Steam-pretreated Softwood Research Articles

Fungal loosenin-like proteins boost the cellulolytic enzyme conversion of pretreated wood fiber and cellulosic pulps

Microbial expansin-related proteins, including loosenins, can disrupt cellulose networks and increase enzyme accessibility to cellulosic substrates. Herein, four loosenins from Phanerochaete carnosa (PcaLOOLs), and a PcaLOOL fused to a family 63 carbohydrate-binding module, were compared for ability to boost the cellulolytic deconstruction of steam pretreated softwood (SSW) and kraft pulps from softwood (ND-BSKP) and hardwood (ND-BHKP). Amending the Cellic® CTec-2 cellulase cocktail with PcaLOOLs increased reducing products from SSW by up to 40 %, corresponding to 28 % higher glucose yield. Amending Cellic® CTec-2 with PcaLOOLs also increased the release of glucose from ND-BSKP and ND-BHKP by 82 % and 28 %, respectively. Xylose release from ND-BSKP and ND-BHKP increased by 47 % and 57 %, respectively, highlighting the potential of PcaLOOLs to enhance hemicellulose recovery. Scanning electron microscopy and fiber image analysis revealed fibrillation and curlation of ND-BSKP after PcaLOOL treatment, consistent with increasing enzyme accessibility to targeted substrates.

Overcoming extended lag phase on optically pure lactic acid production from pretreated softwood solids.

Optically pure lactic acid (LA) is needed in PLA (poly-lactic acid) production to build a crystalline structure with a higher melting point of the biopolymer than that of the racemic mixture. Lignocellulosic biomass can be used as raw material for LA production, in a non-food biorefinery concept. In the present study, genetically engineered P. acidilactici ZP26 was cultivated in a simultaneous saccharification and fermentation (SSF) process using steam pretreated softwood solids as a carbon source to produce optically pure D-LA. Given the low concentrations of identifiable inhibitory compounds from sugar and lignin degradation, the fermentation rate was expected to follow the rate of enzymatic hydrolysis. However, added pretreated solids (7% on weight (w/w) of water-insoluble solids [WIS]) significantly and immediately affected the process performance, which resulted in a long lag phase (more than 40h) before the onset of the exponential phase of the fermentation. This unexpected delay was also observed without the addition of enzymes in the SSF and in a model fermentation with glucose and pretreated solids without added enzymes. Experiments showed that it was possible to overcome the extended lag phase in the presence of pretreated softwood solids by allowing the microorganism to initiate its exponential phase in synthetic medium, and subsequently adding the softwood solids and enzymatic blend to proceed to an SSF with D-LA production.

Two-stage sulfite steam pre-treatment achieves extraordinary bioethanol titer from simultaneously saccharification and fermentation of softwood whole slurries

Softwood biomass, which contains rich portion of readily fermentable sugars, is a type of abundant biomass in Canada and Nordic countries. However, its recalcitrant structure has severely limited its utilization in bioconversion. Sulfite treatment during steam pre-treatment to simultaneously enhance ease of hydrolysis and fermentation of softwood biomass has turned to be an attractive strategy to unlock the possibility of converting softwood to bioethanol. Our previous work showed applying one-stage sulfite treatment during the steam pre-treatment under neutral/alkaline conditions could maximize the sulfonation/delignification and hemicellulose sugar retention, while the use of acidic conditions could enhance the homogenous substrates size reduction. To take the advantages of both the neutral/alkaline and acidic conditions, we developed two-stage sulfite steam pre-treatment to maximize the sugar and ethanol production by utilizing Na2SO3 in the first alkaline steam pre-treatment stage and SO2 in a second acidic steam pre-treatment stage. The resulting substrates showed high sulfonation/delignification extent, nearly 100 % sugar recovery, homogeneous substrates size reduction and sufficient detoxification. Without extra hydrolysate detoxification and concentration, 160 g/L fermentable sugars were obtained from the hydrolysis of two-stage sulfite steam pretreated softwood whole slurry at 25% solid loading. And 80 g/L ethanol was generated by directly fermenting these sugars in the whole slurry hydrolysate without additional detoxification or concentration processes. This work significantly contributed to the development of the industrial relevant bioethanol production from softwood biomass.

