Utilization of lignocellulosic biomass by glycerol organosolv pretreatment for biobutanol production integrated with bioconversion of residual glycerol into value-added products.

Glycerol organosolv pretreatment (GOP) is considered an efficient method to deconstruct lignocellulose for producing fermentable sugars. Herein, the liquid fraction containing glycerol after GOP was utilized for recycled pretreatment of corn stover (CS) for four cycles. Enzymatic yield of glucose after recycled pretreatment was enhanced by 2.4-3.5 folds compared with untreated CS. Meanwhile, residual glycerol was used as carbon source for cultivation of Pichia pastoris to obtain high cell-density, and a final titer of 1.3 g/L human lysozyme was produced by P. pastoris under low temperature methanol induction strategy. Additionally, the pretreated CS was mixed with cassava as fermentable substrates for butanol production by wild-type Clostridium acetobutylicum ATCC 824. Final butanol production of 13.9 g/L was obtained from mixed substrates (25%:75% of CS/cassava) at 10% solids loading by simultaneous saccharification and fermentation. Overall, integration of residual glycerol utilization and butanol production by microbial fermentation provided an efficient strategy for biorefinery.

A facile and efficient synthesis approach of salidroside esters by whole-cell biocatalysts in organic solvents.

Salidroside, the main bioactive compound isolated from the plant source of Rhodiola rosea L, possesses broad-spectrum pharmacological activities, but suffers from the low cell membranes permeability and alimentary absorption due to its high polarity. Therefore, a whole-cell catalytic strategy for the synthesis of salidroside esters was explored to improve its lipophilicity. The results showed that Aspergillus oryzae demonstrated the highest biocatalytic activity among the microbial strains tested. For the synthesis of salidroside caprylate, the optimum conditions of reaction medium, Aspergillus oryzae amount, molar ratio of vinyl caprylate to salidroside and reaction temperature were acetone, 30 mg/ml, 10°C and 40°C, respectively. Under these conditions, the initial reaction rate was 15.36 mM/h, and substrate conversion and regioselectivity all reached 99%. Moreover, the results indicated that although various 6'-monoesters derivatives of salidroside were exclusively obtained with excellent conversions (96%-99%), the reaction rate varied greatly with different chain-length acyl donors. This study details an efficient and cost-effective biocatalytic approach for the synthesis of salidroside esters by using Aspergillus oryzae as a catalyst for the first time. Considering the whole cell catalytic efficiency and operational stability, this strategy may provide a new opportunity to develop green industrial processes production for ester derivatives of salidroside and its analogues.

A new l-cysteine-assisted glycerol organosolv pretreatment for improved enzymatic hydrolysis of corn stover.

Deconstruction of lignocellulose via efficient pretreatment is crucial for producing fermentable sugars. In this study, effects of glycerol organosolv pretreatment (GOP) on main chemical composition of corn stover were investigated. Results indicate that the residual corn stover after 80 wt% glycerol pretreatment (at 220 °C for 0.5 h) yielded 75.97 % glucose and 78.21 % xylose after enzymatic hydrolysis, which were enhanced by 3.39- and 6.08-fold compared to the untreated corn stover. Subsequently, an l-cysteine-assisted GOP was proposed with higher yields of glucose (86.20 %) and xylose (91.13 %). When pretreating corn stover with 80 wt% glycerol containing 0.07 wt% l-cysteine at 220 °C for 0.5 h, higher fermentable sugars of 26.08 g were produced from 100 g feedstock after enzymolysis. Intrinsic mechanisms of the proposed pretreatment for enhancing enzymatic digestibility were elucidated by physiochemical characterization technologies and techno-economic analysis was also studied. This study provides guidance for fermentable sugars production from renewable lignocellulose.

Microbial production of gamma-aminobutyric acid: applications, state-of-the-art achievements, and future perspectives.

