Microwave-Assisted Two-Step Liquefaction of Acetone-Soluble Lignin of Silvergrass Saccharification Residue for Production of Biopolyol and Biopolyurethane.

The application of microwave heating facilitated efficient two-step liquefaction of acetone-soluble lignin obtained from saccharification residue of Miscanthus sacchariflorus (silvergrass), which was prepared by enzymatic hydrolysis, to produce biopolyol with a low acid number and favorable hydroxyl number. The acetone-soluble lignin was liquefied using a crude glycerol and 1,4-butanediol solvent mixture at various solvent blending ratios, biomass loadings, acid loadings, and reaction temperatures. The optimal reaction condition was determined at a solvent blending ratio of crude glycerol to 1,4-butanediol of 1:2, 20% of biomass loading, and 1% of catalyst loading at a reaction temperature of 140 °C for 10 min. Subsequently, the optimal biopolyol was directly used for the preparation of biopolyurethane foam as a value-added product. The chemical and physical properties of biopolyurethane foams derived from acetone-soluble lignin were characterized by Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), and high-resolution scanning electron microscopy (HR-SEM). In addition, mechanical properties of produced biopolyurethane foams, including compressive strength and density, were also characterized to suggest their appropriate applications. The results indicated that the biopolyurethane foam can be used as a green replacement for petroleum-based polyurethane foam due to its comparable thermal properties, mechanical strength, and morphological structure.

Cellulase recycling in high-solids enzymatic hydrolysis of pretreated empty fruit bunches.

BACKGROUND: The lignocellulosic biomass feedstocks such as empty fruit bunches (EFBs) prove to be potential renewable resources owing to their abundance, low prices, and high carbohydrate contents. Generally, the conversion of lignocellulosic biomass into chemicals, fuels, and materials mainly includes pretreatment, enzymatic hydrolysis, fermentation, and recovery of final products. To increase the economic viability of such processes, the cost of cellulase production and enzymatic hydrolysis should be reduced. For this, recycling cellulase can be considered for reducing the saccharification cost of lignocellulose. In this study, cellulase recycling for high-solids enzymatic hydrolysis (i.e., 20%) was evaluated in saccharification of hydrothermally-pretreated EFBs. RESULTS: High-solids (20%) enzymatic hydrolysis of hydrothermally-pretreated empty fruit bunches with 40 FPU of Cellic CTec3/g glucan was carried out for cellulase recycling. In the second round of hydrolysis using a recycled enzyme, only 19.3% of glucose yield was obtained. The most important limiting factors for cellulase recycling of this study were identified as the enzyme inhibition by glucose, the loss of enzyme activities, and the non-productive binding of enzymes to insoluble biomass solids. To overcome these limitations, PEG was added prior to the first-round hydrolysis to reduce non-productive enzyme binding, glucose was removed from the enzyme fraction to be reused in the second-round hydrolysis, and EFB solids from the first-round hydrolysis were used in the second-round hydrolysis. These three additional measures with cellulase recycling resulted in a 3.5 times higher glucose yield (i.e., 68.0%) at the second round than that of the control, the second-round hydrolysis with cellulase recycling but without these measures. CONCLUSIONS: Because of the high obstacles found in this  study in achieving high saccharification yields in the high-solids saccharification of high-lignin lignocellulose with cellulase recycling, effective measures for improving enzymatic saccharification yields need to be accompanied with cellulase recycling.

Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum strains from empty fruit bunch biosugar solution.

