Pretreated Spruce Research Articles

Characterization of cellulases and hemicellulases produced by Trichodermareesei on various carbon sources

Cellulase and hemicellulase enzymes were produced by Trichoderma reesei RUT C30 on steam pretreated spruce, willow, corn stover and delignified lignocellulose (Solka Floc), as a reference. The enzymes produced were characterized by protein and various enzyme activity measurements. On steam pretreated corn stover higher cellulolytic enzyme activities were reached than on Solka Floc, while the activities obtained on steam pretreated spruce and willow were considerably lower. The produced and two commercial cellulases (Celluclast 1.5 L and Econase CE) were compared by determining specific activities. There were minor differences among the enzymes corresponding to their specific cellulase activities. In contrast, within hemicellulase and β-glucosidase activities, the differences were found to be more significant. It should be also noted that commercial cellulases had considerably higher specific acetyl xylan esterase activities than the produced enzymes. According to subsequent hydrolysis experiments, performed to characterize the produced enzyme complexes to evaluate their applicability for hydrolysis and enzyme production, it seems that the application of the enzyme that was produced on the same substrate as was used for hydrolysis can be advantageous in the case of some substrates. As a result, these experiments demonstrated that pretreated corn stover is a good substrate both for enzyme production and hydrolysis, since high cellulolytic activities could be reached using it as carbon source. Moreover, high sugar yields could be obtained in the hydrolysis by the enzyme produced on steam pretreated corn stover.

A comparison between batch and fed-batch simultaneous saccharification and fermentation of steam pretreated spruce

In order to improve the process economy it is important to use as high dry matter content as possible in simultaneous saccharification and fermentation (SSF). However, too high dry matter content often gives rise to severe inhibition of the yeast metabolism, due to the increased levels of toxic compounds. The aim of the present work was to increase the fibrous content in SSF of steam pretreated spruce to 10% by adapting the yeast to the inhibitory substrate and by using a fed-batch process. Both batch and fed-batch approaches were evaluated. The fed-batch experiments were started with a batch fermentation containing 6% dry matter. Fibrous slurry from the pretreatment was then added four times during the first 24 h giving a final dry matter content corresponding to 10%. The yeast used in the fermentation was produced aerobically on the hemicellulose hydrolysate obtained from the pretreatment. SSF batch and fed-batch experiments with a cell mass concentration of 2, 3 and 5 g/L were carried out. When adapted yeast was used, the available hexoses were completely converted within 72 h and the final ethanol concentrations reached 40–44 g/L. No major differences in performance between batch and fed-batch were seen, but the ethanol productivity during the first 24 h was higher in the fed-batch SSF experiments, particularly during the experiments with a cell mass concentration of 2 and 3 g/L.

03/01882 Combustion of polydisperse fuel: Arakelyan, E. K. and Yanukyan, E. G. Izvestiya Akademii Nauk, 2002, (2), 60–67. (In Russian)

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.

Influence of enzyme loading and physical parameters on the enzymatic hydrolysis of steam-pretreated softwood.

Softwood is an interesting raw material for the production of fuel ethanol as a result of its high content of hexoses, and it has attracted attention especially in the Northern hemisphere. However, the enzymatic hydrolysis of softwood is not sufficiently efficient for the complete conversion of cellulose to glucose. Since an improvement in the glucose yield is of great importance for the overall economy of the process, the influence of various parameters on the cellulose conversion of steam-pretreated spruce has been investigated. The addition of beta-glucosidase up to 50 IU g(-)(1) cellulose to the enzymatic hydrolysis process resulted in increased cellulose conversion at a cellulase loading up to 48 FPU g(-)(1) cellulose. Despite very high enzyme loading (120 FPU g(-)(1) cellulose) only about 50% of the cellulose in steam-pretreated spruce was converted to glucose when all of the material following pretreatment was used in the hydrolysis step. The influence of temperature, residence time, and pH were investigated for washed pretreated spruce at a dry matter (DM) content of 5% and a cellulase activity of 18.5 FPU g(-)(1) cellulose. The optimal temperature was found to be dependent on both residence time and pH, and the maximum degree of cellulose conversion, 69.2%, was obtained at 38 degrees C and pH 4.9 for a residence time of 144 h. However, when the substrate concentration was changed from 5% to 2% DM, the cellulose conversion increased to 79.7%. An increase from 5% to 10% DM resulted, however, in a similar degree of cellulose conversion, despite a significant increase in the glucose concentration from 23 g L(-)(1) to 45 g L(-)(1). The deactivation of beta-glucosidase increased with increasing residence time and was more pronounced with vigorous agitation.

Comparison of enzymatic hydrolysis in a worldwide round robin assay

Within the IEA activity on Pretreatment of Lignocellulosic Materials, a number of laboratories performed enzymatic hydrolyses of a standard cellulose and pretreated spruce and aspen wood under strictly standardized conditions. The reproducibility of the time dependence of hydrolysis was examined by incubating Sigmacell 50 (20 g/litre) with a standard cellulase (Celluclast, Trichoderma sp.), 200 FPU/litre, at pH 4·8, 50°C. The liberated glucose was determined by HPLC or by the glucose oxidase method after 6, 24, 48 and 72 h. The individual deviation from the mean values of 11 laboratories was in the range 5–10%. In the enzyme-dependent hydrolysis, Sigmacell 50 was incubated with increasing amounts of Celluclast (5–100 FPU/g, 24 h). Based on the liberated glucose, the maximum digestibility of Sigmacell 50 calculated by 11 laboratories was 64·3±3·7% (mean±standard deviation). Similar reproducibility was obtained with pretreated aspen and spruce wood. This inter-laboratory comparison indicates that both tests, the time-dependent and the enzyme-dependent hydrolysis methods, are reproducible tools to evaluate the enzymatic hydrolysability of lignocellulosics for technical applications.

Comparison of the hydrolytic activity of commercial cellulase preparations

Enzymatic hydrolysis of SO2-impregnated, steam-pretreated aspen and spruce wood fractions was carried out at 2% and 10% substrate concentrations, using five commercial cellulase preparations supplemented with excess of a commercial s-glucosidase in order to obtain a common level of hydrolytic activity. Pretreated aspen wood was found to be more readily hydrolysed than pretreated spruce wood which appeared to contain inhibitory material detrimental to the cellulase enzymes. All five commercial cellulase preparations, when supplemented to the same level of s-glucosidase activity, exhibited similar hydrolysis profiles on the pretreated substrates.