Enzymatic hydrolysis of biomass from wood

Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta‐xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta‐mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2–20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood.     

Enzymatic hydrolysis of cellulose and various pretreated wood fractions

Three strains of Trichoderma-T. reesei C30, T. reesei QM9414, and Trichoderma species E-58-were used to study the enzymatic hydrolysis of pretreated wood substrates. ach of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which had been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis.     

Enzymatic hydrolysis of cellulose in a membrane bioreactor: assessment of operating conditions

The optimization of operating conditions for cellulose hydrolysis was systemically undertaken using an ultra-scaled down membrane bioreactor based on the parameter scanning ultrafiltration apparatus. The bioconversion of cellulose saccharification was carried out with freely suspended cellulase from Aspergillus niger as the biocatalyst. The polyethersulfone ultrafiltration membranes with a molecular weight cutoff of 10 kDa were used to construct the enzymatic membrane bioreactor, with the membrane showing a complete retaining of cellulase and cellobiase. The influence of solution pH, temperature, salt (NaCl) concentration, presence of cellobiase, cellulose-to-enzyme ratio and stirring speed on reducing sugar production was examined. The results showed that the addition of an appropriate amount of NaCl or cellobiase had a positive effect on reducing sugar formation. Under the identified optimal conditions, cellulose hydrolysis in the enzymatic membrane bioreactor was tested for a long period of time up to 75 h, and both enzymes and operation conditions demonstrated good stability. Also, the activation energy (E _a) of the enzymatic hydrolysis, with a value of 34.11 ± 1.03 kJ mol⁻¹, was estimated in this study. The operational and physicochemical conditions identified can help guide the design and operation of enzymatic membrane bioreactors at the industrial scale for cellulose hydrolysis.     

Enzymatic hydrolysis of alpha-chitin

It was shown that the enzymatic preparation Celloviridin G20x can be used for the hydrolysis of alpha-chitin of various origin. The purity of the final product of hydrolysis, N-acetylglucosamine, was monitored using HPLC.     

Enzymatic hydrolysis of molasses

Kinetic studies of the enzymatic hydrolysis of molasses were conducted using glucoamylase. Central Sugar Refinery SDN BHD contains 13-20% glucose. The molasses was diluted and the kinetic experiments were conducted at 67 degrees C with 100-1000 mg/l of glucoamylase. The glucose contents of the molasses were enhanced after hydrolysis of molasses solution with 1000 mg/l glucoamylase. A Lineweaver-Burk plot was obtained based on enzyme kinetic data. The rate constant, Km and maximum reaction rate, Vmax for 500 mg/l of glucoamylase were 100 mmol/l (18 g/l) and 5 mmol/l min (0.9 g/l min), respectively. The maximum reaction rate, Vmax for 1000 mg/l of glucoamylase was doubled, to 100 mmol/l (18 g/l) and the rate constant, Km was the same for 500 mg/l of glucoamylase. The substrate inhibition model was noncompetitive based on the resulting Lineweaver-Burk plot for enzyme concentration of 500 and 1000 mg/l.     

Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor

An ultrafiltration membrane reactor was used to investigate the recovery of biocatalysts during enzymatic hydrolysis of pretreated sallow. Product inhibition could be eliminated by continuous removal of products through the ultrafiltration membrane, thus retaining the macromolecular substrate and enzymes. In this way, the degree of conversion was improved from 40% in a batch hydrolysis to 95% (within 20 h), and the initial hydrolysis rate was increased up to seven times. The recovery studies were focused on mechanical deactivation and irreversible adsorption on to the nonconvertible fraction of the substrate. Cellulase deactivation during mechanical agitation was not significant, and the loss of activity was attributed mainly to strong adsorption of the enzymes onto undigested material. This process was studied in semicontinuous hydrolyses, where fresh substrate was added intermittently. The amount of reducing sugars produced in this experiment was 25.7 g/g enzyme, compared to 4.7 g/g enzyme in a batch hydrolysis.     

Noncatalytic fast hydrolysis of wood

Willow without any pretreatment, and water were studied in an optical micro-reactor, diamond anvil cell by rapid heating (7-10°C/s) to high temperatures and high pressures (up to 403°C and 416 MPa), most of willow (89-99%) dissolved and hydrolyzed in water at 330-403°C within 22 s. It was found that low-density water (e.g., 571 kg/m(3)) solubilized almost all willow with particle size less than 200 μm, and subsequently hydrolyzed to hydrolysates in subcritical water at 354°C and 19 MPa within 9 s. These results were further used to propose a flow process to fast hydrolyze wood in seconds to valuable sugars.     

Enzymatic hydrolysis of gelatin]

The process of gelatin hydrolysis by means of enzymes of the proteolytic action with the aim to determine most effective destructor of gelatin macromolecules for recovering permeability and selective properties of ultrafiltration membranes was investigated. The presence of free alpha-NH2-groups was determined by means of the Lee and Takahashi method. Calculation of the destruction degree of substances in the Lee and Takahashi method during determination of the quantity of free alpha-NH2-groups rose precision of the method by 6-8%. The maximum degree of destruction (48.2% for 1-2 hours) was provided by the enzyme preparation "Pronaz-1" (Str. griseus + Acr. chrysogenum) and by industrial enzymes: alkali proteaze and proteaze C.     

Enzymatic hydrolysis of nucleoside phosphoramidates

The nucleoside phosphoramidate thymidine-5′-phospho-α-naphthylamidate and thymidine-3′-phospho-α-naphthylamidate were prepared as fluorogenic substrates for the study of enzymatic hydrolysis of the PN bond. With these new substrates, the rate and specificity of hydrolysis of the PN bond of the nucleoside phosphoramidate by snake venom and spleen phosphodiesterase could be studied. It was found that the 5′-phosphoramidate was hydrolyzed by snake venom phosphodiesterase and the 3′-phosphoramidate was hydrolyzed only by the spleen phosphodiesterase. Thus, the specificity requirement for PN bond cleavage is similar to that of the P0 bond cleavage, even though the rate is much slower.     

Enzymatic hydrolysis of wheat straw: Kinetic analysis

Pretreated wheat straw was enzymatically hydrolyzed in 250-ml flasks using the culture filtrate of Trichoderma reesei QM-9414. The influence of the initial enzymatic activity in the liquid phase was studied. The unreacted core model was used to analyze the experimental data obtained at 40, 46 and 50°C. The model adequately describes the data for hydrolysis times lower than 10 h.