Boosting enzymatic hydrolysis of steam-pretreated softwood by laccase and endo-β-mannanase enzymes from Streptomyces ipomoeae CECT 3341

Ligninases and hemicellulases are crucial as accessory enzymes to increase the enzymatic hydrolysis of lignocellulose, boosting sugars production from which biofuels and bioproducts could be obtained. In order to find new sources of these accessory enzymes, this study evaluates the potential of laccase and mannanase enzymes from Streptomyces ipomoeae for improving the conventional hydrolysis with commercial cellulases of steam-pretreated softwood. For that, different laccase treatment and mannanase supplementation strategies were performed. S. ipomoeae laccase increased both glucose and xylose production (17.8% and 9.3%, respectively), which was attributed to a removal of phenols of 29%. Moreover, the combination of laccase and alkaline extraction produced a lignin reduction of 16.2%, improving the glucose and xylose production by almost 41.3% and 44.9%, respectively. On the other hand, the supplementation of S. ipomoeae mannanase to the hydrolysis 24 h before the addition of cellulases increased the glucose (18.4%), xylose (12.3%), and mannose (47.2%) production.

Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce

BackgroundTo realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes.ResultsGlucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24–72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation.ConclusionOur study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Comparison of Efficiency and Cost of Methods for Conditioning of Slurries of Steam-Pretreated Softwood

Inhibitors formed during pretreatment impair lignocellulose bioconversion by making enzymatic saccharification and microbial fermentation less efficient, but conditioning of slurries and hydrolysates can improve fermentability and sometimes also enzymatic digestibility. Conditioning of pretreated softwood using four industrial reducing agents (sodium sulfite, sodium dithionite, sodium borohydride, and hydrogen) was compared with standard methods, such as overliming and treatment with activated charcoal. A dosage of approx. 1 mM sulfur oxyanion (sulfite or dithionite) per percent water-insoluble solids (WIS) in the slurry was found to result in good fermentability. Treatment of 10–20% WIS slurries with 15 mM sulfur oxyanion under mild reaction conditions (23°C, pH 5.5) resulted in sulfonation of the solid phase and saccharification improvements of 18–24% for dithionite and 13–16% for sulfite. Among the different conditioning methods studied, treatment of slurries with sodium sulfite was superior with respect to cost-efficient improvement of fermentability. Treatments of slurry or pretreatment liquid with 15 mM sulfite or dithionite resulted in 58–76% reduction of the content of formaldehyde. The comparison indicates that conditioning of pretreated biomass using sulfur oxyanions warrants further attention.

Fabrication of spherical lignin nanoparticles using acid-catalyzed condensed lignins

Valorization of lignin by-products enhances the overall economics of current lignocellulose biorefinery. This work showed the high potential of fabricating acid-catalyzed condensed lignin fragments into high-value lignin nanoparticles (LNPs) with spherical structure. Four condensed lignins, i.e., liquid hot water pretreated hardwood aspen and eucalyptus lignin, steam pretreated softwood lodgepole pine and herbaceous corn stover lignin, were assessed for their abilities for LNPs using facile self-assembly method. Results showed the contents of condensed aromatics (0.20–0.67 mmol g−1) were varied with biomass species and hydrothermal pretreatment methods selected. Those resulting LNPs exhibited yields from 17.5 to 29.4%, particle sizes ranging from 20 to 100 nm and considerable suspension stabilities at pH 4–10. It was proposed that higher content of condensed lignin aromatics could provide more anchors available for their self-assembly through enhanced hydrophobic interactions, thus LNPs with more uniform particle size could be obtained. This work showed the technical opportunity to enhance the value of intractable condensed lignin through LNPs production towards a multi-product lignocellulose biorefinery.

Effects of Biosurfactants on Enzymatic Saccharification and Fermentation of Pretreated Softwood.

The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.

