Biomasa residual de industria del Cannabis, una alternativa para la obtención de productos de alto valor agregado

A medida que como sociedad vamos dando más importancia a lograr una economía circular, se hace importante encontrar fuentes renovables aptas para la producción de biocombustibles y bioquímicos. En los últimos años, diversas fuentes de biomasa lignocelulósica han sido estudiadas para estos propósitos. Dentro de estas fuentes de biomasa se encuentra el cáñamo (Cannabis sativa L.), siendo parte de una industria que ha crecido a pasos agigantados en las últimas décadas, en Colombia, desde su legalización. Específicamente, la industria del cannabis medicinal es responsable de generar una enorme cantidad de residuos en forma de los tallos de la planta, considerados un subproducto de bajo valor. En esta revisión se compila la información de diferentes estudios sobre el aprovechamiento de la fracción de polisacáridos de biomasa cáñamo, mediante transformaciones químicas y bioquímicas, para la obtención de productos de valor agregado. Se encontró que la mayoría de estudios están enfocados en la obtención de bioetanol o biogás; se encontraron también reportes de otras moléculas como ácido succínico, ácido láctico, furfural, polihidroxialcanoatos y bisaboleno. La viabilidad a nivel industrial de todos estos procesos permanece siendo una incógnita, pues los pasos de pretratamiento, hidrólisis y de conversión final utilizados suelen ser costosos. Es necesario que los estudios que realicen en el futuro se enfoquen en optimizar las condiciones de estos procesos y hacerlos verdes y así asegurar que puedan ser escalados.

As we as a society, give more importance to achieving a circular economy, it becomes important to find renewable sources suitable for the production of biofuels and biochemicals. In the last years, several different sources of lignocellulosic biomass have been studied for these purposes. One of these biomass sources is hemp (Cannabis sativa L), being part of an industry that has grown through giant steps in the last decades, in Colombia, since its legalization. Specifically, the industry of medicinal hemp is responsible for the generation of huge amounts of residues in the form of the plant stalks, considered a low value subproduct. This review compiles the information of several studies about the exploitation of the polysaccharide portion of hemp biomass through chemical and biochemical transformations, obtaining value-added products. It was found that most of these studies focus on the production of bioetanol or biogas; reports of other molecules such as succinic acid, furfural, polyhydroxyalkanoates and bisabolene were also found. Industrial viability of these processes remains a question, since pretreatment, hydrolysis and final conversion steps are usually expensive. It necessary that future studies focus on optimizing conditions of these processes as well as making them green, ensuring that they can be scaled.

Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance / 生物工程学报

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

Exploring integrated methodology for phytoremediation and biofuel production potential of Eichhornia crassipes

Eichhornia crassipes (EC) is a well-known invasive weed in different aquatic ecosystems. Its effective and complete eradication remains a challenge. The plant is a heavy metal (HM) hyperaccumulator in water bodies; however, studies regarding its biomass utilisation post-phytoremediation remain limited. The abundant growth rate and biochemical composition make EC a promising lignocellulosic feedstock for biofuel production; hence could be a deterministic approach for solving the twin problems of water pollution and higher energy demand, which are the global pressing issues in today’s scenario. The present study aimed at evaluating the phytoremediation potential of EC followed by proximate and biochemical analysis to investigate its suitability for biofuel production. After two weeks, the EC removed above 90% of Lead (Pb) and 60% of Cadmium (Cd) at all experimental doses. Lower doses of HMs, especially Pb, showed stimulatory effects on E. crassipes leaf biomass (ECLB). The recovered ECLB from Pb contaminated water (1 mg L?1) was further analysed for moisture (89.23±0.86%), dry matter (10.77±0.60%), ash (11.91±1.20%), organic carbon (51.56±1.08%), cellulose (21.89±0.64%), hemicellulose (26.50±1.13%), lignin (5.62±0.83%), total carbohydrate (32.00±1.58%), and protein (20.83±0.52%) content. SEM imaging of harvested ECLB confirmed compact and rigid structure. The recorded peaks in FTIR-spectra (1015.21, 1153.71, 1246.01, 1339.63, 1419.71, 1540.71, 1646.80, 1736.73, 2933.03, and 3263.72 cm?1) indicate the presence of lignocellulosic biomass. XRD peak at 21.55? confirmed the crystalline fraction of cellulose in ECLB. The results of theoretical yields of H2 and CH4 co-generation (HMG) (210.85 mLH2/g DW and 150.28 mL CH4/g DW) and Bioethanol (0.278 g/g DW) derived from cellulose and hemicellulose content of ECLB were comparable to those in reported studies. Overall, this work demonstrates an integrated methodology of phytoremediation followed by biofuel production from the recovered phytobiomass.