BACKGROUND: Recent interest has been focused on the production of platform chemicals from renewable biomass due to increasing concerns on global warming and depletion of fossil fuel reserves. Microbial production of platform chemicals in biorefineries has been suggested to be a promising solution for these problems. Gamma-aminobutyrate (GABA), a versatile bulk chemical used in food and pharmaceutical industry, is also used as a key monomer for nylon 4. GABA can be biologically produced by decarboxylation of glutamate. RESULTS: In this study, we examined high glutamate-producing Corynebacterium glutamicum strains as hosts for enhanced production of GABA from glucose and xylose as carbon sources. An Escherichia coli gadB mutant with a broad pH range of activity and E. coli xylAB genes were expressed under the control of a synthetic H36 promoter. When empty fruit bunch (EFB) solution was used as carbon source (45 g/L glucose and 5 g/L xylose), 12.54 ± 0.07 g/L GABA was produced by recombinant C. glutamicum H36GD1852 expressing E. coli gadB mutant gene and xylAB genes. Batch fermentation of the same strain resulted in the production of 35.47 g/L of GABA when EFB solution was added to support 90 g/L glucose and 10 g/L xylose. CONCLUSIONS: This is the first report of GABA production by recombinant C. glutamicum strains from co-utilization of glucose and xylose from EFB solution. Recombinant C. glutamicum strains developed in this study should be useful for an efficient and sustainable production of GABA from lignocellulosic biomasses.

Production of 5-aminovaleric acid in recombinant Corynebacterium glutamicum strains from a Miscanthus hydrolysate solution prepared by a newly developed Miscanthus hydrolysis process.

This study examined nine expired industrial Corynebacterium glutamicum strains with high lysine producing capability for enhanced production of 5-AVA. C. glutamicum KCTC 1857 exhibiting the highest lysine production was transformed with either original Pseudomonas putida davBA genes, encoding the 5-AVA biosynthesis pathway, or C. glutamicum codon-optimized davBA genes. C. glutamicum KCTC 1857 expressing the original genes had superior cell viability and 5-AVA production capability compared to the other strain. This strain produced 39.93g/L of 5-AVA, which is the highest titer reported to date in fed-batch fermentation from glucose. Indeed, Miscanthus hydrolysate solution prepared from a novel process, comprising pretreatment, hydrolysis, purification, and concentration, was used as feedstock for 5-AVA production. A total of 12.51g/L 5-AVA was produced from the Miscanthus hydrolysate; this value is 34.7% higher than that obtained from glucose in batch fermentation.

Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

BACKGROUND: Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. RESULTS: Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is similar to the initial concentration of xylose during the fed-batch fermentation suggesting that xylose consumption is not inhibited during fermentation. Finally, recombinant R. eutorpha NCIMB11599 expressing the E. coli xylAB gene was examined for the production of P(3HB) from the hydrolysate solution of sunflower stalk. The hydrolysate solution of sunflower stalk was prepared as a model lignocellulosic biomass, which contains 78.8 g/L of glucose, 26.9 g/L of xylose, and small amount of 4.8 g/L of galactose and mannose. When recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes was cultured in a nitrogen limited chemically defined medium containing 23.1 g/L of hydrolysate solution of sunflower stalk, which corresponds to 16.8 g/L of glucose and 5.9 g/L of xylose, it completely consumed glucose and xylose in the sunflower stalk based medium resulting in the production of 7.86 g/L of P(3HB) with a P(3HB) content of 72.53 wt%. CONCLUSIONS: Ralstonia eutropha was successfully engineered to utilize xylose as a sole carbon source as well as to co-utilize it in the presence of glucose for the synthesis of P(3HB). In addition, R. eutropha engineered to utilized xylose could synthesize P(3HB) from the sunflower stalk hydrolysate solution containing glucose and xylose as major sugars, which suggests that xylose utilizing R. eutropha developed in this study should be useful for development of lignocellulose based microbial processes.

Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.

An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein.

Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source.

