids featuring a rare C-7/C-14 cyclized and novel 5/7/7-fused carbon skeleton, along with the known tigliane wallichiioid A, were isolated from the leaves of Croton tiglium. Their structures were determined through spectroscopic methods, X-ray crystallography and ECD analysis. To the best of our knowledge, crotonol B (2) represents the first example of 13,14-seco-tigliane diterpenoids. Crotonols A and B displayed strong cytotoxic activities against the K562 cell line with IC50 values of 0.20 and 0.21 µM, respectively. Furthermore, crotonol A promoted the apoptosis of K562 cells through the cleavage of PARP and the accumulation of bax as well as the degradation of bcl-2.

Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4.

Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol.

Energy efficiency of acetone, butanol, and ethanol (ABE) recovery by heat-integrated distillation.

Acetone, butanol, and ethanol (ABE) is an alternative biofuel. However, the energy requirement of ABE recovery by distillation is considered elevated (> 15.2 MJ fuel/Kg-ABE), due to the low concentration of ABE from fermentation broths (between 15 and 30 g/l). In this work, to reduce the energy requirements of ABE recovery, four processes of heat-integrated distillation were proposed. The energy requirements and economic evaluations were performed using the fermentation broths of several biocatalysts. Energy requirements of the processes with four distillation columns and three distillation columns were similar (between 7.7 and 11.7 MJ fuel/kg-ABE). Double-effect system (DED) with four columns was the most economical process (0.12-0.16 $/kg-ABE). ABE recovery from dilute solutions by DED achieved energy requirements between 6.1 and 8.7 MJ fuel/kg-ABE. Vapor compression distillation (VCD) reached the lowest energy consumptions (between 4.7 and 7.3 MJ fuel/kg-ABE). Energy requirements for ABE recovery DED and VCD were lower than that for integrated reactors. The energy requirements of ABE production were between 1.3- and 2.0-fold higher than that for alternative biofuels (ethanol or isobutanol). However, the energy efficiency of ABE production was equivalent than that for ethanol and isobutanol (between 0.71 and 0.76) because of hydrogen production in ABE fermentation.

Neuroprotective Effects of Butanol Fraction of Cordyceps cicadae on Glutamate-Induced Damage in PC12 Cells Involving Oxidative Toxicity.

The current study was aimed at investigating the neuroprotective effects of the butanol fraction from Cordyceps cicadae (CBU ), which was responsible for the anti-aging effect of this medicine. Glutamate-induced PC12 cells were used as a model to determine the neuroprotective effect against oxidative cell death. Cell viability, cytotoxicity, flow cytometry, mitochondrial transmembrane potential (MMP), reactive oxygen species (ROS), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) levels were analyzed to assess neuronal cell survival or death. The results obtained from the above evaluations showed that CBU was the most effective fraction and even better than pure compounds present in C. cicadae in terms of suppressing glutamate-induced damage in PC12 cells, increasing cell viability, decreasing lactase dehydrogenase (LDH) release, and reduction of apoptosis induced by exposure to glutamate. Furthermore, CBU protected cells against mitochondrial dysfunction and oxidative stress as indicated by the suppression of ROS accumulation and up regulation of the levels of GSH-Px and SOD. In summary, the above results showed that CBU exerted neuroprotective effect against oxidative damage, and this activity could be partly due to the action of nucleosides present in the CBU .

In situ biobutanol recovery from clostridial fermentations: a critical review.