Sulfite Post-Treatment To Simultaneously Detoxify and Improve the Enzymatic Hydrolysis and Fermentation of a Steam-Pretreated Softwood Lodgepole Pine Whole Slurry

Previous work has shown that sulfonation post-treatment employed at a temperature of 130–140 °C modifies the lignin in steam-pretreated softwood to improve the ease of hydrolysis of the resulting “...

Viscosity reduction of pretreated softwood by endoglucanases

AbstractBACKGROUNDCost‐effective processing of lignocellulosic biomass into sugar derived products, such as biofuels or biochemicals, needs to be performed at high water insoluble solid (WIS) loading. However, the difficult rheology of such materials presents significant challenges. The aim of this study was to investigate if a Cel5A endoglucanase can be used to reduce the viscosity of two types of pretreated softwood: steam pretreated Scots pine and sulfite pretreated Norway spruce.RESULTSThe viscosity of steam pretreated pine increased (by more than 60%) during the first 20 min of enzymatic hydrolysis, followed by a gradual decrease. A slightly lower viscosity during prolonged hydrolysis could be obtained by replacing 25% of the protein in Cellic CTec3 with the Cel5A endoglucanase. Very different results were obtained with sulfite pretreated spruce. The viscosity of this material was rapidly reduced by either CTec3 or the Cel5A endoglucanase, without a transient initial increase in viscosity. Even very low doses of Cel5A (0.1 mg protein per g glucan) decreased the viscosity of sulfite pretreated spruce 30‐fold within 6 h.CONCLUSIONLow endoglucanase doses can be used to reduce the viscosity of sulfite pretreated softwood, whereas the viscosity of steam pretreated softwood is less affected by endoglucanase activity. © 2018 Society of Chemical Industry

Unravelling the effect of pretreatment severity on the balance of cellulose accessibility and substrate composition on enzymatic digestibility of steam-pretreated softwood

Pretreatment is essential for effective enzymatic digestion of lignocellulosic biomass. Steam pretreatments increase the digestibility by increasing the accessibility of the carbohydrates to the enzymes. However, they can also cause yield loss and lowered digestibility via increased non-productive binding of enzymes to lignin. The relative importance of these effects is not well defined, especially for softwoods which require more severe pretreatments than other types of biomass. Pinus radiata wood was steam pretreated at 180 °C and 215 °C to Combined Severity Factors of −3.31 or −2.61 and the digestibilities of the washed insoluble fractions examined before and after ball-milling to a common accessibility, as determined by Simons' stain measurements. Pretreatments at 215 °C for 2 min with citric and sulfuric acid catalysts were also investigated. Results showed that the digestibility of the pretreated substrates increased with pretreatment severity, rising from ∼5% with no pretreatment to ∼20% after the most severe pretreatment. However, when the substrates were ball-milled to a common accessibility, the digestibility decreased on increasing pretreatment severity. At a common accessibility and low enzyme dose the digestibility dropped six-fold from ∼30% for the original wood to ∼5% for the most severely pretreated substrate. This showed that while increasing pretreatment severity does lead to greater enzyme inhibition, this was being overridden by increases in the accessibility.

Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks

Softwood bark contains a large amounts of extractives—i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks—Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic digestibility of both steam-pretreated materials. This effect was more pronounced for the spruce than the pine bark, as evidenced by the 30 and 11% glucose yield improvement, respectively, in the enzymatic digestibility. Furthermore, analysis of the chemical composition showed that the acid-insoluble lignin content of the pretreated materials decreased when water-soluble extractives were removed prior to steam pretreatment. This can be explained by a decreased formation of water-insoluble “pseudo-lignin” from water-soluble bark phenolics during the acid-catalyzed pretreatment, which otherwise results in distorted lignin analysis and may also contribute to the impaired enzymatic digestibility of the barks. Thus, this study advocates the removal of extractives as the first step in the processing of bark or bark-rich materials in a sugar platform biorefinery.

