Steam-pretreated Spruce Research Articles

Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins

The cel5C gene, coding for an endoglucanase (Cel5C) of Penicillium brasilianum was cloned and heterologously expressed in Aspergillus oryzae. This is only the second GH5 EG from the genus Penicillium reported in the CAZy database. The promoter region of the gene has putative binding sites for both the carbon catabolite repressor CreA and the activator XlnR. The pH optimum of Cel5C was found to be 4.0 and the temperature optimum was 70 °C. At a typical temperature for lignocellulose hydrolysis Cel5C retained full residual activity after 20 h of incubation at pH 5.0 and 6.0. Adsorption to Avicel and steam pretreated spruce, was found to follow the Langmuir isotherm, and the maximum adsorption was similar for both substrates, 40 and 49 mg/g, respectively. The affinity for Avicel was 10 times higher than for steam pretreated spruce, 0.040 and 0.0035 L/mg, respectively. Non-productive binding of cellulolytic enzymes to lignin is an important obstacle to overcome for commercial biomass to ethanol production. Therefore, the adsorption on residual lignin produced from various biomass samples was investigated. Both substrate and pretreatment conditions resulted in different adsorptions of Cel5C to the residual lignin.

Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride

Cellulase and β-glucosidase production on steam pretreated spruce (SPS) with Trichoderma reesei Rut C30, Trichoderma atroviride TUB F-1505 and TUB F-1663 was investigated. The enzymes were compared in term of activity, temperature optima and hydrolytic capacity. The T. atroviride cellulases proved to have lower temperature optima for filter paper activity (FPA) assay (50 °C) and for hydrolysis of SPS (40 °C) than the Rut C30 enzymes (60 °C for FPA and 50 °C for hydrolysis). Due to high levels of extracellular β-glucosidases, the T. atroviride enzyme supernatants hydrolyzed the washed SPS to glucose more efficiently than the enzymes produced by T. reesei. On the other hand, when the whole fermentation broths were used instead of the supernatants, thus the mycelium bound enzymes were also present, the hydrolytic capacity of T. reesei Rut C30 was enhanced by approximately 200%, while an improvement of only 15% was observed in case of the T. atroviride isolates.

Enhanced enzymatic conversion of softwood lignocellulose by poly(ethylene glycol) addition

Ethanol production from lignocellulose has great potential and is an important step in changing fuel consumption to a more environmentally friendly alternative. Lignocellulose is a large source of biomass. However, with lignocellulose and softwood lingocellulose in particular, high conversion of cellulose into fermentable sugars requires large amounts of enzymes. Addition of surfactants is known to increase the enzymatic conversion and decrease the amount of enzymes needed. Surfactants and polymers with various amount of ethylene oxide (EO) content were used to study the conversion of steam-pretreated spruce lignocellulose. Increasing conversion was obtained with longer EO chains on the non-ionic surfactants. Similar results were obtained by using only the hydrophilic part of the surfactant, i.e. by addition of ethylene oxide polymers such as poly(ethylene glycol) (PEG) to the hydrolysis mixture. Interactions of enzymes and PEG with substrate was monitored with 14C-labeled PEG 4000 and 3H-labeled Cel7A (CBH I), the dominating cellulase from Trichoderma reesei. Addition of PEG to enzyme hydrolysis of lignocellulose increased the conversion from 42% without addition to 78% in 16 h. Adsorption of Cel7A decreased from 81 to 59%. No effect of PEG was seen on a delignified substrate. By addition of PEG it was possible to perform hydrolysis at 50 °C leading to both high cellulose conversion (80%) and shorter process time (48 h). Two different interactions are proposed in PEG adsorption on lignocellulose, hydrogen bonding and hydrophobic interactions. Our conclusions from experiments on lignocellulose and delignified substrate are that EO containing surfactants and polymers, such as PEG, bind to lignin by hydrophobic interaction and hydrogen bonding and reduce the unproductive binding of enzymes.