In this study, a biosugar obtained from empty fruit bunch (EFB) of oil palm by hot water treatment and subsequent enzymatic saccharification was used for undecylprodigiosin production, using Streptomyces coelicolor. Furfural is a major inhibitor present in EFB hydrolysate (EFBH), having a minimum inhibitory concentration (MIC) of 1.9mM, and it reduces utilization of glucose (27%), xylose (59%), inhibits mycelium formation, and affects antibiotic production. Interestingly, furfural was found to be a good activator of undecylprodigiosin production in S. coelicolor, which enhanced undecylprodigiosin production by up to 52%. Optimization by mixture analysis resulted in a synthetic medium containing glucose:furfural:ACN:DMSO (1%, 2mM, 0.2% and 0.3%, respectively). Finally, S. coelicolor was cultured in a fermenter in minimal medium with EFBH as a carbon source and addition of the components described above. This yielded 4.2µg/mgdcw undecylprodigiosin, which was 3.2-fold higher compared to that in un-optimized medium.

Use of mechanical refining to improve the production of low-cost sugars from lignocellulosic biomass.

Mechanical refining is widely used in the pulp and paper industry to enhance the end-use properties of products by creating external fibrillation and internal delamination. This technology can be directly applied to biochemical conversion processes. By implementing mechanical refining technology, biomass recalcitrance to enzyme hydrolysis can be overcome and carbohydrate conversion can be enhanced with commercially attractive levels of enzymes. In addition, chemical and thermal pretreatment severity can be reduced to achieve the same level of carbohydrate conversion, which reduces pretreatment cost and results in lower concentrations of inhibitors. Refining is versatile and a commercially proven technology that can be operated at process flows of ∼ 1500 dry tons per day of biomass. This paper reviews the utilization of mechanical refining in the pulp and paper industry and summarizes the recent development in applications for biochemical conversion, which potentially make an overall biorefinery process more economically viable.

Efficient ethanol production from dried oil palm trunk treated by hydrothermolysis and subsequent enzymatic hydrolysis.

BACKGROUND: Oil palm trunk (OPT) is a valuable bioresource for the biorefinery industry producing biofuels and biochemicals. It has the distinct feature of containing a large amount of starch, which, unlike cellulose, can be easily solubilized by water when heated and hydrolyzed to glucose by amylolytic enzymes without pretreatment for breaking down the biomass recalcitrance. Therefore, it is suggested as beneficial to extract most of the starch from OPT through autoclaving and subsequent amylolytic hydrolysis prior to pretreatment. However, this treatment requires high capital and operational costs, and there could be a high probability of microbial contamination during starch processing. In terms of biochemical conversion of OPT, this study aimed to develop a simple and efficient ethanol conversion process without any chemical use such as acids and bases or detoxification. RESULTS: For comparison with the proposed efficient ethanol conversion process, OPT was subjected to hydrothermal treatment at 180 °C for 30 min. After enzymatic hydrolysis of PWS, 43.5 g of glucose per 100 g dry biomass was obtained, which corresponds to 81.3 % of the theoretical glucose yield. Through subsequent alcohol fermentation, 81.4 % ethanol yield of the theoretical ethanol yield was achieved. To conduct the proposed new process, starch in OPT was converted to ethanol through enzymatic hydrolysis and subsequent fermentation prior to hydrothermal treatment, and the resulting slurry was subjected to identical processes that were applied to control. Consequently, a high-glucose yield of 96.3 % was achieved, and the resulting ethanol yield was 93.5 %. CONCLUSIONS: The proposed new process was a simple method for minimizing the loss of starch during biochemical conversion and maximizing ethanol production as well as fermentable sugars from OPT. In addition, this methodology offers the advantage of reducing operational and capital costs due to minimizing the process for ethanol production by excluding expensive processes related to detoxification prior to enzymatic hydrolysis and fermentation such as washing/conditioning and solid-liquid separation of pretreated slurry. The potential future use of xylose-digestible microorganisms could further increase the ethanol yield from the proposed process, thereby increasing its effectiveness for the conversion of OPT into biofuels and biochemicals.

Fermentative l-lactic acid production from pretreated whole slurry of oil palm trunk treated by hydrothermolysis and subsequent enzymatic hydrolysis.