Metagenomics approach to identify lignocellulose-degrading enzymes in the gut microbiota of the Chinese bamboo rat cecum

BACKGROUND: Lignocellulose is considered a renewable organic material, but the industrial production of biofuel from lignocellulose is challenging because of the lack of highly active hydrolytic enzymes. The guts of herbivores contain many symbiotic microorganisms that have evolved to hydrolyze plant lignocellulose. Chinese bamboo rats mainly consume high-fiber foods, indicating that some members of the intestinal tract microbiota digest lignocellulose, providing these rats with the energy required for growth. RESULTS: Here, we used metagenomics to analyze the diversity and functions of the gut microbiota in Chinese bamboo rats. We identified abundant populations of lignocellulose-degrading bacteria, whose main functions involved carbohydrate, amino acid, and nucleic acid metabolism. We also found 587 carbohydrate-active enzyme genes belonging to different families, including 7 carbohydrate esterase families and 21 glycoside hydrolase families. The glycoside hydrolase 3, glycoside hydrolase 1, glycoside hydrolase 43, carbohydrate esterase 4, carbohydrate esterase 1, and carbohydrate esterase 3 families demonstrated outstanding performance. CONCLUSIONS: The microbes and enzymes identified in our study expand the existing arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. This study also describes a powerful approach for targeting gut microbes and enzymes in numerous industries.

Biodegradation of furan aldehydes in lignocellulose hydrolysates / 生物工程学报

Lignocellulose is the most abundant renewable organic carbon resource on earth. However, due to its complex structure, it must undergo a series of pretreatment processes before it can be efficiently utilized by microorganisms. The pretreatment process inevitably generates typical inhibitors such as furan aldehydes that seriously hinder the growth of microorganisms and the subsequent fermentation process. It is an important research field for bio-refining to recognize and clarify the furan aldehydes metabolic pathway of microorganisms and further develop microbial strains with strong tolerance and transformation ability towards these inhibitors. This article reviews the sources of furan aldehyde inhibitors, the inhibition mechanism of furan aldehydes on microorganisms, the furan aldehydes degradation pathways in microorganisms, and particularly focuses on the research progress of using biotechnological strategies to degrade furan aldehyde inhibitors. The main technical methods include traditional adaptive evolution engineering and metabolic engineering, and the emerging microbial co-cultivation systems as well as functional materials assisted microorganisms to remove furan aldehydes.

Progress in detoxification of inhibitors generated during lignocellulose pretreatment / 生物工程学报

Lignocellulose can be hydrolyzed by cellulase into fermentable sugars to produce hydrogen, ethanol, butanol and other biofuels with added value. Pretreatment is a critical step in biomass conversion, but also generates inhibitors with negative impacts on subsequent enzymatic hydrolysis and fermentation. Hence, pretreatment and detoxification methods are the basis of efficient biomass conversion. Commonly used pretreatment methods of lignocellulose are chemical and physic-chemical processes. Here, we introduce different inhibitors and their inhibitory mechanisms, and summarize various detoxification methods. Moreover, we propose research directions for detoxification of inhibitors generated during lignocellulose pretreatment.

Lytic polysaccharide monooxygenase of soil actinomycete with potential use for lignocellulose biodegradation

