Efficient butanol recovery from acetone-butanol-ethanol fermentation cultures grown on sweet sorghum juice by pervaporation using silicalite-1 membrane.

We investigated butanol recovery by pervaporation separation, using a silicalite-1 membrane, from batch cultures of butanol-producing Clostridium beijerinckii SBP2 grown on sweet sorghum juice as a fermentation medium. The pervaporation system yielded 73% (w/v) butanol from intact feed cultures containing 1% (w/v) butanol, and had a butanol permeation flux of 11 g m(-2) h(-1). Upon neutralization and activated charcoal treatment of the feed cultures, butanol yield and total flux increased to 82% (w/v) and 40 g m(-2) h(-1), respectively. This system is applicable to refining processes for practical biobutanol production from a promising energy crop, sweet sorghum.

Effect of membrane-bound aldehyde dehydrogenase-encoding gene disruption on glyceric acid production in Gluconobacter oxydans.

Gluconobacter oxydans IFO12528 is able to produce glyceric acid (GA) from glycerol through the action of a membrane-bound alcohol dehydrogenase (mADH), which is required for GA production. To determine whether membrane-bound aldehyde dehydrogenase (mALDH) also plays a role in GA production in G. oxydans, we constructed an aldH-disrupted mutant of G. oxydans (ΔaldH). ΔaldH was unable to produce acetic acid from ethanol, but was able to produce GA at a level approximately half that of the wildtype strain, suggesting the involvement of another ALDH in GA production. We also investigated the enantiomeric composition of GA produced by the IFO12528 and ΔaldH strains. No difference in GA composition was evident in the ΔaldH mutant, with ~73% D-GA enantiomeric excess observed in both strains.

Stepwise synthesis of 2,3-O-dipalmitoyl-D-glyceric acid and an in vitro evaluation of its cytotoxicity.

2,3-O-Dipalmitoyl-D-glyceric acid (PA2-DGA) was synthesized from D-glyceric acid calcium salt and palmitoyl chloride with improved yield. Direct condensation between the D-glyceric acid calcium salt and palmitoyl chloride produced PA2-DGA with a yield of <10%, whereas stepwise synthesis yielded this compound at up to 24% of overall yield. PA2-DGA was then subjected to a cytotoxic test using normal human dermal fibroblasts and primary normal human dermal microvascular endothelial cells. This compound had no toxic effects on human cells in vitro at concentrations up to 34 µM.

Synthesis and interfacial properties of monoacyl glyceric acids as a new class of green surfactants.

Glyceric acid (GA) is one of the most promising functional hydroxyl acids, and it is abundantly obtained from glycerol by a bioprocess using acetic acid bacteria. In this study, several monoacyl GAs were synthesized by esterification of GA and saturated fatty acyl chlorides (C12, C14, C16, and C18), forming a new class of bio-based surfactants. By the present method, a mixture of two isomers, namely 2-O-acyl and 3-O-acyl GAs, was produced, in which the 2-O-acyl derivatives were obtained as a major product. These isomers were isolated, and their surface-active properties were investigated for the first time. The surface tensions of 2-O-acyl GAs with different chain lengths were determined by the Wilhelmy method. At concentrations below 10(-4) M, the 2-O-acyl GAs exhibited higher surface-active properties compared to commercially available synthetic surfactants. For example, 2-O-lauroyl GA reduced the surface tension of water to around 25 mN/m above the critical micelle concentration (3.0×10(-4) M). In addition, 2-O-acyl derivatives showed higher surface-tension-lowering activity than 3-O-acyl GAs. The monoacyl GAs synthesized herein can potentially be used as "green surfactants."

Membrane-assisted extractive butanol fermentation by Clostridium saccharoperbutylacetonicum N1-4 with 1-dodecanol as the extractant.