A simple and cost-effective biochemical conversion process consisting of hydrothermal treatment, enzymatic hydrolysis and fermentation of pretreated whole slurry (PWS) was developed for producing l-lactic acid (L-LA) from oil palm trunk (OPT). When OPT was hydrothermally treated at optimal condition capable of achieving maximum yield of hemicellulosic sugars after enzymatic hydrolysis, the enzymatic digestibility of the PWS afforded a yield of 81.4% of the theoretical glucose yield (TGY). However, glucose yield from washed pretreated solid (WPS) was only 43.5% of TGY. The use of two hydrolysates from PWS and WPS for fermentation by Lactobacillus paracasei engineered to selectively produce L-LA afforded yields of 89.5% and 45.8% of the theoretical LA yield (TLY), respectively. This study confirmed the inevitable extensive sugar loss during washing of pretreated slurry due to loss of soluble starch. Alternatively, the proposed design process is considered suitable for converting OPT to L-LA without such starch loss.

Development of rice bran treatment process and its use for the synthesis of polyhydroxyalkanoates from rice bran hydrolysate solution.

Rice bran treatment process for the production of 43.7 kg of hydrolysate solution containing 24.41 g/L of glucose and small amount of fructose from 5 kg of rice bran was developed and employed to produce polyhydroxyalkanoates in recombinant Escherichia coli and Ralstonia eutropha strains. Recombinant E. coli XL1-Blue expressing R. eutropha phaCAB genes and R. eutropha NCIMB11599 could produce poly(3-hydroxybutyrate) with the polymer contents of 90.1 wt% and 97.2 wt%, respectively, when they were cultured in chemically defined MR medium and chemically defined nitrogen free MR medium containing 10 mL/L of rice bran hydrolysate solution, respectively. Also, recombinant E. coli XL1-Blue and recombinant R. eutropha 437-540, both of which express the Pseudomonas sp. phaC1437 gene and the Clostridium propionicum pct540 gene could produce poly(3-hydroxybutyrate-co-lactate) from rice bran hydrolysate solution. These results suggest that rice bran may be a good renewable resource for the production of biomass-based polymers by recombinant microorganisms.

Sugar yields from sunflower stalks treated by hydrothermolysis and subsequent enzymatic hydrolysis.

To produce fermentable sugar with fewer microbial inhibitors via few processes, batch-type hydrothermal treatments of sunflower stalks were performed, followed by enzymatic hydrolysis of pretreatment slurries with Cellic CTec2 and Cellic HTec2 (9:1, v/v, 0.1 ml/g dry biomass, 8.1FPU). The yields of hemicellulosic sugars were maximized at the pretreatment condition of 180°C for 30 min, while the furfural and 5-hydroxymethyl-2-furaldehyde (HMF) concentrations remained low. The glucose yield, however, was only 67.0% of the theoretical glucose yield (TGY). Either the treatment of raw biomass prior to pretreatment or the post-treatment of pretreated residue prior to enzymatic hydrolysis increased the glucose yield as follows: washing the pretreated solid with solvents (90% TGY)>partial removal of liquid fraction from the pretreatment slurry (PS, 83%)>removal of hot-water extractives from the biomass prior to pretreatment (77%)>prewetting of the biomass (70%).

Effect of additives in aqueous formulation on the foliar uptake of dimethomorph by cucumbers.