Aims@#Lytic polysaccharide monooxygenase (LPMO) is an enzyme capable of cleaving glycoside bonds of recalcitrant polysaccharides through an oxidative mechanism. LPMO activity, in synergy with hydrolytic enzymes, increases the production of monomer sugars from the biodegradation of lignocellulose. This study was aimed at evaluating actinomycete S2 strain LPMO activity based on the release of xylose as one of reducing sugar and hydrogen peroxide (H2O2) in the course of lignocellulosic biodegradation. @*Methodology and results@#The oxidation activity of LPMO from actinomycete S2 strain was measured by using the substrate of Avicel supplemented with ascorbic acid and copper ions (Cu2+) to identify its effect on the release of xylose as one of reducing sugar. The optimum incubation time for the LPMO production was also conducted. Further, H2O2 quantitative analysis was performed as by-product of LPMO activity and 16S rRNA gene sequence of actinomycete S2 strain were subsequently determined. We found that supplementation of 1 mM ascorbic acid and 0.2 mM Cu2+ increased xylose as one of reducing sugar production by up to 5-fold from 255.03 to 1290 μg/mL after an optimal incubation period of 6 days. Based on H2O2 production, the LPMO activity of actinomycete S2 strain was 0.019 ± 0.001 U/mL. There is likelihood that LPMO activity derived from actinomycete S2 strain has a synergistic effect with the activity of other lignocellulose-degrading enzymes. This actinomycete showed 99% similarity to the 16S rRNA gene sequence of Streptomyces avermitilis strain EAAG80. @*Conclusion, significance, and impact of study@#LPMO enzyme activity from actinomycete S2 strain as determined by the production of reducing sugar and H2O2 was greatly increased by supplementation with ascorbic acid as an electron donor and Cu2+ ions. To the best of our knowledge, this is the first elucidation of LPMO activity from an indigenous Indonesian actinomycete.

Hydrothermal Pretreatment as a Strategy for the Improvement of Sugarcane Bagasse Saccharification by Fungal Enzyme Blend

Abstract Obtaining low cost lignocellulolytic enzymes and efficient biomass pretreatment are key to increase the competitiveness of second-generation ethanol in comparison with fossil fuels. The enzymatic cocktail produced by the Chrysoporthe cubensis fungus as well as the mixture prepared with the cocktails of the Chrysoporthe cubensis and Penicillium pinophilum fungi have already proven to be efficient for hydrolyzing biomass pretreated with alkali. In this study, they were evaluated in saccharification of sugarcane bagasse pretreated with dilute acid or hot water at 121°C using an enzyme loading equal to 8 filter paper units per gram of biomass. The most promising results were obtained from the hydrolysis of biomass pretreated with hot water by the C. cubensis-P. pinophilum enzymes blend. In this condition, the glucose and xylose production were 25.2 g.L-1 and 4.6 g.L-1, respectively, that resulted in the conversion of 68% of glucan and 23% of xylan in only 48 hours. This study shows that the hydrothermal pretreatment is a promising alternative to improve the enzymes performance, produced by the fungi C. cubensis and P. pinophilum, in the sugarcane bagasse hydrolysis without the need of chemical compounds, generally used in the acid and alkali pretreatments. Furthermore, the hydrothermal pretreatment for 60 min allowed all cocktails applied to convert the cellulose efficiently with only 24 h of saccharification, which contributes to the energy savings employed in the process.

Isolation, identification, and optimization of laccase from Alternaria alternata

Laccase, lignin peroxidase, and manganese peroxidase have a synergistic effect on a wide range of recalcitrantcompounds. Among them, laccase is polyphenol oxidase widely available in fungi, plant species, and insects.Laccase has an important role in effluent decoloration, detoxification of pulp bleaching, and bioremediationprocess. Screening was carried out to find new fungal isolate for the presence of laccase activity with guaiacolas indicator compound. Sixteen fungal isolates were obtained from biodeteriorated agro waste and the woodsamples were collected from North Gujarat region of India. Among these isolates, one of the fungal isolateswas observed with good laccase activity and identified as Alternaria alternata. Laccase activity was determinedusing 2,2’-azinobis-(3-ethylbenzethiazoline-6-sulfonate) as substrate. Various production parameters such aspH, temperature, various carbon sources, nitrogen source, inducers, and cations were used to select the optimumcondition for further increase in the production of this enzyme. Maximum laccase activity was obtained withglucose and sucrose as carbon source, 0.2% ammonium sulfate as nitrogen source, and 0.06% Cu+2 with 1.5 mMveratryl alcohol as inducer under optimized condition.

Isolation of thermotolerant Bacillus subtilis DCH4 from Chancos hot spring (Carhuaz, Peru) with potential to degrade lignocellulosic agriculture wastes / Aislamiento de Bacillus subtilis DCH4 termotolerante de la fuente termal Chancos (Carhuaz, Perú) con potencial para degradar residuos lignocelulósicos agrícolas

