Succinic acid production from softwood with genome-edited Corynebacterium glutamicum using the CRISPR-Cpf1 system.

Corynebacterium glutamicum is a useful microbe that can be used for producing succinic acid under anaerobic conditions. In this study, we generated a knock-out mutant of the lactate dehydrogenase 1 gene (ΔldhA-6) and co-expressed the succinic acid transporter (Psod:SucE- ΔldhA) using the CRISPR-Cpf1 genome editing system. The highly efficient HPAC (hydrogen peroxide and acetic acid) pretreatment method was employed for the enzymatic hydrolysis of softwood (Pinus densiflora) and subsequently utilized for production of succinic acid. Upon evaluating a 1%-5% hydrolysate concentration range, optimal succinic acid production with the ΔldhA mutant was achieved at a 4% hydrolysate concentration. This resulted in 14.82 g L-1 succinic acid production over 6 h. No production of acetic acid and lactic acid was detected during the fermentation. The co-expression transformant, [Psod:SucE-ΔldhA] produced 17.70 g L-1 succinic acid in 6 h. In the fed-batch system, 39.67 g L-1 succinic acid was produced over 48 h. During the fermentation, the strain consumed 100% and 73% of glucose and xylose, respectively. The yield of succinic acid from the sugars consumed was approximately 0.77 g succinic acid/g sugars. These results indicate that the production of succinic acid from softwood holds potential applications in alternative biochemical processes.

Optimizing bioconversion processes of rice husk into value-added products: D-psicose, bioethanol, and lactic acid.

Rice husk, rich carbon content, is an agricultural waste produced globally at an amount of 120 million tons annually, and it has high potential as a biorefinery feedstock. Herein, we investigated the feasibility of producing various products as D-psicose, bioethanol and lactic acid from rice husk (RH) through a biorefinery process. Alkali-hydrogen peroxide-acetic acid pretreatment of RH effectively removed lignin and silica, resulting in enzymatic hydrolysis yield of approximately 86.3% under optimal hydrolysis conditions. By using xylose isomerase as well as D-psicose-3-epimerase with borate, glucose present in the RH hydrolysate was converted into D-psicose with a 40.6% conversion yield in the presence of borate. Furthermore, bioethanol (85.4%) and lactic acid (92.5%) were successfully produced from the RH hydrolysate. This study confirmed the high potential of RH as a biorefinery feedstock, and it is expected that various platform chemicals and value-added products can be produced using RH.

Converting textile waste into value-added chemicals: An integrated bio-refinery process.

The rate of textile waste generation worldwide has increased dramatically due to a rise in clothing consumption and production. Here, conversion of cotton-based, colored cotton-based, and blended cotton-polyethylene terephthalate (PET) textile waste materials into value-added chemicals (bioethanol, sorbitol, lactic acid, terephthalic acid (TPA), and ethylene glycol (EG)) via enzymatic hydrolysis and fermentation was investigated. In order to enhance the efficiency of enzymatic saccharification, effective pretreatment methods for each type of textile waste were developed, respectively. A high glucose yield of 99.1% was obtained from white cotton-based textile waste after NaOH pretreatment. Furthermore, the digestibility of the cellulose in colored cotton-based textile wastes was increased 1.38-1.75 times because of the removal of dye materials by HPAC-NaOH pretreatment. The blended cotton-PET samples showed good hydrolysis efficiency following PET removal via NaOH-ethanol pretreatment, with a glucose yield of 92.49%. The sugar content produced via enzymatic hydrolysis was then converted into key platform chemicals (bioethanol, sorbitol, and lactic acid) via fermentation or hydrogenation. The maximum ethanol yield was achieved with the white T-shirt sample (537 mL/kg substrate), which was 3.2, 2.1, and 2.6 times higher than those obtained with rice straw, pine wood, and oak wood, respectively. Glucose was selectively converted into sorbitol and LA at a yield of 70% and 83.67%, respectively. TPA and EG were produced from blended cotton-PET via NaOH-ethanol pretreatment. The integrated biorefinery process proposed here demonstrates significant potential for valorization of textile waste.

Utilization of bamboo as biorefinery feedstock: Co-production of xylo-oligosaccharide with succinic acid and lactic acid.