Gamma-aminobutyric acid (GABA) is an important non-protein amino acid with wide-ranging applications. Currently, GABA can be produced by a variety of methods, including chemical synthesis, plant enrichment, enzymatic methods, and microbial production. Among these methods, microbial production has gained increasing attention to meet the strict requirements of an additive in the fields of food, pharmaceutical, and livestock. In addition, renewable and abundant resources, such as glucose and lignocellulosic biomass can also be used for GABA microbial production under mild and environmentally friendly processing conditions. In this review, the applications, metabolic pathways and physiological functions of GABA in different microorganisms were firstly discussed. A comprehensive overview of the current status of process engineering strategies for enhanced GABA production, including fermentation optimization and whole-cell conversion from different feedstocks by various host strains is also provided. We also presented the state-of-the-art achievements in strain development strategies for industrial lactic acid bacteria (LAB), Corynebacterium glutamicum and Escherichia coli to enhance the performance of GABA bioproduction. In order to use bio-based GABA in the fields of food and pharmaceutical, some Generally Recognized as Safe (GRAS) strains such as LAB and C. glutamicum will be the promising chassis hosts. Toward the end of this review, current challenges and valuable research directions/strategies on the improvements of process and strain engineering for economic microbial production of GABA are also suggested.

Prediction of phenolic compounds and glucose content from dilute inorganic acid pretreatment of lignocellulosic biomass using artificial neural network modeling.

Dilute inorganic acids hydrolysis is one of the most promising pretreatment strategies with high recovery of fermentable sugars and low cost for sustainable production of biofuels and chemicals from lignocellulosic biomass. The diverse phenolics derived from lignin degradation during pretreatment are the main inhibitors for enzymatic hydrolysis and fermentation. However, the content features of derived phenolics and produced glucose under different conditions are still unclear due to the highly non-linear characteristic of biomass pretreatment. Here, an artificial neural network (ANN) model was developed for simultaneous prediction of the derived phenolic contents (CPhe) and glucose yield (CGlc) in corn stover hydrolysate before microbial fermentation by integrating dilute acid pretreatment and enzymatic hydrolysis. Six processing parameters including inorganic acid concentration (CIA), pretreatment temperature (T), residence time (t), solid-to-liquid ratio (RSL), kinds of inorganic acids (kIA), and enzyme loading dosage (E) were used as input variables. The CPhe and CGlc were set as the two output variables. An optimized topology structure of 6-12-2 in the ANN model was determined by comparing root means square errors, which has a better prediction efficiency for CPhe (R2 = 0.904) and CGlc (R2 = 0.906). Additionally, the relative importance of six input variables on CPhe and CGlc was firstly calculated by the Garson equation with net weight matrixes. The results indicated that CIA had strong effects (22%-23%) on CPhe or CGlc, then followed by E and T. In conclusion, the findings provide new insights into the sustainable development and inverse optimization of biorefinery process from ANN modeling perspectives.

Significantly Enhanced Synthesis of Aromatic Esters of Arbutin Catalyzed by Immobilized Lipase in Co-solvent Systems.

Highly efficient and regioselective synthesis of pharmacologically interesting aromatic esters of arbutin catalyzed by immobilized lipase from Penicillium expansum in co-solvent systems was successfully carried out. As compared to tetrahydrofuran solvent, the initial rate and substrate conversion of arbutin vanilylation were markedly enhanced in tetrahydrofuran-isopropyl ether (20%, v/v). Moreover, the effects of three reaction parameters (enzyme amount, temperature and substrate molar ratio of vinyl vanillic acid to arbutin) on 6'-O-vanilloyl-arbutin synthesis were scrutinized and the key process parameters were optimized using response surface methodology (RSM). The experimental data were fitted well to a second order polynomial model by using multiple regression analysis. The best combination of variables was 50°C, 93 U/mL and 11 for the reaction temperature, the enzyme amount and mole ratio of arbutin to vinyl vanilic acid, respectively, and which the reaction rate, substrate conversion and regioselectivity were as high as 8.2 mM/h, 93 and 99%. It was worth noting that a variety of aromatic esters of arbutin were obtained with much higher conversion (93-99%) at these optimal conditions.

Efficient bio-butanol production from lignocellulosic waste by elucidating the mechanisms of Clostridium acetobutylicum response to phenolic inhibitors.

Lignocellulosic biomass is considered abundant renewable feedstock to constitute a green and environmentally friendly approach for biofuels (bio-butanol) production as an effective substitute for fossil resources. However, a variety of fermentable inhibitors can be generated in hydrolysates during the biomass pretreatment process. Among them, phenolics including phenolic acids and phenolic aldehydes are the most toxic inhibitors to solventogenic clostridia for bio-butanol production. This study elucidates the physiological mechanism of Clostridium acetobutylicum ATCC 824 response to phenolic inhibitors by the integration of kinetics and transcriptional analysis. Butanol fermentations were stressed by 0.4 g/L phenolic acids or 0.4 g/L phenolic aldehydes at 12 h at the beginning of solventogenesis. With post-stress for 12 h, butanol titer was 7.01 g/L in fermentation with phenolic acid stress, while only 5.82 g/L butanol was produced in the case of phenolic aldehydes stress. Reductions in the two fermentations were 27.6% and 40.0% in comparison with the control (without stress), indicated that phenolic aldehydes had a stronger inhibitory effect on solvents synthesis in C. acetobutylicum than phenolic acids. Additionally, the transcriptional analysis revealed that phenolics altered the gene expression profiles related to membrane transporters such as ATP-binding cassette (ABC)-transporter and phosphotransferase system (PTS), glycolysis, and heat shock proteins. The lower expression levels of PTS-related genes might result in reduced glucose consumption and finally inhibited solvents synthesis under phenolic aldehydes stress. Some genes encoding histidine kinase (CA_C0323, CA_C0903, and CA_C3319) were also affected by phenolics, which might inhibit sporulation. In conclusion, our results provide valuable guidance for the construction of robust strain to efficiently produce bio-butanol from lignocellulosic biomass.

Effects of Dietary Supplementation with Mulberry (Morus alba L.) Leaf Polysaccharides on Immune Parameters of Weanling Pigs.

In this study, the effect of dietary supplementation of mulberry leaf polysaccharides (MLPs) on the immune parameters-i.e., the immune organ weight, serum immunoglobulins, cytokines, nitric oxide (NO) production, and insulin-Like growth factor-1 (IGF1) mRNA expression-of weanling pigs as a model animal was investigated. A total of 120 healthy weanling pigs (aged 28 ± 2 d) with the same body weights were randomly divided into four groups: (1) Control treatment (CT), basal diet (BD), (2) MLP low-dose treatment (MLT), 0.6 g/kg MLP + BD, (3) MLP high-dose treatment (MHT), 1.2 g/kg MLP + BD, and (4) antibiotic treatment (AT), 0.15 g/kg chlortetracycline + BD. The results revealed that the thymus and spleen indices were significantly increased (P < 0.05) in both MLT and MHT groups in comparison with the CT group, while the serum levels of immunoglobulin G (IgG), interleukin (IL)-1ß, IL-2, IL-8, and interferon (IFN-γ) in the MLT group and IL-2, IL-6, and IFN-γ in the MHT group were also considerably greater (P < 0.05) than the corresponding levels in the CT group. The serum contents of IgG, IL-1ß, IL-2, and IL-8 in the MLT group and IL-2 and IL-6 in the MHT group were significantly increased in comparison with the corresponding contents in the AT group (P < 0.05). The transformation rate of lymphocytes in the MLT and MHT groups was higher compared to the CT and AT groups. However, a notable difference was found between the MLT group and the two control groups. The peripheral lymphocyte NO production in the MLT, MHT, and AT groups was significant relative to the CT group. The expression levels of IGF1 mRNA in the liver and muscle longissimus tissues of both the MLT and MHT groups showed significant improvement (P < 0.05) over those in the CT group. Moreover, the IGF1 mRNA expression in the muscle longissimus from the MLT group was significantly higher than in the AT group. In conclusion, the results suggest that incorporating MLPs into the diets of weanling pigs improves the animals' metabolisms and immune functions, and the effects of the MLT group were superior to those of both the MHT and AT groups.