Abstract It was isolated bacteria strains from three different types of samples: fresh water, in situ baits and ex situ enrichment. Serial dilutions were prepared and culture was carried at 50 °C using a Basal-Saline medium. Isolated strains were screened for endoglucanase and xylanase activities with qualitative (Congo Red) and quantitative (DNS) methods. Molecular 16S rDNA sequencing analysis was performed for taxonomic identification. It was isolated 31 strains of which 14 showed hydrolytic activities and belonged to Bacillus subtilis and Bacillus licheniformis species. Moreover, the strain B. subtilis DCH4 showed the highest endoglucanase activity at 45°C and pH 5, and xylanase activity at 55°C and pH 6. Then, DCH4 was cultivated by submerged fermentation with two different media supplemented with sugar cane bagasse, wheat straw, or quinoa stalk to evaluate its saccharification capability. Likewise, it was screening its xylanase and cellulase genes employing specific primers; the amplicons obtained were sequenced, and analyzed. It was found that, enzymatic extracts of DCH4 prepared with cane bagasse or quinoa stalk media achieved the highest endoglucanase and xylanase activities. According to molecular analysis of genes involved in the hydrolytic process, the endoglucanase and xylanase activities exhibited by DCH4 could be attributed to a bifunctional cellulase conformed by endo-beta-1,4-glucanase (GH5) joined to cellulose binding domain 3 (CBM3), and an endo-1,4-beta-xylanase (GH11), respectively. Further transcriptomic experiments would be considered to accomplish optimization strategies for biofuel production from lignocellulosic biomass.

Resumen Se aislaron cepas de bacterias provenientes de tres tipos de muestras: agua fresca, cebos enriquecidos in situ y ex situ. Se prepararon diluciones seriadas y el cultivo fue a 50 °C usando un medio Salino-Basal. Las cepas aisladas fueron tamizadas para las actividades endoglucanasa y xilanasa con métodos cualitativos (Rojo Congo) y cuantitativos (DNS). Se usó el análisis molecular 16S rDNA para la identificación taxonómica. Se aislaron 31 cepas, de las cuales 14 mostraron actividades hidrolíticas y pertenecían a Bacillus subtilis y Bacillus licheniformis. Además, B. subtilis DCH4 mostró la mayor actividad endoglucanasa a 45 °C y pH 5, y xilanasa a 55 °C y pH 6. Entonces, DCH4 se cultivó por fermentación sumergida con dos medios diferentes suplementado con bagazo de caña de azúcar, paja de trigo o tallo de quinua para evaluar su capacidad de sacarificación. También, se exploraron los genes de xilanasa y celulasa mediante cebadores específicos; los amplicones obtenidos fueron secuenciados y analizados. Se encontró que los extractos enzimáticos de DCH4 preparados con bagazo de caña o tallos de quinua mostraron las actividades endoglucanasa y xilanasa más elevadas. De acuerdo a los análisis moleculares de los genes involucrados en el proceso hidrolítico, las actividades de endoglunacasa y xilanasa exhibidas por DCH4 podrían atribuirse a una celulasa bifuncional conformada por una endo-beta-1,4-glucanasa (GH5) unida al dominio celulosa 3 (CBM3), y una endo-1,4-beta-xilanasa (GH11), respectivamente. Posteriores experimentos transcriptómicos podrían ser considerados para lograr estrategias de optimización para la producción de biocombustibles a partir de biomasa lignocelulósica.

Progress in applying surfactants to lignocellulose hydrolysis for sugar production / 生物工程学报

Lignocellulose is a major biomass resource for the production of biofuel ethanol. Due to its abundance, environmental friendliness and renewability, the utilization of lignocellulose is promising to solve energy shortage. Surfactant can effectively promote the enzymatic hydrolysis of lignocellulose. By discussing the influence and mechanism of different surfactants on the enzymatic hydrolysis, we provide references for finding appropriate surfactants in enzymatic hydrolysis process.

Advances in the synthesis of biobutanol by consolidated bioprocessing from lignocellulose / 生物工程学报

Consolidated bioprocessing (CBP) is a multi-step process in a bioreactor, which completes hydrolase production, enzymatic hydrolysis, and microbial fermentation. It is considered to be the most promising process for the production of second-generation biofuels because of its simple steps and low cost. Due to the complexity of lignocellulose degradation and the butanol synthesis pathway, few wild microorganisms can directly utilize lignocellulose to synthesize butanol. With the development of synthetic biology, single-bacterium directly synthesizes butanol using lignocellulose by introducing a butanol synthesis pathway in the cellulolytic Clostridium. However, there are still some problems such as heavy metabolic load of single bacterium and low butanol yield. Co-culture can relieve the metabolic burden of single bacterium through the division of labor in different strains and can further improve the efficiency of butanol synthesis. This review analyzes the recent research progress in the synthesis of biobutanol using lignocellulose by consolidated bioprocessing from both the single-bacterium strategy and co-culture strategy, to provide a reference for the research of butanol and other biofuels.