Herein, we investigated the possibility of co-producing xylo-oligosaccharides (XOSs) from bamboo, as value-added products, along with succinic and lactic acids, as platform chemicals. Xylan was extracted from bamboo using the alkali method under mild conditions. From xylan, XOSs were produced by partial enzymatic hydrolysis at a conversion rate of 83.9%, and all reaction conditions resulted in similar degrees of polymerization. Hydrogen peroxide-acetic acid (HPAC) pretreatment effectively removed lignin from NaOH-treated bamboo, and the enzymatic hydrolytic yield of NaOH and HPAC-treated bamboo was 84.3% of the theoretical yield. The production of succinic and lactic acids from the hydrolysate resulted in conversion rates of approximately 63.2% and 91.3% of the theoretical yield using Corynebacterium glutamicum Δldh and Actinobacillus succinogenes, respectively, under facultative anaerobic conditions. This study demonstrates that bamboo has a high potential to produce value-added products using a biorefinery process and is an alternative resource for compounds typically derived from petroleum.

Comparative Evaluation of Adsorption of Major Enzymes in a Cellulase Cocktail Obtained from Trichoderma reesei onto Different Types of Lignin.

Cellulase adsorption onto lignin decreases the productivity of enzymatic hydrolysis of lignocellulosic biomass. Here, adsorption of enzymes onto different types of lignin was investigated, and the five major enzymes-cellobiohydrolases (CBHs), endoglucanase (Cel7B), ß-glucosidase (Cel3A), xylanase (XYNIV), and mannanase (Man5A)-in a cellulase cocktail obtained from Trichoderma reesei were individually analyzed through SDS-PAGE and zymogram assay. Lignin was isolated from woody (oak and pine lignin) and herbaceous (rice straw and kenaf lignin) plants. The relative adsorption of CBHs compared to the control was in the range of 14.15-18.61%. The carbohydrate binding motif (CBM) of the CBHs contributed to higher adsorption levels in oak and kenaf lignin, compared to those in pine and rice lignin. The adsorption of endoglucanase (Cel7B) by herbaceous plant lignin was two times higher than that of woody lignin, whereas XYNIV showed the opposite pattern. ß-glucosidase (Cel3A) displayed the highest and lowest adsorption ratios on rice straw and kenaf lignin, respectively. Mannanase (Man5A) was found to have the lowest adsorption ratio on pine lignin. Our results showed that the hydrophobic properties of CBM and the enzyme structures are key factors in adsorption onto lignin, whereas the properties of specific lignin types indirectly affect adsorption.

An integrated process for conversion of spent coffee grounds into value-added materials.

Spent coffee grounds (SCG) are inexpensive materials with a complex composition that makes them promising feedstocks for a biorefinery.Here, conversion of SCG into a wide range of high value-added products (coffee oil, bio-ethanol, D-mannose, manno-oligosaccharide (MOS), cafestol and kahweol) using a novel integrated system was evaluated. The process involves oil extraction, MOS production by mannanase obtained from Penicillium purpurogenum, NaOH (Na) and hydrogen peroxide (HP) pretreatment for the degradation of lignin and phenolic compounds, diterpenes extraction, enzymatic hydrolysis, and fermentation, which can be performed using environmentally friendly technologies. Approximately 97 mL of coffee oil, 164 g of D-mannose, 102 g of MOS, 99 g of bioethanol and a dash of cafestol/kahweol were produced from 1 kg of dry SCG. Producing high-value co-products from SCG using an integrated approach as demonstrated here may be an efficient strategy to reduce waste generation, while improving the economics of the biorefinery production process.

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation.

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

Hydrolysis pattern analysis of xylem tissues of woody plants pretreated with hydrogen peroxide and acetic acid: rapid saccharification of softwood for economical bioconversion.

