Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 µmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Unrevealing the influence of reagent properties on disruption and digestibility of lignocellulosic biomass during alkaline pretreatment.

Beyond the conventional consideration of pretreatment severity (PS) responsible for biomass disruption, the influence of reagent properties on biomass (LCB) disruption is often overlooked. To investigate the LCB disruption as a function of reagent properties, reagents with distinct cations (NaOH and KOH) and significantly higher delignification potential were chosen. NaOH solution (3 % w/v) with a measured pH of 13.05 ± 0.01 is considered the reference, against which a KOH solution (pH = 13.05 ± 0.01) was prepared for LCB pretreatment under the same PS. Despite comparable lignin content, varying glucose yield of NaOH (68.76 %) and KOH (46.88 %) pretreated residues indicated the presence of heterogeneously disrupted substrate. Holocellulose extracted from raw poplar (ASC, control) and alkaline pretreated residues (C-NaOH and C-KOH) were analyzed using HPLC, XRD, SEM, TGA/DTG, XPS, and 13CP MAS NMR to investigate the pretreatment-induced structural modification. Results revealed that, despite the same pretreatment severity, better disruption in C-NaOH (higher accessible fibril surface and less-ordered region) leading to higher digestibility than C-KOH, likely due to the smaller ionic radius of Na+, facilitates better penetration into dense LCB matrix. This study elucidates the importance of considering the reagent properties during LCB pretreatment, eventually enhancing consciousness while selecting reagents for efficient LCB utilization.

Unraveling interplay between lignocellulosic structures caused by chemical pretreatments in enhancing enzymatic hydrolysis.

To investigate the interplay between substrate structure and enzymatic hydrolysis (EH) efficiency, poplar was pretreated with acidic sodium-chlorite (ASC), 3 % sodium-hydroxide (3-SH), and 3 % sulfuric acid (3-SA), resulting in different glucose yields of 94.10 %, 74.35 %, and 24.51 %, respectively, of pretreated residues. Residues were fractionated into cellulose, lignin and unhydrolyzed residue after EH (for lignin-carbohydrate complex (LCC) analysis) and analyzed using HPLC, FTIR, XPS, CP MAS 13C NMR and 2D-NMR (Lignin and LCC analysis). After delignification, holocellulose exhibited a dramatic increase in glucose yield (74.35 % to 90.82 % for 3-SH and 24.51 % to 80.0 % for 3-SA). Structural analysis of holocellulose suggested the synergistic interplay among cellulose allomorphs to limit glucose yield. Residual lignin analysis from un/pretreated residues indicated that higher ß-ß' contents and S/G ratios were favorable to the inhibitory effect but unfavourable to the holocellulose digestibility and followed the trend in the following order: 3-SA (L3) > 3-SH (L2) > native-lignin (L1). Analysis of enzymatically unhydrolyzed pretreated residues revealed the presence of benzyl ether (BE1,2) LCC and phenyl glycoside (PG) bond linking to xylose (X) and mannose (M), which yielded a xylan-lignin-glucomannan network. The stability, steric hindrance and hydrophobicity of this network may play a central role in defining poplar recalcitrance.

Simultaneous saccharification of hemicellulose and cellulose of corncob in a one-pot system using catalysis of carbon based solid acid from lignosulfonate.

The drive towards sustainable chemistry has inspired the development of active solid acids as catalysts and ionic liquids as solvents for an efficient release of sugars from lignocellulosic biomass for future biorefinery practices. Carbon-based solid acid (SI-C-S-H2O2) prepared from sodium lignosulfonate, a waste of the paper industry, was used with water or ionic liquid to hydrolyze corncob in this study. The effects of various reaction parameters were investigated in different solvent systems. The highest xylose yield of 83.4% and hemicellulose removal rate of 90.6% were obtained in an aqueous system at 130 °C for 14 h. After the pretreatment, cellulase was used for the hydrolysis of residue and the enzymatic digestibility of 92.6% was obtained. Following these two hydrolysis steps in the aqueous systems, the highest yield of total reducing sugar (TRS) was obtained at 88.1%. Further, one-step depolymerization and saccharification of corncob hemicellulose and cellulose to reducing sugars in an IL-water system catalyzed by SI-C-S-H2O2 was conducted at 130 °C for 10 h, with a high TRS yield of 75.1% obtained directly. After recycling five times, the solid acid catalyst still showed a high catalytic activity for sugar yield in different systems, providing a green and effective method for lignocellulose degradation.