Qualitative analyses of lignocellulolytic enzymes produced by Emericella sp strain HST9 under diverse culture conditions

Aim: Optimization of cultural conditions for improved lignocellulolytic enzyme production by an ascomycete HST9 isolated from leaf and litter waste. Methodology: The fungus HST9 was isolated from leaf and litter waste collected from Chaudhary Charan Singh Haryana Agricultural University, Hisar, India. Culture experiments were conducted at different temperature, pH, incubation periods and aeration conditions. Effects of addition of different concentrations of various metal ions, different carbon complexes and nitrogen salts on enzyme production were also studied under submerged culture condition. Enzyme activities were measured by standard protocols using spectrophotometer. Results: HST9 grew well and produced optimum enzymes at 30oC on 7th day of incubation at stationary conditions and pH- 6. Overall enzyme activities decreased after addition of metal salts. Carboxymethyl cellulose (300 mg l-1) and alkali lignin (200 mg l-1) were observed to be the best carbon complexes for cellulolytic and ligninolytic activities. Ammonium sulfate was found to be a better nitrogen source compared to others. Under optimum conditions, different enzyme activities observed were 0.011 IU m l-1 FPase, 0.015 IU ml-1 CMCase, 6.5 IU m l-1 Lac, 57.5 IU m l-1 LiP and 4 IU ml-1 MnP. Molecular phylogenetic analysis of the strain confirmed that strain HST9 showed closeness with genus Emericella. Interpretation: Lignocellulolytic enzyme activity of Emericella isolate HST9 enhanced at optimum culture conditions, signifying that it can be used as a biological agent to degrade lignocellulosic waste.

Assessment of wasteland derived biomass for bioethanol production

Background: The bioethanol produced from biomass is a promising alternative fuel. The lignocellulose from marginal areas or wasteland could be a promising raw material for bioethanol production because it is present in large quantities, is cheap, renewable and has favorable environmental properties. Despite these advantages, lignocellulosic biomass is much more difficult to process than cereal grains, due to the need for intensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Therefore, there is a need to develop an efficient and cost-effective method for the degradation and fermentation of lignocellulosic biomass to ethanol. Results: The usefulness of lignocellulosic biomass from wasteland for the production of bioethanol using pretreatment with the aid of ionic liquids of 1-ethyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium chloride was evaluated in this study. The pretreatment process, enzymatic hydrolysis and alcoholic fermentation lasted a total of 10 d. The largest amounts of bioethanol were obtained from biomass originating from agricultural wasteland, in which the dominant plant was fireweed (Chamaenerion angustifolium) and from the field where the common broom (Cytisus scoparius) was the dominant. Conclusions: The plants such as fireweed, common broom, hay and goldenrod may be useful for the production of liquid biofuels and it would be necessary in the further stage of research to establish and optimize the conditions for the technology of ethyl alcohol producing from these plant species. Enzymatic hydrolysis of biomass from agricultural wastelands results in a large increase in fermentable sugars, comparable to the enzymatic hydrolysis of rye, wheat, rice or maize straw.

Improvement of ethanol production from birch and spruce pretreated with 1-H-3-methylmorpholinium chloride

Background: Pretreatment is the critically important step for the production of ethanol from lignocelluloses. In this study, hardwood birch (Betula pendula) and softwood spruce (Norway spruce) woods were pretreated with a newly synthesized morpholinium ionic liquid, 1-H-3-methylmorpholinium chloride ([HMMorph][Cl]), followed by enzymatic hydrolysis and fermentation to ethanol. Results: [HMMorph][Cl] was synthesized using inexpensive raw materials, i.e., hydrochloric acid and N-methyl morpholine, following a simple process. The influence of pretreatment time (2, 3, 5, and 8 h) and temperature (120 and 140°C) in terms of hydrolysis efficiency was investigated. Glucose yields from enzymatic hydrolysis were improved from 13.7% to 45.7% and 12.9% to 51.8% after pretreatment of birch and spruce woods, respectively, under optimum pretreatment conditions (i.e., at 140°C for 3 h) as compared to those from pristine woods. Moreover, the yields of ethanol production from birch and spruce were increased to 34.8% and 44.2%, respectively, while the yields were negligible for untreated woods. Conclusions: This study demonstrated the ability of [HMMorph][Cl] as an inexpensive agent to pretreat both softwood and hardwood.