Co-production of solvents and organic acids in butanol fermentation by Clostridium acetobutylicum in the presence of lignin-derived phenolics.

Co-production of solvents (butanol, acetone, and ethanol) and organic acids (butyrate and acetate) by Clostridium acetobutylicum using lignocellulosic biomass as a substrate could further enlarge the application scope of butanol fermentation. This is mainly because solvents and organic acids could be used for production of fine chemicals such as butyl butyrate, butyl oleate, etc. However, many phenolic fermentation inhibitors are formed during the pretreatment process because of lignin degradation. The present study investigated the effects of five typical lignin-derived phenolics on the biosynthesis of solvents and organic acids in C. acetobutylicum ATCC 824. Results obtained in 100 mL anaerobic bottles indicated that butanol concentration was enhanced from 10.29 g L-1 to 11.36 g L-1 by the addition of 0.1 g L-1 vanillin. Subsequently, a pH-control strategy was proposed in a 5 L anaerobic fermenter to alleviate the "acid crash" phenomenon and improve butanol fermentation performance, simultaneously. Notably, organic acid concentration was enhanced from 6.38 g L-1 (control) to a high level of 9.21-12.57 g L-1 with vanillin or/and vanillic acid addition (0.2 g L-1) under the pH-control strategy. Furthermore, the butyrate/butanol ratio reached the highest level of 0.80 g g-1 with vanillin/vanillic acid co-addition, and solvent concentration reached 13.85 g L-1, a comparable level to the control (13.69 g L-1). The effectiveness and robustness of the strategy for solvent and organic acid co-production was also verified under five typical phenolic environments. In conclusion, these results suggest that the proposed process strategy would potentially promote butanol fermentative products from renewable biomass.

Recent advances and strategies in process and strain engineering for the production of butyric acid by microbial fermentation.

Butyric acid is an important platform chemical, which is widely used in the fields of food, pharmaceutical, energy, etc. Microbial fermentation as an alternative approach for butyric acid production is attracting great attention as it is an environmentally friendly bioprocessing. However, traditional fermentative butyric acid production is still not economically competitive compared to chemical synthesis route, due to the low titer, low productivity, and high production cost. Therefore, reduction of butyric acid production cost by utilization of alternative inexpensive feedstock, and improvement of butyric acid production and productivity has become an important target. Recently, several advanced strategies have been developed for enhanced butyric acid production, including bioprocess techniques and metabolic engineering methods. This review provides an overview of advances and strategies in process and strain engineering for butyric acid production by microbial fermentation. Additionally, future perspectives on improvement of butyric acid production are also proposed.

Effect of iron(III) ion on moso bamboo pyrolysis under microwave irradiation.

The effect of iron(III) ion on microwave pyrolysis of moso bamboo was investigated. Hydrofluoric acid washing was used as a pilot process to demineralize moso bamboo in order to eliminate the influences of the other inorganics contained in moso bamboo itself. The results indicated that the addition of iron(III) ion increased the maximal reaction temperatures under microwave condition dependent on the amount of the added iron(III) ion. The production of the non-condensable gases was promoted by the addition of iron(III) ion mainly at the expense of liquid products. Iron(III) ion exhibited the positive effect for syngas production and inhibited the formation of CO2 and CH4. The formation of Fe2O3 and Fe3O4 was found during microwave pyrolysis and the mechanism of the two metallic oxides formation was described in this work.

Enzymatic Synthesis of Sorboyl-Polydatin Prodrug in Biomass-Derived 2-Methyltetrahydrofuran and Antiradical Activity of the Unsaturated Acylated Derivatives.