Life cycle environmental benefits of recycling waste liquor and chemicals in production of lignocellulosic bioethanol.

In recent decades, numerous bioconversion processes and techniques have been developed to utilize lignocellulosic biomass as feedstock in the production of bio-based fuels and materials. However, waste treatment, an important sub-system, is seldom considered in the life cycle assessment of lignocellulose derived products. This study comprehensively investigated the environmental impacts of bioethanol and electricity cogeneration from sugarcane bagasse, with a focus on recycling techniques adopted in waste treatment. A life cycle assessment indicated that high recycle rate of black liquor, acid and waste washing water can substantially reduce the consumption of fresh water, related chemicals and energy by 70-80%. Environmental impacts relating to global warming, acidification potential and primary energy demand can be decreased by 5-10 times or even entirely eliminated. These study outcomes demonstrate significant environmental benefits of integrating waste recycling techniques into lignocellulose biorefinery process, providing a solid foundation for future industrial development.

Combination of surfactants and enzyme cocktails for enhancing woody biomass saccharification and bioethanol production from lab-scale to pilot-scale.

Converting woody biomass to bioethanol might be more affordable, environmentally friendly, and efficient for making biofuel commercially feasible, but it would still need a significant optimization process and expand pilot-scale research. A combination of commercial low enzymes loading at 10 FPU/g glucan and compound additives utilizing Tween 80, PEG8000 and sophorolipid applied from lab-scale to pilot-scale have been studied in this work at economically viable dosages for enhancing bioethanol production. In lab-scale saccharification and fermentation, pretreated poplar at a high solid loading of 20% yielded the highest ethanol titers of 30.96 g/L and theoretical ethanol yield of 92.79%. Additionally, pilot-scale operation was used to investigate the bioethanol amplification, a final volume of 33 m3 which yielded the greatest ethanol amount of 599.6 kg from poplar wood while gaining on-site value-added production of hemicellulosic and cellobiose liquor 1122 kg and lignin residues 2292 kg.

N-glycosylation as an effective strategy to enhance characteristics of Rhizomucor miehei lipase for biodiesel production.

The construction of methanol-resistant lipases with high catalytic activity is world-shattering for biodiesel production. A semi-rational method has been constructed to enhance the properties of Rhizomucor miehei lipase with propeptide (ProRML) by introducing N-glycosylation sites in the Loop structure. The enzyme activities of the mutants N288 (1448.89 ± 68.64 U/mg) and N142 (1073.68 ± 33.87 U/mg) increased to 56.09 and 41.56 times relative to that of wild type ProRML (WT, 25.83 ± 0.73 U/mg), respectively. After incubation in 50 % methanol for 2.5 h, the residual activities of N314 and N174-1 were 95 % and 85%, which were higher than the WT (27 %). Additionally, the biodiesel yield of all mutants was increased after a one-time addition of methanol for 24 h. Among them, N288 increased the quantity of biodiesel from colza oil from 9.49 % to 88 %, and N314 increased the amount of biodiesel from waste soybean oil from 8.44% to 70%. This study provides an effective method to enhance the properties of lipase and improve its application potential in biodiesel production.

Impact of Alkaline Pretreatment Condition on Enzymatic Hydrolysis of Sugarcane Bagasse and Pretreatment Cost.

A combined severity factor (RCSF) which is usually used to evaluate the effectiveness of hydrothermal pretreatment at above 100 °C had been developed to assess the influence of temperature, time, and alkali loading on pretreatment and enzymatic hydrolysis of lignocellulose. It is not suitable for evaluating alkaline pretreatment effectiveness at lower than 100 °C. According to the reported deducing process, this study modified the expression of [Formula: see text] as [Formula: see text] which is easier and more reasonable to assess the effectiveness of alkaline pretreatment. It showed that RCSF exhibited linear trend with lignin removal, and quadratic curve relation with enzymatic hydrolysis efficiency (EHE) at the same temperature. The EHE of alkali-treated SCB could attain the maximum value at lower RCSF, which indicated that it was not necessary to continuously enhance strength of alkaline pretreatment for improving EHE. Within a certain temperature range, the alkali loading was more important than temperature and time to influence pretreatment effectiveness and EHE. Furthermore, the contribution of temperature, time, and alkali loading to pretreatment cost which was seldom concerned was investigated in this work. The alkali loading contributed more than 70% to the pretreatment cost. This study laid the foundation of further optimizing alkaline pretreatment to reduce cost for its practical application.

Improving ß-glucosidase and xylanase production in a combination of waste substrate from domestic wastewater treatment system and agriculture residues.

Cellulase and hemicellulase activities are considered to the major bottlenecks in the lignocellulosic biorefinery process, especially in an enzyme cocktail lacking ß-glucosidase (BGL) and xylanase (XYL). In view of this issue, higher levels of BGL and XYL activities were obtained in the presence of wastewater and activated sludge as an induction medium mixed with 5% of rice straw by Hypocrea sp. W63. The analysis of the ionic content showed that a relatively low sludge dose could enhance the production of BGL and XYL. Most importantly, compared to a medium using freshwater, the proportion of 1:10 sludge to wastewater, which contained nutrient elements, led to 3.4-fold BGL and 3.7-fold XYL production improvements. This research describes the reuse of substrates that are largely and continuously generated from domestic wastewater treatment systems and agriculture residues, which consequently leads to the development of a simultaneous enzyme production process for sustainable biorefinery practices.

Comparative study on the properties of lignin isolated from different pretreated sugarcane bagasse and its inhibitory effects on enzymatic hydrolysis.

Five sugarcane bagasse lignin samples, namely, dilute sulfuric acid (DSAL), sodium hydroxide (SHL), ethanol (EL), hot liquid water (HLWL)-pretreated residual solids, and raw material (cellulolytic enzyme lignin, CEL), were extracted. Comparative studies on the physicochemical properties of isolated lignin, nonproductive adsorption of cellulase by lignin, and its effect on enzymatic hydrolysis was performed. Results showed that the molecular weight and homogeneity of lignin remarkably decreased after pretreatment compared with CEL. Lignin with low negative zeta potential, high phenolic hydroxyl group content and hydrophobicity exhibited strong nonproductive adsorption performance to cellulase. This phenomenon was positively correlated with it's inhibitory effect on enzymatic hydrolysis. Compared with the control (without lignin), the Avicel conversion rate (40 mg lignin/200 mg Avicel) decreased by 10.74%, 9.28%, 8.73%, 4.22%, and 2.80% after digestion of Avicel for 72 h with the presence of EL, SHL, CEL, HLWL, and DSAL, respectively.

Lignin prepared from different alkaline pretreated sugarcane bagasse and its effect on enzymatic hydrolysis.

Sugarcane bagasse (SCB) was pretreated by sodium hydroxide (SH), alkaline ethanol (AE) and alkaline hydrogen peroxide (AHP), and the solid residue with similar lignin content after different pretreatment was selected. Enzymatic saccharification with different substrates was performed at 2%, 10%, and 20% (w/v) solid loading. After that, the lignin from different substrate was extracted and its structure was characterized. Furthermore, the adsorption capability of isolated lignin to cellulase and its effect on enzymatic hydrolysis were studied. The results showed that, as the substrate content increased from 2% (w/v) to 20% (w/v), the glucose yield after digestion of SH, AE, and AHP pretreated SCB reduced by 41.8%, 35.4%, and 28.7%, respectively. The inhibitory effect of different prepared lignin on the digestibility of Avicel was as follows, SH pretreated lignin (SHL) > cellulase enzymatic hydrolysis lignin (CEL) > AE pretreated lignin (AEL) > AHP pretreated lignin (AHPL), which exhibited positive correlation with its non-productive adsorption capability to cellulase. Lignin samples with low negative zeta potential, high hydrophobicity and high ration of syringyl (S) to guaiacyl (G) were unfavorable for the enzymatic saccharification of cellulose.