Influence of strain and cultivation procedure on the performance of simultaneous saccharification and fermentation of steam pretreated spruce

Yeast to be used in simultaneous saccharification and fermentation (SSF) of lignocelluloses materials has to be prepared in a separate cultivation step. The effects of the cultivation procedure on the performance of SSF of steam pretreated softwood were studied in the current work. The yeast used in the SSF was either directly commercially available Baker's yeast (as packaged yeast) or the same strain of yeast produced from the hydrolysate obtained in the pretreatment of the softwood material. A second strain of Saccharomyces cerevisiae TMB3000, isolated from spent sulphite liquor, was also compared with the commercial Baker's yeast. The strains were tested in SSF at substrate loads of 3, 5 and 8% dry weight of water insoluble material. Final ethanol yields were above 85% of the theoretical (based on the available hexoses) in all cases, except for the package yeast for the 8% substrate load, in which case the final yield was less than 65%. The cultivation procedure was found to have a significant impact on the performance during SSF, as well as in small-scale fermentations of hydrolysate liquor without solid material. The Baker's yeast cultivated on the hydrolysate from the steam pretreatment had in all cases a higher productivity, in particular at the highest substrate load. Cultivated Baker's yeast had a slightly higher productivity than TMB3000. The results suggest that the adaptation of the yeast to the inhibitors present in the medium is an important factor that must be considered in the design of SSF processes.

Production of cellulases by Penicillium brasilianum IBT 20888—Effect of substrate on hydrolytic performance

The fungus Penicillium brasilianum IBT 20888 was cultivated on three different carbon sources to investigate the effect of the carbon source on the enzyme production. The carbon sources used were Sigmacell cellulose (SC), steam pretreated spruce (SPS) and a mixture of SC, oat spelts xylan and birchwood xylan (SCXX). Enzymatic assays and capillary electrophoresis revealed clear differences among three enzyme preparations produced—both in activity levels and in the distribution between enzymes within the same class. The hydrolysis efficiency of the resulting enzyme preparations was studied on SC and SPS. The three enzyme preparations performed equally well on SPS using an enzyme loading of 25 FPU (g cellulose) −1. The enzyme preparation produced on SCXX resulted in 15% less glucose released from SC compared to the two other enzyme preparations. A lower total cellobiohydrolase level could in part explain this difference. Adsorption studies revealed that three-fold more protein was adsorbed onto SPS compared to SC due to the presence of lignin in SPS. The binding onto lignin was demonstrated to be non-specific as most proteins in the enzyme preparations were adsorbed. It was also demonstrated by the use of CE that some degradation of the enzymes occurred during the hydrolysis.

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.

Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose

Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of adsorption of the dominating cellulase of Trichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90% adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei.

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

Modeling simultaneous saccharification and fermentation of softwood.

Simultaneous saccharification and fermentation (SSF) of wood has been modeled for the past 15-20 years, but the substrates used for model evaluation have so far not included pretreated softwood. In the present study, data from lab-scale batch SSF of SO2-impregnated, steam-pretreated spruce chips were used to evaluate a model found in the literature. The model, which was somewhat modified, consists of a number of nonlinear, coupled ordinary differential equations, which were solved numerically. Some parameter values were fitted to data by use of least-squares minimization. A difficulty in parameter estimation was the lack of cellobiose measurements, something that was relieved by adding assumptions about parameter relations. The simulated concentration profiles agreed well with the measured concentrations of glucose and ethanol. It is therefore concluded that the basic model features apply to softwood SSF. The model predicts rate saturation with respect to enzyme concentration at concentrations above 60 FPU/g cellulose, although this was not observed in the experimental data, which only comprised enzyme concentrations up to 32 FPU/g cellulose.

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.