Efficient and highly regioselective synthesis of the potential 6''-O-sorboyl-polydatin prodrug in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was achieved using Candida antarctica lipase B for the first time. Under the optimal conditions, the initial reaction rate, maximum substrate conversion, and 6''-regioselectivity were as high as 8.65 mM/h, 100%, and 100%, respectively. Kinetic and operational stability investigations evidently demonstrated excellent enzyme compatibility of the 2-MeTHF compared to the traditional organic solvents. With respect to the antioxidant properties, three unsaturated ester derivatives showed slightly lower DPPH radical scavenging activities than the parent agent. Interestingly, further studies also revealed that the antiradical capacities of the acylates decreased with the elongation of the unsaturated aliphatic chain length from C4 to C11. The reason might be attributed to the increased steric hindrance derived from the acyl residues in derivatives.

Efficient Regioselective Synthesis of the Crotonyl Polydatin Prodrug by Thermomyces lanuginosus Lipase: a Kinetics Study in Eco-friendly 2-Methyltetrahydrofuran.

Bio-based solvents have recently been discussed as sustainable green and promising alternatives to conventional organic media for enzymatic processes. In this paper, highly regioselective synthesis of the 6″-O-crotonyl-polydatin catalyzed by Thermomyces lanuginosus lipase (TLL) in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was successfully performed for the first time. The results indicated that TLL lipase displayed significantly improved catalytic performance in 2-MeTHF than in other traditional solvents. Under the optimal conditions, the initial reaction rate, 6″-regioselectivity, and maximum substrate conversion were as high as 12.38 mM h(-1), 100 %, and 100 %, respectively. Moreover, further investigations on the operational stability, kinetic parameters like V max, K m, V max/K m, and E a revealed that 2-MeTHF exhibited excellent biocompatibility and rendered the greener process of the enzymatic acylation.

The halo-substituent effect on Pseudomonas cepacia lipase-mediated regioselective acylation of nucleosides: A comparative investigation.

In this work, comparative experiments were explored to investigate the substrate specificity of Pseudomonas cepacia lipase in regioselective acylation of nucleosides carrying various substituents (such as the H, F, Cl, Br, I) at 2'- and 5-positions. Experimental data indicated that the catalytic performance of the enzyme depended very much on the halo-substituents in nucleosides. The increased bulk of 2'-substituents in ribose moiety of the nucleoside might contribute to the improved 3'-regioselectivity (90-98%, nucleosides a-d) in enzymatic decanoylation, while the enhancement of regioselectivity (93-99%) in 3'-O-acylated nucleosides e-h could be attributable to the increasing hydrophobicity of the halogen atoms at 5-positions. With regard to the chain-length selectivity, P. cepacia lipase displayed the highest 3'-regioselectivity toward the longer chain (C14) as compared to shorter (C6 and C10) ones. The position, orientation and property of the substituent, specific structure of the lipase's active site, and acyl structure could account for the diverse results.

Chemical Composition and Immunomodulatory Activity of Mycelia of the Hairy Bracket Mushroom, Trametes hirsuta (Higher Basidiomycetes).

Trametes hirsuta is a medicinal mushroom that produces laccase. Its mycelium is a by-product when this species is used for laccase production. Aiming to develop its potential medicinal value, we investigated the chemical composition and immunomodulatory activity of T. hirsuta mycelia (THM). Dried THM contained 26.06% protein, 1.15% fat, 57.87% carbohydrates, and 5.47% ash. Sixteen free amino acids (2.63% total content) and 6 5'-nucleotides (adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, uridine 5'-monophosphate, xanthosine 5'-monophosphate, and inosine 5'-monophosphate) constituting 0.275% were detected. Dominant sugars and polyols were fructose (2.47%), mannitol (2.03%), and glucose (1.8%); trehalose and arabinose contents were less than 0.10%. Evaluation of immunomodulatory activity in mice showed that THM could improve macrophage phagocytic function and serum hemolysin concentrations, but only the low-dose group significantly enhanced the natural killer cell activity and increased the spleen index, and only the middle-dose group remarkably increased the thymus index. Therefore, T. hirsuta mycelia could enhance immune function in mice and have immunomodulatory activity.

Highly efficient and enzymatic regioselective undecylenoylation of gastrodin in 2-methyltetrahydrofuran-containing systems.