Enhanced Enzymatic Hydrolysis of Corncob by Synthesized Enzyme-Mimetic Magnetic Solid Acid Pretreatment in an Aqueous Phase.

A novel magnetic carbon-based solid acid catalyst (C350-Cl) was synthesized through a simple impregnation-carbonization process and used for the pretreatment of corncob in an aqueous medium. Under the optimized pretreatment reaction conditions, the yield of pentose reached 91.6% with a hemicellulose removal rate of 91.7%, and the subsequent enzymatic digestibility of the pretreated corncob residue reached 90.0% at 48 h. C350-Cl is a magnetic enzyme-mimetic solid acid catalyst, and its catalytic behavior is similar to those of enzymes. In addition, the catalyst is also an excellent carrier for Fe and Cl in that the Fe3+ and Cl-can be released slowly in the pretreatment to assist the hydrolysis of lignocellulose. Compared with the traditional method with other catalysts, this hydrolysis process is suitable for the effective and sustainable saccharification of lignocellulose for producing fermentable sugar.

A study of CO/syngas bioconversion by Clostridium autoethanogenum with a flexible gas-cultivation system.

Bioconversion of CO/syngas to produce ethanol is a novel route in bioethanol production, which can be accomplished by some acetogens. Specific culture vessels and techniques are needed to cultivate these microorganisms since they are anaerobic and substrates are gaseous. In this work, gas-sampling bag was applied as a gas-cultivation system to study CO/syngas bioconversion by Clostridium autoethanogenum and was demonstrated to be efficient because of its flexibility and excellent ability to maintain the headspace atmosphere. C. autoethanogenum can use CO as the sole carbon and energy source to produce ethanol, acetate as well as CO2. In the experimental range, higher ethanol production was favored by higher yeast extract concentrations, and the maximum ethanol concentration of 3.45g/L was obtained at 1.0g/L of yeast extract. Study with various bottled gases showed that C. autoethanogenum preferred to use CO other than CO2 and produced the highest level of ethanol with 100% CO as the substrate. C. autoethanogenum can also utilize biomass-generated syngas (36.2% CO, 23.0% H2, 15.4% CO2, 11.3% N2), but the process proceeded slowly and insufficiently due to the presence of O2 and C2H2. In our study, C. autoethanogenum showed a better performance in the bioconversion of CO to ethanol than Clostridium ljungdahlii, a strain which has been most studied, and for both strains, ethanol production was promoted by supplementing 0.5g/L of acetate.

Production of d-psicose from d-glucose by co-expression of d-psicose 3-epimerase and xylose isomerase.

d-Psicose has been drawing increasing attention in recent years because of its medical and health applications. The production of d-psicose from d-glucose requires the co-expression and synergistic action of xylose isomerase and d-psicose 3-epimerase. To co-express these genes, vector pET-28a(+)-dual containing two T7 promoters and RBS sites and an Multiple Cloning Sites was constructed using the Escherichia coli expression plasmid pET-28a(+). The xylose isomerase gene from E. coli MG1665 and the d-psicose 3-epimerase gene from Agrobacterium tumefaciens CGMCC 1.1488 were cloned and co-expressed in E. coli BL21(DE3). After 24h incubation with the dual enzyme system at 40°C, the sugar conversion ratio from d-glucose to d-psicose reached 10%. The optimal conditions were 50°C, pH 7.5 with Co2+ and Mg2+. The d-psicose yields from sugarcane bagasse and microalgae hydrolysate were 1.42 and 1.69g/L, respectively.