A polytetrafluoroethylene (PTFE) membrane was used in membrane-assisted extractive (MAE) fermentation of acetone-butanol-ethanol (ABE) by Clostridium saccharoperbutylacetonicum N1-4. The growth inhibition effects of 1-dodecanol, which has a high partition coefficient for butanol, can be prevented by employing 1-dodecanol as an extractant when using a PTFE membrane. Compared to conventional fermentation, MAE-ABE fermentation with 1-dodecanol decreased butanol inhibition and increased glucose consumption from 59.4 to 86.0 g/L, and total butanol production increased from 16.0 to 20.1g/L. The maximum butanol production rate increased from 0.817 to 0.979 g/L/h. The butanol productivity per membrane area was remarkably high with this system, i.e., 78.6g/L/h/m(2). Therefore, it is expected that this MAE fermentation system can achieve footprint downsizing.

Silage produces biofuel for local consumption.

BACKGROUND: In the normal process of bioethanol production, biomass is transported to integrated large factories for degradation to sugar, fermentation, and recovery of ethanol by distillation. Biomass nutrient loss occurs during preservation and degradation. Our aim was to develop a decentralized ethanol production system appropriate for farm or co-operative level production that uses a solid-state fermentation method for producing bio-ethanol from whole crops, provides cattle feed, and produces no wastes. The idea is to incorporate traditional silage methods with simultaneous saccharification and fermentation. Harvested, fresh biomass is ensiled with biomass-degrading enzymes and yeast. Multiple parallel reactions for biomass degradation and ethanol and lactic acid production are induced in solid culture in hermetically sealed containers at a ranch. After fermentation, ethanol is collected on site from the vapor from heated fermented products. RESULTS: The parallel reactions of simultaneous saccharification and fermentation were induced efficiently in the model fermentation system. In a laboratory-scale feasibility study of the process, 250 g of freshly harvested forage rice with 62% moisture was treated with 0.86 filter paper units/g dry matter (DM) of cellulase and 0.32 U/g DM of glucoamylase. After 20 days of incubation at 28°C, 6.4 wt.% of ethanol in fresh matter (equivalent to 169 g/kg DM) was produced. When the 46 wt.% moisture was gathered as vapor from the fermented product, 74% of the produced ethanol was collected. Organic cellular contents (such as the amylase and pronase degradable fractions) were decreased by 63% and organic cell wall (fiber) content by 7% compared to silage prepared from the same material. CONCLUSIONS: We confirmed that efficient ethanol production is induced in nonsterilized whole rice plants in a laboratory-scale solid-state fermentation system. For practical use of the method, further study is needed to scale-up the fermentation volume, develop an efficient ethanol recovery method, and evaluate the fermentation residue as an actual cattle feed.

Effect of glyceric acid calcium salt on the viability of ethanol-dosed gastric cells.

D-Glyceric acid (D-GA) calcium has been reported to accelerate ethanol oxidation in vivo in rats (Eriksson et al., Metabolism, 56, 895-898 (2007)). However, no other reports have shown that D-GA can reduce the harmful effects of ethanol. In this study, the effects of D-, L-, and DL-GA calcium on ethanol-dosed gastric cell viability were investigated using human gastric carcinoma cells (Kato III) and normal rat gastric mucosa cells (RGM1). Addition of 2% and 3 % ethanol to Kato III and RGM1 cells, respectively, decreased their cell viability by approximately 20-50 % after 24 or 72 h of cultivation. In 2 % ethanol-dosed Kato III cells cultivated for 24 h, addition of 0.002-20 µg/mL D- and L-GA calcium did not affect cell viability. Similarly, addition of less than 20 µg/mL DL-GA calcium did not affect cell viability. However, when 20 µg/mL DL-GA calcium was added, cell viability increased by 35.7 % after 72 h of incubation, compared to the viability of control cells without ethanol or GA. Addition of 20 µg/mL DL-GA calcium to 3 % ethanol-dosed RGM1 cells cultivated for 24 or 72 h also increased cell viability up to those observed in control cells. These results suggest that a racemic mixture of GA may have the strongest effect on enhancing the viability of ethanol-exposed cells.

Synthesis of dilinoleoyl-D-glyceric acid and evaluation of its cytotoxicity to human dermal fibroblast and endothelial cells.