Highly efficient and regioselective acylation of pharmacologically interesting gastrodin with vinyl undecylenic acid has been firstly performed through an enzymatic approach. The highest catalytic activity and regioselectivity towards the acylation of 7'-hydroxyl of gastrodin was obtained with Pseudomonas cepacia lipase. In addition, it was observed the lipase displayed higher activity in the eco-friendly solvent 2-methyltetrahydrofuran-containing systems than in other organic solvents. In the co-solvent mixture of tetrahydrofuran and 2-methyltetrahydrofuran (3/1, v/v), the reaction rate was 60.6 mM/h, substrate conversion exceeded 99%, and 7'-regioselectivity was 93%. It was also interesting that the lipase-catalyzed acylation couldn't be influenced by the benzylic alcohol in gastrodin. However, pseudomonas cepacia lipase displayed different regioselectivity towards gastrodin and arbutin.

Novel and highly efficient regioselective route to helicid esters by lipozyme TLL.

Highly regioselective acylation of helicid with fatty acid vinyl esters catalyzed by the lipase from Thermomyces lanuginosus has been successfully performed for the first time. For the enzymatic caproylation of helicid, under the optimal conditions, initial reaction rate was 33.2 mM/h, and substrate conversion and regioselectivity were greater than 99%. In addition, the acyl recognition of the enzyme in the regioselective acylation of helicid was investigated. The results showed that although 6'-O-acyl derivatives of helicid were exclusively obtained with all the tested acyl donors, the enzymatic reaction rate varied widely with different acyl donors, presumably owing to their different interactions with the active site of the lipase. It is also interesting that the different configuration of only one hydroxyl group at C-3 in helicid couldn't affect the lipase-catalyzed esterification and helicid has the same regioselectivity as that of D-glucose and arbutin.

Enhanced mycelial biomass production of the hairy bracket mushroom, Trametes hirsuta (Higher Basidiomycetes), by optimizing medium component with Plackett-Burman design and response surface methodology.

Statistical analyses based on experimental designs were applied to optimize the medium components for mycelial biomass production by Trametes hirsuta in shake flask cultivation. First, the effects of different carbon resources (glucose, sucrose, lactose, maltose, fructose, soluble starch and potato), nitrogen resources (yeast extract, peptone, (NH4)2SO4, NH4NO3, NH4Cl, peanut powder, soybean powder) and mineral elements (CaCl2, ZnSO4·7H2O, FeSO4·7H2O, MnSO4·H2O, CuSO4·7H2O) on mycelial biomass production were investigated using a univariate design. Second, a Plackett-Burman design was applied to identify the significant variables that principally influenced the mycelial biomass production, and the path of steepest ascent was pursued to approach the regions of optimal value of the significant variables. Subsequently, these significant variables were optimized using the Box-Behnken design of response surface methodology. Ultimately, the optimized medium conditions were composed of sucrose 25.65 g·L-1, MgSO4·7H2O 1.24 g·L-1, and FeSO4·7H2O 3.36 g·L-1, and the yield of mycelial biomass reached 15.45 g·L-1, which represents an approximately 1.6-fold increase above the initial yield.

A highly regioselective route to arbutin esters by immobilized lipase from Penicillium expansum.

Immobilized lipase from Penicillium expansum, a novel and inexpensive enzyme preparation that we immobilized in our laboratory, was an excellent catalyst for highly regioselective acylation of arbutin with fatty acid vinyl esters. For the enzymatic butanoylation of arbutin, under the optimal conditions, initial reaction rate was 75.1 mM/h, and substrate conversion and regioselectivity were greater than 99%. In addition, a variety of 6'-esters of arbutin were prepared with high conversion (>99%) and excellent regioselectivity (>99%). It was found that the enzymatic reaction rate varied widely with different acyl donors, presumably owing to their different interactions with the active site of the lipase. The immobilized lipase from P. expansum displayed highest catalytic activity with medium-length straight-chain acyl donors. Acyl donors bearing a substituent or a conjugate double bond gave reduced reaction rates.