Current pretreatment technologies for the development of cellulosic ethanol and biorefineries

Lignocellulosic materials, such as forest, agriculture, and agroindustrial residues, are among the most important resources for biorefineries to provide fuels, chemicals, and materials in such a way to substitute for, at least in part, the role of petrochemistry in modern society. Most of these sustainable biorefinery products can be produced from plant polysaccharides (glucans, hemicelluloses, starch, and pectic materials) and lignin. In this scenario, cellulosic ethanol has been considered for decades as one of the most promising alternatives to mitigate fossil fuel dependence and carbon dioxide accumulation in the atmosphere. However, a pretreatment method is required to overcome the physical and chemical barriers that exist in the lignin–carbohydrate composite and to render most, if not all, of the plant cell wall components easily available for conversion into valuable products, including the fuel ethanol. Hence, pretreatment is a key step for an economically viable biorefinery. Successful pretreatment method must lead to partial or total separation of the lignocellulosic components, increasing the accessibility of holocellulose to enzymatic hydrolysis with the least inhibitory compounds being released for subsequent steps of enzymatic hydrolysis and fermentation. Each pretreatment technology has a different specificity against both carbohydrates and lignin and may or may not be efficient for different types of biomasses. Furthermore, it is also desirable to develop pretreatment methods with chemicals that are greener and effluent streams that have a lower impact on the environment. This paper provides an overview of the most important pretreatment methods available, including those that are based on the use of green solvents (supercritical fluids and ionic liquids)

Preliminary Characterization of a New Processive Endoglucanase from Clostridium alkalicellulosi DSM17461

The Clostridium alkalicellulosi DSM17461T genome contains several glucoside hydrolase encoding genes essential for cellulose degradation. Herein, the family 9 glycoside hydrolase enzyme (CalGH9_2089) was cloned and expressed. The enzyme contains one GH9 catalytic module, a family 3 carbohydrate-binding module, and one Type I dockerin at its C-termini. The optimal pH and temperature for CalGH9_2089 to hydrolyze CMC were 55 °C and pH 6.0, with the remaining activity of more than 60% at pH 10.0. CalGH9_2089 produced a series of cello-oligomers (G2-G6) from CMC, suggesting that the enzyme has an endo-acting capability. When regenerated amorphous cellulose was hydrolyzed with CalGH9_2089, the ratio of reducing ends in the soluble fraction to that in the insoluble pellets was 4.8, suggesting that this enzyme acts processively on RAC. This work extends our knowledge of the behavior of the GH9 endoglucanase from the microorganism living in an alkaline environment

CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.</jats:p

CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance