Mehdi Dashtban

Cellulase activities in biomass conversion: measurement methods and comparison

Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

Recent advancements in the production of hydroxymethylfurfural

Lignocellulosic materials can be utilized in the production of platform chemicals such as hydroxymethylfurfural (HMF). HMF production has been investigated in various aqueous, solvent, biphasic and ionic liquid systems. Aqueous medium usually suffers from a low HMF yield (usually 50 to 60% while using fructose as feedstock) due to the production of by-products and the decomposition of HMF. A higher HMF production was achieved by applying biphasic systems, however, these systems face some technical challenges including solvent recovery, process complexity and environmental issues, which prevent its practical implementations at industrial scales. The unique properties of ionic liquids (IL)s make them promising solvents for producing HMF from polysaccharides. In this review, the effects of various parameters such as catalysts, solvents, and process conditions on the production of HMF from various lignocellulosic feedstocks as well as systems developed for purifying HMF after production are discussed. Generally, the yield of HMF production in the IL systems was higher than 80% when fructose was used as the raw material, but was less than 50% when cellulose or other polysaccharides were used. However, the IL system is complicated and has a challenging recovery process. The proposed IL systems are also not environmentally friendly. The main emphasis of this paper is on the industrial applicability of proposed processes for producing HMF.

Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

BackgroundTrichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH.ResultsWe successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain.ConclusionsThe recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for β-glucosidase production as well as improving the biomass conversion using cellulases.

Acidification of prehydrolysis liquor and spent liquor of neutral sulfite semichemical pulping process

Acidification has been commercialized for producing kraft lignin from black liquor of kraft pulping process. This work intended to evaluate the effectiveness of acidification in extracting lignocelluloses from the spent liquor of neutral sulfite semichemical pulping (NSSC) process and from prehydrolysis liquor (PHL) of kraft-based dissolving pulp production process. The results showed that the NSSC and PHL spent liquors had some lignin-carbohydrate complexes (LCC), and that the square weighted counts of particles with a chord length of 50-150μm in the spent liquors were significantly increased as pH dropped to 1.5. Interestingly, the acidification reduced the lignosulfonate/lignin content of NSSC and PHL by 13% or 20%, while dropped their oligosugars content by 75% and 38%, respectively. On a dry basis, the precipitates had more carbon, hydrogen and a high heating value of 18-22MJ/kg, but less oxygen, than spent liquors. The precipitates of PHL could be used as fuel.

Lignin in Paper Mill Sludge is degraded by White-Rot Fungi in Submerged Fermentation

This study investigated biological treatment of paper mill sludge and lignin degradation by fungi. Four different Basidiomycetes white-rot fungi (WRF) were used: Coriolus versicolor, Tyromyces albidus, Trametes gallica and Pleurotus ostreatus. The fungi were cultured in submerged fermentation cultures with paper mill sludge. The pH values of the cultures and ligninolytic enzymes production profiles of the four fungi were monitored and reported here. The highest laccase activity of 202 U/L was obtained in T. albidus at day 25 while the maximum manganese-dependent peroxidase (MnP) activity of 50 U/L was obtained in C. versicolor from day 15 to 25. The P. ostreatus showed highest total cellulase activity with the peak of 0.26 μmol at day 15 while the other three WRF showed negligible total cellulase activity. The lignin contents were significantly decreased in the four WRF-treated sludge samples and were confirmed by acetyl bromide and FTIR analyses. The results suggested that lignin was preferentially used and significantly degraded or converted in all the four WRF-treated sludge samples.

Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei.

T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.