BACKGROUND: Woody plants with high glucose content are alternative bioresources for the production of biofuels and biochemicals. Various pretreatment methods may be used to reduce the effects of retardation factors such as lignin interference and cellulose structural recalcitrance on the degradation of the lignocellulose material of woody plants. RESULTS: A hydrogen peroxide-acetic acid (HPAC) pretreatment was used to reduce the lignin content of several types of woody plants, and the effect of the cellulose structural recalcitrance on the enzymatic hydrolysis was analyzed. The cellulose structural recalcitrance and the degradation patterns of the wood fibers in the xylem tissues of Quercus acutissima (hardwood) resulted in greater retardation in the enzymatic saccharification than those in the tracheids of Pinus densiflora (softwood). In addition to the HPAC pretreatment, the application of supplementary enzymes (7.5 FPU cellulase for 24 h) further increased the hydrolysis rate of P. densiflora from 61.42 to 91.94% whereas the same effect was not observed for Q. acutissima. It was also observed that endoxylanase synergism significantly affected the hydrolysis of P. densiflora. However, this synergistic effect was lower for other supplementary enzymes. The maximum concentration of the reducing sugars produced from 10% softwood was 89.17 g L-1 after 36 h of hydrolysis with 15 FPU cellulase and other supplementary enzymes. Approximately 80 mg mL-1 of reducing sugars was produced with the addition of 7.5 FPU cellulase and other supplementary enzymes after 36 h, achieving rapid saccharification. CONCLUSION: HPAC pretreatment removed the interference of lignin, reduced structural recalcitrance of cellulose in the P. densiflora, and enabled rapid saccharification of the woody plants including a high concentration of insoluble substrates with only low amounts of cellulase. HPAC pretreatment may be a viable alternative for the cost-efficient production of biofuels or biochemicals from softwood plant tissues.

Enhanced Biomass Yield of and Saccharification in Transgenic Tobacco Over-Expressing ß-Glucosidase.

Here, we report an increase in biomass yield and saccharification in transgenic tobacco plants (Nicotiana tabacumL.) overexpressing thermostable ß-glucosidase from Thermotoga maritima, BglB, targeted to the chloroplasts and vacuoles. The transgenic tobacco plants showed phenotypic characteristics that were significantly different from those of the wild-type plants. The biomass yield and life cycle (from germination to flowering and harvest) of the transgenic tobacco plants overexpressing BglB were 52% higher and 36% shorter than those of the wild-type tobacco plants, respectively, indicating a change in the genome transcription levels in the transgenic tobacco plants. Saccharification in biomass samples from the transgenic tobacco plants was 92% higher than that in biomass samples from the wild-type tobacco plants. The transgenic tobacco plants required a total investment (US$/year) corresponding to 52.9% of that required for the wild-type tobacco plants, but the total biomass yield (kg/year) of the transgenic tobacco plants was 43% higher than that of the wild-type tobacco plants. This approach could be applied to other plants to increase biomass yields and overproduce ß-glucosidase for lignocellulose conversion.

Bioconversion of biomass waste into high value chemicals.

Dwindling petroleum resources and increasing environmental concerns have stimulated the production of platform chemicals via biochemical processes through the use of renewable carbon sources. Various types of biomass wastes, which are biodegradable and vastly underutilized, are generated worldwide in huge quantities. They contain diverse chemical constituents, which may serve as starting points for the manufacture of a wide range of valuable bio-derived platform chemicals, intermediates, or end products via different conversion pathways. The valorization of inexpensive, abundantly available, and renewable biomass waste could provide significant benefits in response to increasing fossil fuel demands and manufacturing costs, as well as emerging environmental concerns. This review explores the potential for the use of available biomass waste to produce important chemicals, such as monosaccharides, oligosaccharides, biofuels, bioactive molecules, nanocellulose, and lignin, with a focus on commercially viable technologies.

Use of coffee flower as a novel resource for the production of bioactive compounds, melanoidins, and bio-sugars.

Although coffee beans have been widely studied, application of coffee flower (CF) has not been previously investigated. Here, we evaluated the use of CF for the production of bioactive compounds, melanoidins, and bio-sugars through the green process. Pressurized hot water extraction was found to be the most appropriate method for extracting bioactive compounds from CF, which contain high values of total phenolic content and have antioxidant properties. Caffeine and trigonelline were the main compounds in CF with yields of 1070.8 mg and 1092.8 mg/100 g dry weight (DW), respectively. Melanoidins were also identified and quantified in the CF extracts that is approximately 30.2% were efficiently recovered in the initial extracts of CF. Bio-sugar was also obtained from cellulase and pectinase at a 92.8% conversion rate. The aim of this study is to promote a novel approach using high amounts of CFs in the production of functional healthy foods and beverages.