BACKGROUND: The efficacy enhancement of dimethomorph formulation by several adjuvants is thought to be through increased foliar uptake. In order to identify the most effective adjuvants, the adjuvancy of 36 additives was examined in aqueous formulations in relation to the absorption of dimethomorph by cucumber leaves. RESULTS: Polyethylene glycol monohexadecyl ethers with ethylene oxide (EO) contents of between 7 and 20, polyethylene glycol monooctadecyl ethers with EO contents of between 10 and 20 and polyethylene glycol monooctadecenyl ethers with EO contents of between 6 and 20 were effective adjuvants for promoting dimethomorph uptake from both aqueous acetone solutions and aqueous wettable powder (WP) suspensions into cucumber leaves. Polyethylene glycol monododecyl ethers with EO contents of between 7 and 9 were effective in promoting dimethomorph uptake from aqueous WP suspensions but less effective relative to the other adjuvants tested with aqueous acetone solutions. Foliar uptake of dimethomorph was also facilitated by the addition of methyl hexadecanoate, methyl octadecenoate and methyl octadecadienoate. CONCLUSIONS: Although the foliar uptake of dimethomorph from both aqueous WP suspensions and aqueous acetone solutions was greatest in the presence of fatty alcohol ethoxylates generally having a C(16) or C(18) lipophile, uptake from aqueous surfactant-acetone solutions was, on average, 7.6-fold greater than that from aqueous WP suspensions containing surfactant.

Co-lyophilization with D-proline greatly enhances peroxidase's stereoselectivity in a non-aqueous medium.

The impact of co-lyophilizing horseradish peroxidase (HRP) with numerous amino acids and other chiral excipients on the enzyme's subsequent stereoselectivity [E(S/R)] in the sulfoxidation of thioanisole in 2-propanol was systematically investigated. While many improved the stereoselectivity of (and significantly activated) HRP, the greatest effect was observed with D-proline which enhanced the E(S/R) value by over an order of magnitude from synthetically meaningless to useful.

Activation of protein kinase C by 1-beta-D-arabinofuranosylcytosine conjugates of phospholipid.

1-beta-D-arabinofuranosylcytosine (ara-C) conjugates of phospholipid were shown to be highly antineoplastic against various tumor cells. In this study, we report that these conjugates are potent activators of protein kinase C (PKC, EC) in vitro. Although required Ca2+, PKC activation by the conjugates occurred even in the absence of phospholipid and diacylglycerol. Among the conjugates, 1-beta-D-arabino-furanosylcytosine 5'-diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol [(ara-CDP-DL-PBA); ara-C conjugate of ether phospholipid], was employed to investigate its mode of activation, since ether phospholipid has been reported to be a regulator of the PKC. When PKC was activated by ara-CDP-DL-PBA, diacylglycerol enhanced its activity with 3-fold reduction of an apparent Ka value for ara-CDP-DL-PBA and no change in the Vmax. During the PKC activation by phosphatidylserine, ara-CDP-DL-PBA exhibited a synergistic effect on the activation. Studies on the relationship between the structures of ara-CDP-DL-PBA and their effects on PKC activity showed that phosphate group of ether lipid was important for its activation of PKC, and that conjugation of ara-C and ether lipid further enhanced the enzyme activity. These results suggest that the ara-C conjugate of phospholipid activates PKC in a co-operative manner with diacylglycerol and/or phosphatidylserine, however, the exact mechanism of the antineoplastic effect of ara-CDP-DL-PBA through PKC activation still remains speculative.

Characteristics of the squalene synthase inhibitors produced by a Streptomyces species isolated from soils.

Microorganisms producing squalene synthase inhibitors were screened from soils. A high producer was selected and identified as a Streptomyces species. Two active inhibitors were obtained from culture broths via a series of purification processes involving solvent extraction, WK-10 cation-exchange column chromatography, HP-20 adsorption column chromatography, silica-gel column chromatography, preparative HPLC, and crystallization. The inhibitors were confirmed as macrolactins A and F with molecular weights of 402 by UV-absorption spectrometry, fast atom bombardment mass spectometry, and 13C- and 1H-NMR analyses. Kinetic results for macrolactins A and F showed that they appear to be noncompetitive inhibitors of rat liver squalene synthase with IC50 values of 1.66 and 1.53 micromol/L, respectively. Since mammalian squalene synthase was used, these inhibitors have significant potential as therapeutic agents for hyperlipemia and suppression of cholesterol biosynthesis.