Butanol is a precursor of many industrial chemicals, and a fuel that is more energetic, safer and easier to handle than ethanol. Fermentative biobutanol can be produced using renewable carbon sources such as agro-industrial residues and lignocellulosic biomass. Solventogenic clostridia are known as the most preeminent biobutanol producers. However, until now, solvent production through the fermentative routes is still not economically competitive compared to the petrochemical approaches, because the butanol is toxic to their own producer bacteria, and thus, the production capability is limited by the butanol tolerance of producing cells. In order to relieve butanol toxicity to the cells and improve the butanol production, many recovery strategies (either in situ or downstream of the fermentation) have been attempted by many researchers and varied success has been achieved. In this article, we summarize in situ recovery techniques that have been applied to butanol production through Clostridium fermentation, including liquid-liquid extraction, perstraction, reactive extraction, adsorption, pervaporation, vacuum fermentation, flash fermentation and gas stripping. We offer a prospective and an opinion about the past, present and the future of these techniques, such as the application of advanced membrane technology and use of recent extractants, including polymer solutions and ionic liquids, as well as the application of these techniques to assist the in situ synthesis of butanol derivatives.

Applications of high-resolution recycling liquid chromatography: From small to large molecules.

A twin-column recycling separation process (TCRSP) is assembled and used to generate higher speed and/or higher resolution levels than those of the usual non-recycling process at the same back pressure. It enables the users to solve very challenging separation problems caused by too small selectivity factors and/or too low column efficiencies. The relative gain in speed-resolution performance increases with increasing the number of cycles in the TCRSP, decreasing the maximum allowable pressure imposed by the LC system, decreasing the column permeability, and with reducing the separation speed. TCRSP is then particularly attractive for conventional LC systems (5000psi maximum) and columns packed with sub-2µm to 3.5µm particles. The performance of the real TCRSP was compared to that of the ideal TCRSP for which the retention factor is strictly pressure-independent. A broad range of separation problems encountered in conventional non-recycling chromatography can be easily solved by using a TCRSP assembly based on two 15cm long columns. Under adsorption conditions, the TCRSP enables the full baseline separation of polycyclic aromatic hydrocarbon (PAH) isomers (benzo[a]anthracene and chrysene) on a 3.5µm XSelect-HSS T3 phase, the complete or improved resolution of racemic mixtures (4-phenylbutanol and bromacil) using the same 2.5µm cellulose-1 chiral stationary phase, and the full resolution of isotopic compounds (benzene/1,3,5-benzene-d3/benzene-d6) on a 2.7µm Cortecs-C18 phase. Under non-adsorption conditions or in size-exclusion chromatography (SEC), the fractionation of a polystyrene standard mixture (molecular weights of 35, 66, 130, 277, 552, 1210, and 2500kDa) was completed after only 8 cycles on a 1.7µm BEH 200Åphase. Similarly, a mixture of intact proteins with molecular weights of 16.7, 66.4, 150, 660, and 1320kDa was fully resolved on a 2.5µm BEH 450Åphase after only 6 cycles. Finally, TCRSP enables the complete separation of a few high-molecular-weight species (monoclonal antibody aggregates, small relative abundance of 1 for 250) from the intact monomeric monoclonal antibody (Vectibix).

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.

Developing new platform chemicals: what is required for a new bio-based molecule to become a platform chemical in the bioeconomy?

This paper proposes a framework with six dimensions that can be useful for evaluating the potential and the current stage of a bio-based platform chemical. The framework considers the technological and strategic challenges to be fulfilled by a company that intends to lead a platform based on a bio-based chemical. A platform chemical should be an intermediate molecule, with a structure able to generate a number of derivatives, that is produced at a competitive cost, capable of allowing exploitation of the scale and scope economies, and inserted within a complete innovation ecosystem that is able to create value with governance mechanisms that are capable of allowing coordination of the innovation process and facilitation of the value capture by the focal company leading the platform, in our case the producer of the platform molecule. Based on these six dimensions, three potential platform chemicals - succinic acid, butanol and farnesene - are compared and discussed. It is possible to identify important differences concerning the technological dimensions and the strategic dimensions as well. Two of the molecules - farnesene and succinic acid - adhere to most of the conditions required to structure a platform chemical. However, the innovation ecosystem is not complete and the governance mechanisms are still under development, so it is not clear if they will be capable of allowing a favorable position for value capture by the platform leader. Butanol structuring for a platform does not seem promising. The potential of the molecule is apparently not high and the strategic initiatives are in general not focused on innovation ecosystem structuring.