Cellulase enzyme production by various fungal strains on different carbon sources

In the process of making ethanol from lignocellulosics, enzyme production is still the most crucial and costly step. Trichoderma reesei Rut C-30 is the best known cellulase producer. This fungus is very good in endo- and exoglucanase production, however, the amount of β-glucosidase excreted is not sufficient for hydrolysis. In addition, T. reesei Rut C-30 is sensitive to inhibitors generated during steam-pretreatment of wood. In the present study, two good cellulase producers were selected from 16 fungal strains and investigated regarding filter paper activity (FPU) and β-glucosidase activity using inexpensive lignocellulosic carbon sources for cultivation. T. reesei Rut C-30 proved to be a good cellulase producer, resulting in a maximum FPU of 1.39 FPU ml Ś1 on washed, steam-pretreated willow, but the β-glucosidase activity was insufficient. High β-glucosidase activities were reached with T. viride OKI B1 on all substrates, with a maximum activity of 1.62 IU ml Ś1 on steam-pretreated spruce.

Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production

Economic optimization of the production of ethanol by simultaneous saccharification and fermentation (SSF) requires knowledge about the influence of substrate and enzyme concentration on yield and productivity. Although SSF has been investigated extensively, the optimal conditions for SSF of softwoods have yet not been determined. In this study, SO2-impregnated and steam-pretreated spruce was used as substrate for the production of ethanol by SSF. Commercial enzymes were used in combination with the yeast Saccharomyces cerevisiae. The effects of the concentration of substrate (2% to 10% w/w) and of cellulases (5 to 32 FPU/g cellulose) were investigated. SSF was found to be sensitive to contamination because lactic acid was produced. The ethanol yield increased with increasing cellulase loading. The highest ethanol yield, 68% of the theoretical based on the glucose and mannose present in the original wood, was obtained at 5% substrate concentration. This yield corresponds to 82% of the theoretical based on the cellulose and soluble glucose and mannose present at the start of SSF. A higher substrate concentration caused inefficient fermentation, whereas a lower substrate concentration, 2%, resulted in increased formation of lactic acid, which lowered the yield. Compared with separate hydrolysis and fermentation, SSF gave a higher yield and doubled the productivity. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 204–210, 2000.

Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production

Economic optimization of the production of ethanol by simultaneous saccharification and fermentation (SSF) requires knowledge about the influence of substrate and enzyme concentration on yield and productivity. Although SSF has been investigated extensively, the optimal conditions for SSF of softwoods have yet not been determined. In this study, SO2-impregnated and steam-pretreated spruce was used as substrate for the production of ethanol by SSF. Commercial enzymes were used in combination with the yeast Saccharomyces cerevisiae. The effects of the concentration of substrate (2% to 10% w/w) and of cellulases (5 to 32 FPU/g cellulose) were investigated. SSF was found to be sensitive to contamination because lactic acid was produced. The ethanol yield increased with increasing cellulase loading. The highest ethanol yield, 68% of the theoretical based on the glucose and mannose present in the original wood, was obtained at 5% substrate concentration. This yield corresponds to 82% of the theoretical based on the cellulose and soluble glucose and mannose present at the start of SSF. A higher substrate concentration caused inefficient fermentation, whereas a lower substrate concentration, 2%, resulted in increased formation of lactic acid, which lowered the yield. Compared with separate hydrolysis and fermentation, SSF gave a higher yield and doubled the productivity.

Cellulase production of Trichoderma reesei Rut C 30 using steam-pretreated spruce. Hydrolytic potential of cellulases on different substrates.

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C 30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hemicellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and Iogen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C 30. However, the conversion of cellulose using these enzymes was higher than that obtained with Iogen or Celluclast cellulases using steam-pretreated spruce as substrate.

Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol.

Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42 degrees C, using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate, at a cellulase loading of 37 filter paper units/g of cellulose, and a beta-glucosidase loading of 38 IU/g of cellulose. The detoxification of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37 degrees C, owing to the cessation of ethanol fermentation after the first 10 h.