Scale-up of high-solid enzymatic hydrolysis of steam-pretreated softwood: the effects of reactor flow conditions

The importance of flow conditions during scale-up of high-solid enzymatic hydrolysis of steam-pretreated spruce was demonstrated by comparing hydrolysis rates between laboratory (2 L) and demonstration (4 m3) scale. A positive effect of increased agitation speed on the rate of enzymatic hydrolysis was found regardless of scale. Importantly, the hydrolysis rate was higher at the larger scale when compared at similar specific power inputs. Changes in the rheological properties of the pretreated material during the hydrolysis were followed by off-line measurements of apparent viscosity. This information was used to estimate the flow conditions in the reactors, i.e., average Reynolds numbers, which together with measured mixing power consumptions enabled a more detailed comparison between the scales. The hydrolysis yields correlated better with average Reynolds numbers than specific power input over the different scales. This indicates that mass transport limitations, caused by insufficient bulk flow, likely play a decisive role in determining the rate of enzymatic hydrolysis.

Simultaneous saccharification and fermentation process for ethanol production from steam-pretreated softwood: Recirculation of condensate streams

Wastewater from fuel ethanol production plant represents a considerable potential pollutant. Treatment is an essential operation in the overall conversion of lignocellulosic to ethanol. This significantly reduces the effluent volume, and reduces the need of fresh water. Present work concerns a simultaneous saccharification and fermentation (SSF) process for ethanol production from steam-pretreated softwood. Within this process, the effect of recirculation of the condensate from the evaporation of the stillage stream was investigated. The condensate employed in this study represents the condensate of four evaporators connected in series, with each evaporator producing a condensate corresponding to 14% of the process stream after distillation of the SSF broth. The investigation demonstrated that it was possible to replace 100% of the fresh water used in the process (except for the steam used in the pretreatment), by recirculation of all the condensate fractions together, without affecting the ethanol yield and the productivity in SSF. A significant factor was the absence of lactic acid production, which is sometimes observed when the pretreated material is diluted with fresh water. The prospect of using condensate to replace fresh water offers an attractive means of reducing the cost of wastewater management and thereby improving the overall economic feasibility of the process.

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignin’s restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.

Inhibition of enzymatic hydrolysis by residual lignins from softwood—study of enzyme binding and inactivation on lignin‐rich surface

Lignin-derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin-rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin-bound enzymes. Severe inactivation of the lignin-bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS-PAGE analysis of the lignin-bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat-induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature.

Influence of steam pretreatment severity on post‐treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings

It is recognized that some form of post-treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post-treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post-treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post-treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post-treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post-treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post-treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post-treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used.

Utilization of Soft Wood Wastes as a Feed Stock to Produce Fuel Ethanol

Problem statement: The current research investigated the utilization of soft wood waste as a feedstock to produce a value-added product-fuel ethanol. Approach: The main issue in converting soft wood waste to fuel ethanol is the accessibility of the polysaccharides for breaking down into monosaccharides. This study focused on the use of steam as the pretreatment method. The governing factors for the effectiveness of steam pretreatment are steam temperature and retention times. Following steam pretreatment, soft wood waste was subjected to acid hydrolysis. The sugars released by acid hydrolysis were fermented in series chemical reactions that convert sugars to ethanol. The fermentation reaction was caused by yeast, which feed on the sugars. Results: Steam pretreatment was able to improve both glucose yields from acid hydrolysis and ethanol yields from fermentation. The results obtained from this study showed that steam pretreated soft wood waste are a heterogeneous material. So biomass goes through a size-reduction step to make it easier to handle and to make the ethanol production process more efficient. Steam treatment on soft wood waste increased the hydrolysis of cellulose by acid hydrolysis. Following 24 h of diluted or concentrated acid hydrolysis, a maximum cellulose conversion of 20.5% was obtained. Similarly, sugars to ethanol conversions were improved by steam treatment. Maximum sugar to ethanol conversion of 40.7% was observed. Conclusion: It was recommended that the hydrolysis process be done for 40 min to obtain the maximum sugars yield in a reasonable period of time.

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.

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.