Elucidation of the possible mechanism of analgesic actions of butanol leaf fraction of Olax subscorpioidea Oliv.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparations of Olax subscorpioidea have been used traditionally for the management of pains, inflammatory diseases, yellow fever, cancer and rheumatism. Previously, the analgesic activity of its leaf extract have been reported. Furthermore, an analgesic assay guided fractionation showed that the butanol soluble fraction is the most active. However, the mechanism of this activity remains to be elucidated. This present study investigated the possible pharmacological mechanisms involved in the analgesic activity of the butanol leaf fraction of Olax subscorpioidea (BFOS) using the acetic acid induced writhing test in mice. MATERIALS AND METHODS: Animals were orally administered distilled water (10ml/kg), BFOS (1,000mg/kg) and morphine (10mg/kg) 60minutes before i.p administration of acetic acid and the resulting writhing were counted for 10minutes. To establish the possible mechanism(s) of action of BFOS, separate group of animals were pretreated with naloxone (2mg/kg, i.p), prazosin (1mg/kg, i.p), yohimbine (1mg/kg, i.p), propranolol (20mg/kg, i.p), metergoline (2mg/kg, i.p), glibenclamide (5mg/kg, i.p) and l-arginine (50mg/kg, i.p) 15minutes before BFOS. RESULTS: BFOS and morphine showed marked analgesic activities (p<0.001); the pretreatment of animals with naloxone, metergoline and l-arginine significantly (p<0.05 and p<0.001) reduced the analgesic activity of BFOS; however, pretreatment with prazosin, yohimbine, propranolol and glinbenclamide showed no effect on its analgesic activity. CONCLUSION: Results obtained in this study suggest the involvement of opioidergic, serotonergic and nitric oxide-l-arginine pathways in the analgesic effect of butanol leaf fraction of Olax subscorpioidea.

Towards improved butanol production through targeted genetic modification of Clostridium pasteurianum.

Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1,3-propanediol, when grown on glycerol. Further increases in the yields of n-butanol produced by C. pasteurianum could be accomplished through rational metabolic engineering of the strain. Accordingly, in the current report we have developed and exemplified a robust tool kit for the metabolic engineering of C. pasteurianum and used the system to make the first reported in-frame deletion mutants of pivotal genes involved in solvent production, namely hydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase). We were, for the first time in C. pasteurianum, able to eliminate 1,3-propanediol synthesis and demonstrate its production was essential for growth on glycerol as a carbon source. Inactivation of both rex and hydA resulted in increased n-butanol titres, representing the first steps towards improving the utilisation of C. pasteurianum as a chassis for the industrial production of this important chemical.

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium.

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2g/L of AECC and produce 22.1g/L of solvents (4.25g/L acetone, 11.5g/L butanol and 6.37g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.

Two-stage pervaporation process for effective in situ removal acetone-butanol-ethanol from fermentation broth.

Two-stage pervaporation for ABE recovery from fermentation broth was studied to reduce the energy cost. The permeate after the first stage in situ pervaporation system was further used as the feedstock in the second stage of pervaporation unit using the same PDMS/PVDF membrane. A total 782.5g/L of ABE (304.56g/L of acetone, 451.98g/L of butanol and 25.97g/L of ethanol) was achieved in the second stage permeate, while the overall acetone, butanol and ethanol separation factors were: 70.7-89.73, 70.48-84.74 and 9.05-13.58, respectively. Furthermore, the theoretical evaporation energy requirement for ABE separation in the consolidate fermentation, which containing two-stage pervaporation and the following distillation process, was estimated less than ∼13.2MJ/kg-butanol. The required evaporation energy was only 36.7% of the energy content of butanol. The novel two-stage pervaporation process was effective in increasing ABE production and reducing energy consumption of the solvents separation system.

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum.

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicuml-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37g/l 2-methyl-1-butanol and 2.76g/l 3-methyl-1-butanol in defined medium within 48h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.