Production optimization, purification, expression, and characterization of a novel α-L-arabinofuranosidase from Paenibacillus polymyxa

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.

Chemical pretreatment of Arundo donax L. for second-generation ethanol production

Background: Pretreatment of lignocellulosic biomass is essential for using it as a raw material for chemical and biofuel production. This study evaluates the effects of variables in the chemical pretreatment of the Arundo biomass on the glucose and xylose concentrations in the final enzymatic hydrolysate. Three pretreatments were tested: acid pretreatment, acid pretreatment followed by alkaline pretreatment, and alkaline pretreatment. Results: The amounts of glucose and xylose released by the enzymatic hydrolysis of the Arundo biomass obtained from acid pretreatment ranged from 6.2 to 19.1 g/L and 1.8 to 3.1 g/L, respectively. The addition of alkaline pretreatment led to a higher yield from the enzymatic hydrolysis, with the average glucose concentration 3.5 times that obtained after biomass hydrolysis with an acid pretreatment exclusively. The use of an alkaline pretreatment alone resulted in glucose and xylose concentrations similar to those obtained in the two-step pretreatment: acid pretreatment followed by alkaline pretreatment. There was no significant difference in 5-hydroxymethylfurfural, furfural, or acetic acid concentrations among the pretreatments. Conclusion: Alkaline pretreatment was essential for obtaining high concentrations of glucose and xylose. The application of an alkaline pretreatment alone resulted in high glucose and xylose concentrations. This result is very significant as it allows a cost reduction by eliminating one step.

Production of thermostable ß-glucosidase and CMCase by Penicillium sp: LMI01 isolated from the Amazon region

Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and ß-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of ß-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the ß-glucosidase activity was optimal at pH 6.0. Both CMCase and ß-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and ß-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.

Colour changes as an indicator for estimating fungal growth in solid state fermentation

@#Aims:In this study, measurement of colour changes during solid state fermentation (SSF) are presented as one of the potential techniques that can be used to describe growth, complementary to other biomass estimation, such as weight of fungus, spores concentration, organic matter loss, glucosamine and enzyme activity, which is directly related to growth.Methodology and results: In this study, fungal fermentation of Aspergillus awamoriand A.oryzaeare carried out on complex heterogeneous solid media; wheat bran, soybean hull and rapeseed meal, which are constituted of various soluble and insoluble solid particles. Fermented mass was extracted using distilled water to obtain a cell free extract, which canbe determined quickly and accurately using UV-Visible spectrophotometermeasured absorbance at 300 nm. The results showed a significant correlation between colours produced from fungal SSF and the concentration of spores, weight of fungus cells, organic matter loss, glucosamine and enzyme activity. We found that the colour density proportionally increased when the studied parameters mentioned above increased.For the growth of A. awamoriand A. oryzaeon wheat bran, soybean hulls and rapeseed meal, it was confirmed that colour production was directly proportional to fungal growth. In general, colour-based methods seem to be the most promising approach for biomass estimation in SSF.Conclusions, significance and impacts of study:The theory of measuring colour changes in SSF by UV-visible spectroscopy demonstrates that the colour method gives some valuableinformation than just obtaining a visual observation or spore count to describe growth of fungal mycelium in SSF. The ideas obtained from this exercise might provide a quick and convenient method for quality control of fungal growth. The advantages of this method are that the procedure is simple to carry out, it is non-destructive and no special and expensive reagents are required and the process is very cheap.This newapproach is an important complimentation to the existing techniques especially for basic studies.

Progress in lytic polysaccharide monooxygenase / 生物工程学报

Lignocellulose is the most abundant renewable biomass resource. Enzymatic breakdown of lignocellulose into oligosaccharides or monosaccharides is the key to exploit lignocellulosic biomass. However, traditional glycoside hydrolases are insufficient to degrade lignocellulose. The emergence of lytic polysaccharide monooxygenase, a novel enzyme for lignocellulose degradation, has enriched the deconstruction schema and accelerated the enzymatic conversion of polysaccharides, by introducing new chain breaks that allow hydrolases to initiate further degradation. Here, we review the discovery, classification and catalytic mechanism of the enzyme, as well as the methods for assaying its activity. The prospect for its application in feed additive, functional food and biofuel development is further discussed.