Production of D-tagatose and bioethanol from onion waste by an intergrating bioprocess.

The rapid increase of agricultural waste is becoming a burgeoning problem and considerable efforts are being made by numerous researchers to convert it into a high-value resource material. Onion waste is one of the biggest issues in a world of dwindling resource. In this study, the potential of onion juice residue (OJR) for producing valuable rare sugar or bioethanol was evaluated. Purified Paenibacillus polymyxaL-arabinose isomerase (PPAI) has a molecular weight of approximately 53kDa, and exhibits maximal activity at 30°C and pH 7.5 in the presence of 0.8mM Mn2+. PPAI can produce 0.99g D-tagatose from 10g OJR. In order to present another application for OJR, we produced 1.56g bioethanol from 10g OJR through a bioconversion and fermentation process. These results indicate that PPAI can be used for producing rare sugars in an industrial setting, and OJR can be converted to D-tagatose and bioethanol.

Simultaneous production of bioethanol and value-added d-psicose from Jerusalem artichoke (Helianthus tuberosus L.) tubers.

In this study, the production of bioethanol and value added d-psicose from Jerusalem artichoke (JA) was attempted by an enzymatic method. An enzyme mixture used for hydrolysis of 100mgmL-1 JA. The resulting concentrations of released d-fructose and d-glucose were measured at approximately 56mgmL-1 and 15mgmL-1, respectively. The d-psicose was epimerized from the JA hydrolyzate, and the conversion rate was calculated to be 32.1%. The residual fructose was further converted into ethanol at 18.0gL-1 and the yield was approximately 72%. Bioethanol and d-psicose were separated by pervaporation. This is the first study to report simultaneous d-psicose production and bioethanol fermentation from JA.

Strategy for dual production of bioethanol and d-psicose as value-added products from cruciferous vegetable residue.

In this study, fermentable sugars and d-fructose were produced from cruciferous vegetable residue by enzymatic method without the use of either chemical or mechanical mechanisms. Production of d-psicose was effectively converted from hydrolyzed d-fructose in cabbage residue by d-psicose-3 epimerase; the presence of the borate increased the conversion rate by about two fold, and ethanol production yield was 85.7% of the theoretical yield. Both products, bioethanol and d-psicose, were successfully separated and purified by pervaporation and cation exchange chromatography, and their recovery yields were approximately 87% and 86.2%, respectively.

Cellulosic bioethanol production from Jerusalem artichoke (Helianthus tuberosus L.) using hydrogen peroxide-acetic acid (HPAC) pretreatment.

Jerusalem artichoke (JA) is recognized as a suitable candidate biomass crop for bioethanol production because it has a rapid growth rate and high biomass productivity. In this study, hydrogen peroxide-acetic acid (HPAC) pretreatment was used to enhance the enzymatic hydrolysis and to effectively remove the lignin of JA. With optimized enzyme doses, synergy was observed from the combination of three different enzymes (RUT-C30, pectinase, and xylanase) which provided a conversion rate was approximately 30% higher than the rate with from treatment with RUT-C30 alone. Fermentation of the JA hydrolyzates by Saccharomyces cerevisiae produced a fermentation yield of approximately 84%. Therefore, Jerusalem artichoke has potential as a bioenergy crop for bioethanol production.

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum.

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co(2+). Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co(2+) stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.

Isolation and characterization of lignin from the oak wood bioethanol production residue for adhesives.