A novel derivative of glyceric acid (GA), dilinoleoyl-D-glyceric acid (LA2-DGA), was synthesized from D-GA calcium salt and linoleoyl chloride and evaluated for cytotoxicity. The D-GA calcium salt was first reacted with 4-methoxybezylchloride, and the resulting compound was esterified with linoleoyl chloride. This reaction was followed by hydrolysis of the 4-methoxybenzyl moiety, yielding LA2-DGA. LA2-DGA was then subjected to cytotoxicity testing using normal human dermal fibroblasts and primary normal human dermal microvascular endothelial cells. LA2-DGA showed no significant toxic effects in either type of cell.

Membrane-bound alcohol dehydrogenase is essential for glyceric acid production in Acetobacter tropicalis.

Acetobacter tropicalis NBRC16470 can produce highly enantiomerically pure D-glyceric acid (D-GA; >99 % enantiomeric excess) from glycerol. To investigate whether membrane-bound alcohol dehydrogenase (mADH) is involved in GA production in A. tropicalis, we amplified part of the gene encoding mADH subunit I (adhA) using polymerase chain reaction and constructed an adhA-disrupted mutant of A. tropicalis (ΔadhA). Because ΔadhA did not produce GA, we confirmed that mADH is essential for the conversion of glycerol to GA. We also cloned and sequenced the entire region corresponding to adhA and adhB, which encodes mADH subunit II. The sequences showed high identities (84-86 %) with the equivalent mADH subunits from other Acetobacter spp.

Bioprocessing of glycerol into glyceric Acid for use in bioplastic monomer.

Utilization of excess glycerol supplies derived from the burgeoning biodiesel industry has recently become very important. Glyceric acid (GA) is one of the most promising glycerol derivatives, and it is abundantly obtained from glycerol by a bioprocess using acetic acid bacteria. In this study, a novel branched-type poly(lactic acid) (PLA) was synthesized by polycondensation of lactide in the presence of GA. The resulting branched PLA had lower crystallinity and glass transition temperatures than the conventional linear PLA, and the peak associated with the melting point of the branched PLA disappeared. Moreover, in a blend of the branched polymer, the crystallization of the linear PLA occurred at a lower temperature. Thus, the branched PLA containing GA synthesized in this study could potentially be used as a novel bio-based modifier for PLA.

Synthesis and evaluation of dioleoyl glyceric acids showing antitrypsin activity.

Previously, Lesová et al. reported the isolation and identification of metabolite OR-1, showing antitrypsin activity, produced during fermentation by Penicillium funiculosum. The structure of OR-1 was a mixture of glyceric acid (GA), esterified with C(14)-C(18) fatty acids, and oleic acid (C18:1) as the most predominant fatty acid (Folia Microbiol. 46, 21-23, 2001). In this study, dioleoyl D-GA and dioleoyl L-GA were synthesized via diesterification with oleoyl chloride, and their antitrypsin activities were evaluated using both a disk diffusion method and spectral absorption measurements. The results show that both compounds and their equivalent mixtures possess antitrypsin activities; however, their IC(50) values (approximately 2 mM) are much higher than that of OR-1 (4.25 µM), suggesting that dioleoyl GA does not play a major role in the OR-1 antitrypsin activity.

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol.

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.

Two-stage electrodialytic concentration of glyceric acid from fermentation broth.

The aim of this research was the application of a two-stage electrodialysis (ED) method for glyceric acid (GA) recovery from fermentation broth. First, by desalting ED, glycerate solutions (counterpart is Na+) were concentrated using ion-exchange membranes, and the glycerate recovery and energy consumption became more efficient with increasing the initial glycerate concentration (30 to 130 g/l). Second, by water-splitting ED, the concentrated glycerate was electroconverted to GA using bipolar membranes. Using a culture broth of Acetobacter tropicalis containing 68.6 g/l of D-glycerate, a final D-GA concentration of 116 g/l was obtained following the two-stage ED process. The total energy consumption for the D-glycerate concentration and its electroconversion to D-GA was approximately 0.92 kWh per 1 kg of D-GA.

Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans.

Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant DeltaadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. DeltaadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting DeltaadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on DeltaadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

Microbial production of glyceric acid, an organic acid that can be mass produced from glycerol.

Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

Glycerol conversion to D-xylulose by a two-stage microbial reaction using Candida parapsilosis and Gluconobacter oxydans.

A yeast strain, 25N-2B, that produces D-arabitol from glycerol, was identified as Candida parapsilosis based on phylogenetic, morphological, physiological, and biochemical analyses. It produced 32.2 g/L D-arabitol from 170 g/L glycerol in a jar fermentor. The D-arabitol in the reaction mixture was then completely converted to D-xylulose using Gluconobacter oxydans NBRC3293. The product was isolated from the reaction mixture and confirmed to be D-xylulose by (1)H and (13)C-NMR and optical rotation.

Production of glyceric acid by Gluconobacter sp. NBRC3259 using raw glycerol.

Gluconobacter sp. NBRC3259 converted glycerol to glyceric acid (GA). The enantiomeric composition of the GA produced was a mixture of DL-forms with a 77% enantiomeric excess of D-GA. After culture conditions, such as initial glycerol concentration, types and amounts of nitrogen sources, and initial pH, were optimized, Gluconobacter sp. NBRC3259 produced 54.7 g/l of GA as well as 33.7 g/l of dihydroxyacetone (DHA) from 167 g/l of glycerol during 4 d of incubation in a jar fermentor with pH control. GA production from raw glycerol samples, the main by-product of the transesterification process in the biodiesel production and oleochemical industries, was also evaluated after proper pretreatment of the samples. Using a raw glycerol sample with activated charcoal pretreatment, 45.9 g/l of GA and 28.2 g/l of DHA were produced from 174 g/l of glycerol.

Biotechnological production of D-glyceric acid and its application.

Glycerol is currently produced in large amounts as a by-product during fat splitting and biodiesel fuel production. Over the past decade, both chemical and biotechnological processes to convert glycerol to value-added chemicals have been increasingly explored. This mini-review provides recent information about the biotechnological production of a glycerol derivative, D-glyceric acid (D-GA), and its possible applications. Little is known about GA as a bioproduct, but it is naturally found in different kinds of plants as a phytochemical constituent and is reported to have some biological activity. A racemic mixture of DL-GA can be obtained from glycerol via chemical oxidation; however, D-GA is mainly biotechnologically produced with the aid of bacteria. Under aerobic conditions, some acetic acid bacteria transform glycerol into D-GA, and optimization of initial glycerol concentration and aeration rate provided a yield of more than 80 g/l D-GA, using a strain of Gluconobacter frateurii.

Application of electrodialysis to glycerate recovery from a glycerol containing model solution and culture broth.

Glyceric acid is produced by the conversion of glycerol via bioprocesses. The glycerate recovery from model solutions and from real culture broth was demonstrated by a desalting electrodialysis (ED) method. The addition of several impurities in glycerate model solutions, such as polypepton or yeast extract, did not have significant adverse effects on the whole ED process, and more than 93% of the glycerol added in the model solutions (50-150 g/l) was excluded. Using culture broth of Acetobacter tropicalis containing 14.6 g/l D-glycerate, the D-glycerate recovery and the energy consumption were 99.4% and 0.24 kWh/kg, respectively.

Detection of acetyl monoglyceride as a metabolite of newly isolated glycerol-assimilating bacteria.

Thirty-five glycerol-assimilating bacteria have recently been isolated from soil samples. Amplified ribosomal DNA restriction analysis revealed that these strains are grouped into four genetically different types of bacteria. Gas chromatography-mass spectrometry analysis of glycerol metabolites produced by the three selected strains (strains HH7, HH12, and HH31) revealed that extracts of culture liquid with ethyl acetate contains acetyl monoglyceride (monoacetin), which has not previously been reported as a glycerol metabolite and is used as a solvent, plasticizer, and food additive, as well as for other industrial purposes. The sequence analyses of the 16S rRNA genes from the selected strains showed that all of them belong to the Enterobacteriaceae.