Comparative effects of EtOH consumption and thiamine deficiency on cognitive impairment, oxidative damage, and ß-amyloid peptide overproduction in the brain.

The effects of chronic EtOH consumption, associated or not with thiamine deficiency (TD), on cognitive impairment, oxidative damage, and ß-amyloid (Aß) peptide accumulation in the brain were investigated in male C57BL/6 mice. We established an alcoholic mouse model by feeding an EtOH liquid diet, a TD mouse model by feeding a thiamine-depleted liquid diet, and an EtOH treatment associated with TD mouse model by feeding a thiamine-depleted EtOH liquid diet for 7 weeks. The learning and memory functions of the mice were detected through the Y-maze test. Biochemical parameters were measured using corresponding commercial kits. The Aß expression in the hippocampus was observed by immunohistochemical staining. Several results were obtained. First, EtOH significantly reduced cognitive function by significantly decreasing the Glu content in the hippocampus; increasing the AChE activity in the cortex; and reducing the thiamine level, and superoxide dismutase (SOD), glutathione peroxidase (GPx), and choline acetyltransferase (ChAT) activities in both the hippocampus and cortex. The treatment also increased the levels of malondialdehyde (MDA), protein carbonyl, 8-hydroxydeoxyguanosine (8-OHdG), and nitric oxide (NO) and the activities of total nitric oxide synthase (tNOS), inducible nitric oxide synthase (iNOS), and monoamine oxidase B (MAO-B). Furthermore, EtOH enhanced the expression levels of Aß1-42 and Aß1-40 in the hippocampus. Second, TD induced the same dysfunctions caused by EtOH in the biochemical parameters, except for learning ability, 8-OHdG content, and GPx, tNOS, and AChE activities in the cortex. Third, the modification of MDA, protein carbonyl and NO levels, and GPx, iNOS, ChAT, and MAO-B activities in the brain induced by chronic EtOH treatment associated with TD was greater than that induced by EtOH or TD alone. The synergistic effects of EtOH and TD on Aß1-40 and Glu release, as well as on SOD activity, depended on their actions on the hippocampus or cortex. These findings suggest that chronic EtOH consumption can induce TD, cognitive impairment, Aß accumulation, oxidative stress injury, and neurotransmitter metabolic abnormalities. Furthermore, the association of chronic EtOH consumption with TD causes dramatic brain dysfunctions with a severe effect on the brain.

Metagenomic analysis for the microbial consortium of anaerobic CO oxidizers.

Metagenomics analysis has been applied to identify the dominant anaerobic microbial consortium of the carbon monoxide (CO) oxidizers in anaerobic sludge. Reads from the hypervariable V6 region in the bacterial 16s rDNA were aligned and finally clustered into operational taxonomic units (OTUs). The OTUs from different stages in anaerobic CO condition were classified. Alphaproteobacteria, clostridia, betaproteobacteria and actinobacteria were the most abundant groups, while alphaproteobacteria, betaproteobacteria and actinobacteria were variable groups. CO consumption and production efficiency of the microbial consortium were studied. Semi-continuous trials showed that these anaerobic CO oxidizers formed a stable microbial community, and the CO conversion rate was at over 84%, with the highest CO consumption activity of 28.9 mmol CO/g VSS●day and methane production activity at 7.6 mmol CH4 /g VSS●day during six cycles.

Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production.

Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The optimal initial solids loading, feeding time and quantities were examined. The hydrolysis system was initiated with 12% (w/v) solids loading in flasks, where 7% fresh solids were fed consecutively at 6h, 12h, 24h to get a final solids loading of 33%. All the requested cellulase loading (10 FPU/g substrate) was added completely at the beginning of hydrolysis reaction. After 120 h of hydrolysis, the maximal concentrations of cellobiose, glucose and xylose obtained were 9.376 g/L, 129.50 g/L, 56.03 g/L, respectively. The final total glucan conversion rate attained to 60% from this fed-batch process.