Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Bioethanol production from agricultural wastes: An overview

Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review

Antioxidant and prooxidant properties of flavonoids.

This article reviews developments in the technology for ethanol production from lignocellulosic materials by “enzymatic” processes. Several methods of pretreatment of lignocelluloses are discussed, where the crystalline structure of lignocelluloses is opened up, making them more accessible to the cellulase enzymes. The characteristics of these enzymes and important factors in enzymatic hydrolysis of the cellulose and hemicellulose to cellobiose, glucose, and other sugars are discussed. Different strategies are then described for enzymatic hydrolysis and fermentation, including separate enzymatic hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), non-isothermal simultaneous saccharification and fermentation (NSSF), simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing (CBP). Furthermore, the by-products in ethanol from lignocellulosic materials, wastewater treatment, commercial status, and energy production and integration are reviewed.

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Acid-based hydrolysis processes for ethanol from lignocellulosic materials: a review.

The production of β-glucosidase by soil enrichment cultures grown on cellobiose and carboxymethylcellulose and several experimental conditions were investigated in this work. The highest rates of induced enzyme activity were found in the soil cultures in which 0.4% cellobiose at pH 8.0 was used as the sole C and energy source. A bacterial strain, producing significant amounts of enzyme associated with the cell extracts and which was identified as Pseudomonas pickettii , was isolated from these soil cultures. In a comparative way, induction and location of β-glucosidase in Aspergillus niger cultures (using 0.4% cellobiose as C substrate) were also studied. In contrast with the results obtained with the bacterial cultures, only low levels of activity were detected when no N source was used. However, an appreciable amount of enzyme was assayed when 0.2% peptone was included in the culture medium with more than 70% of the total activity located in the extramycelial fraction.

Induction of β-glucosidase in fungal and soil bacterial cultures

An alkaline phosphatase (EC 3.1.3.1) from Escherichia coli ATCC27257 was immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retained about 74% of the original enzymatic activity. The pH and temperature profile of the immobilized and free enzyme revealed a similar behavior. Kinetic parameters were determined: K(m) and K(i) values were 2.44 and 0.423 mM, respectively, for the phosphatase-polyresorcinol complex and 1.07 and 0.069 mM, respectively, for free phosphatase. The thermal and storage stabilities of the immobilized phosphatase were higher than those of the native one. On addition to soil, free enzyme was completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex was comparatively stable. Barley seed coated with the immobilized enzyme exhibited higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus was detected when the seed was encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot was observed.

Alkaline phosphatase-polyresorcinol complex: characterization and application to seed coating.

Three methods of wheat straw treatment (with NaOH, H2O2 and butylamine) and its subsequent saccharification by Trichoderma reesei cellulases and Aspergillus niger β-glucosidase are reported. The treatment of straw with NaOH for 1 h in the autoclave (120°C) caused a great loss of the hemicellulose content and a partial removal of lignin, provoking an increase of the cellulose content (from 24% to 69%) in the residue. When the straw was pre-treated with H2O2 at 25°C for 20 h, the relative content of cellulose in the straw increased (from 24% to 52%) due to the solubilisation of hemicellulose.The effect of varying the hydrolysis parameters (enzyme and substrate concentration, temperature and pH) was studied in order to maximise the yield of sugars. Under the best conditions and after 48 h with a mixture of 2:1 (w/w) cellulase/β-glucosidase (with a concentration of 7 and 0.1 U ml-1, respectively) the native, NaOH-treated and H2O2-treated straw material were degraded to reducing sugars for 28%, 89% and 97% res...

Enzymatic saccharification of pretreated wheat straw by T. reesei cellulases and A. niger β-glucosidase

Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co-flocculation with soil humates in the presence of Ca2+. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate–phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of Km (2.22 mM) and activation energy (33.4 kJ mol−1) similar to those of the free enzyme (2.00 mM and 27.6 kJ mol−1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80–100 °C, while the free enzyme had its highest activity at 60 °C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates.

© 2003 Society of Chemical Industry

Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

Fractional factorial screening design and response surface methodology were applied to optimize the entrapment of glucose oxidase in liposomes by the dehydration–rehydration vesicle (DRV) method. Phosphatidylcholine from different sources, cholesterol:phosphatidylcholine (Ch:PC) and enzyme:lipid (E:L) ratios, buffer pH, sonication frequency and trehalose concentration were the parameters selected for this study. The type of phosphatidylcholine was found to be the most important factor followed by the trehalose concentration, Ch:PC ratio, sonication frequency and E:L ratio. The pH did not play an important role in the response. By treating liposomes with trehalose, as cryoprotectant, the activity of entrapped enzyme decreased by 16%. Two of the factors (cholesterol:phosphatidylcholine and enzyme:lipid ratios) were further studied in a 32 central composite design. The optimized liposomal formulation with an entrapment efficiency of 24% was obtained for egg yolk PC with Ch:PC and E:L ratios of 0.95 and 14.69, respectively, at pH 6 and applying a sonication frequency of 150 W. Copyright © 2004 Society of Chemical Industry

Manuel Perez-Mateos

Papers

Induction of β-glucosidase in fungal and soil bacterial cultures

Alkaline phosphatase-polyresorcinol complex: characterization and application to seed coating.

Enzymatic saccharification of pretreated wheat straw by T. reesei cellulases and A. niger β-glucosidase

Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

Application of experimental design to the formulation of glucose oxidase encapsulation by liposomes