Lignin was isolated from the residue of bioethanol production with oak wood via alkaline and catalyzed organosolv treatments at ambient temperature to improve the purity of lignin for the materials application. The isolated lignins were analyzed for their chemical composition by nitrobenzene oxidation method and their functionality was characterized via wet chemistry method, element analysis, (1)H NMR, GPC and FTIR-ATR. The isolated lignin by acid catalyzed organosolv treatment (Acid-OSL) contained a higher lignin content, aromatic proton, phenolic hydroxyl group and a lower nitrogen content that is more reactive towards chemical modification. The lignin-based adhesives were prepared and the bond strength was measured to evaluate the enhanced reactivity of lignin by the isolation. Two steps of phenolation and methylolation were applied for the modification of the isolated lignins and their tensile strengths were evaluated for the use as an adhesive. The acid catalyzed organosolv lignin-based adhesives had comparable bond strength to phenol-formaldehyde adhesives. The analysis of lignin-based adhesives by FTIR-ATR and TGA showed structural similarity to phenol adhesive. The results demonstrate that the reactivity of lignin was enhanced by isolation from hardwood bioethanol production residues at ambient temperature and it could be used in a value-added application to produce lignin-based adhesives.

Improving lignocellulose degradation using xylanase-cellulase fusion protein with a glycine-serine linker.

The fungal hydrolytic system efficiently degrades lignocellulosics efficiently. We previously characterized two hydrolytic enzymes from Gloeophyllum trabeum, namely, endoglucanase (Cel5B) and xylanase (Xyl10g). To enhance lignocellulosic degradation, we designed a fusion protein (Xyl10g GS Cel5B) using a glycine-serine (GS) linker and expressed it in Pichia pastoris GS115, which produced a hydrolytic fusion enzyme for the degradation of lignocellulosics. Purified Xyl10g GS Cel5B protein has a molecular weight of approximately 97 kDa and shows a lower specific activity than Xyl10g or Cel5B. However, Xyl10g GS Cel5B can degrade popping-pretreated rice straw, corn stover, kenaf, and oak more efficiently than the mixture of Xyl10g and Cel5B, by about 1.41-, 1.37-, 1.32-, and 1.40-fold, respectively. Our results suggest that Xyl10g GS Cel5B is an efficient hydrolytic enzyme and a suitable candidate for degrading lignocellulosics to produce fermentable sugar.

Efficient approach for bioethanol production from red seaweed Gelidium amansii.

Gelidium amansii (GA), a red seaweed species, is a popular source of food and chemicals due to its high galactose and glucose content. In this study, we investigated the potential of bioethanol production from autoclave-treated GA (ATGA). The proposed method involved autoclaving GA for 60min for hydrolysis to glucose. Separate hydrolysis and fermentation processing (SHF) achieved a maximum ethanol concentration of 3.33mg/mL, with a conversion yield of 74.7% after 6h (2% substrate loading, w/v). In contrast, simultaneous saccharification and fermentation (SSF) produced an ethanol concentration of 3.78mg/mL, with an ethanol conversion yield of 84.9% after 12h. We also recorded an ethanol concentration of 25.7mg/mL from SSF processing of 15% (w/v) dry matter from ATGA after 24h. These results indicate that autoclaving can improve the glucose and ethanol conversion yield of GA, and that SSF is superior to SHF for ethanol production.

Heterologous expression of endo-1,4-ß-xylanase A from Schizophyllum commune in Pichia pastoris and functional characterization of the recombinant enzyme.

Endo-1,4-ß-xylanase A (XynA) from Schizophyllum commune was cloned into pPCZαA and expressed in Pichia pastoris GS115. The open reading frame of the xynA gene is composed of 684 bp, encoding 278 amino acids with a molecular weight of 26 kDa. Based on sequence similarity, XynA belongs to the CAZy glycoside hydrolase family 11. The optimal activity of XynA was at pH 5 and 50 °C on beechwood xylan. Under these conditions, the K(m), V(max) and specific activity of XynA were 5768 units mg(-1), 4 mg ml(-1) and 9000 µmol min(-1)mg(-1), respectively. XynA activity was enhanced in the presence of cations, such as K(+), Na(+), Li(2+), Cd(2+), and Co(2+). However, in the presence of EDTA, Hg(2+) and Fe(3+), xylanase activity was significantly inhibited. This enzyme effectively degraded approximately 45% of unsubstituted xylans in the cell wall from poplar stems. The high level of XynA activity might increase the yield of enzyme hydrolysis from biomass. Thus, XynA could be used as a major component of a lignocellulosic degrading enzyme cocktail.