The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei.

BACKGROUND: On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes' hydrolytic strength towards the same substrates. RESULTS: Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. CONCLUSION: Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable.

Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules.

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

The influence of feedstock characteristics on enzyme production in Trichoderma reesei: a review on productivity, gene regulation and secretion profiles.

Biorefineries, designed for the production of lignocellulose-based chemicals and fuels, are receiving increasing attention from the public, governments, and industries. A major obstacle for biorefineries to advance to commercial scale is the high cost of the enzymes required to derive the fermentable sugars from the feedstock used. As summarized in this review, techno-economic studies suggest co-localization and integration of enzyme manufacturing with the cellulosic biorefinery as the most promising alternative to alleviate this problem. Thus, cultivation of Trichoderma reesei, the principal producer of lignocellulolytic enzymes, on the lignocellulosic biomass processed on-site can reduce the cost of enzyme manufacturing. Further, due to a complex gene regulation machinery, the fungus can adjust the gene expression of the lignocellulolytic enzymes towards the characteristics of the feedstock, increasing the hydrolytic efficiency of the produced enzyme cocktail. Despite extensive research over decades, the underlying regulatory mechanisms are not fully elucidated. One aspect that has received relatively little attention in literature is the influence the characteristics of a lignocellulosic substrate, i.e., its chemical and physical composition, has on the produced enzyme mixture. Considering that the fungus is dependent on efficient enzymatic degradation of the lignocellulose for continuous supply of carbon and energy, a relationship between feedstock characteristics and secretome composition can be expected. The aim of this review was to systematically collect, appraise, and aggregate data and integrate results from studies analyzing enzyme production by T. reesei on insoluble cellulosic model substrates and lignocellulosic biomass. The results show that there is a direct effect of the substrate's complexity (rated by structure, composition of the lignin-carbohydrate complex, and recalcitrance in enzymatic saccharification) on enzyme titers and the composition of specific activities in the secretome. It further shows that process-related factors, such as substrate loading and cultivation set-up, are direct targets for increasing enzyme yields. The literature on transcriptome and secretome composition further supports the proposed influence of substrate-related factors on the expression of lignocellulolytic enzymes. This review provides insights into the interrelation between the characteristics of the substrate and the enzyme production by T. reesei, which may help to advance integrated enzyme manufacturing of substrate-specific enzymes cocktails at scale.

Sequential fractionation of the lignocellulosic components in hardwood based on steam explosion and hydrotropic extraction.

BACKGROUND: The forest biorefinery plays an important part in the evolving circular bioeconomy due to its capacity to produce a portfolio of bio-based and sustainable fuels, chemicals, and materials. To tap into its true potential, more efficient and environmentally benign methods are needed to fractionate woody biomass into its main components (cellulose, hemicellulose, and lignin) without reducing their potential for valorization. This work presents a sequential fractionation method for hardwood based on steam pretreatment (STEX) and hydrotropic extraction (HEX) with sodium xylene sulfonate. By prehydrolyzing the hemicellulose (STEX) and subsequently extract the lignin from the cellulose fraction (HEX), the major wood components can be recovered in separate process streams and be further valorized. RESULTS: Using autocatalyzed STEX and HEX, hemicellulose (> 70%) and lignin (~ 50%) were successfully fractionated and recovered in separate liquid streams and cellulose preserved (99%) and enriched (~ twofold) in the retained solids. Investigation of pretreatment conditions during HEX showed only incremental effects of temperature (150-190 °C) and hold-up time (2-8 h) variations on the fractionation efficiency. The hydrolyzability of the cellulose-rich solids was analyzed and showed higher cellulose conversion when treated with the combined process (47%) than with HEX alone (29%), but was inferior to STEX alone (75%). Protein adsorption and surface structure analysis suggested decreased accessibility due to the collapse of the fibrillose cellulose structure and an increasingly hydrophobic lignin as potential reasons. CONCLUSION: This work shows the potential of sequential STEX and HEX to fractionate and isolate cellulose, hemicellulose, and a sulfur-free lignin in separate product streams, in an efficient, sustainable, and scalable process.

Short-term adaptation during propagation improves the performance of xylose-fermenting Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation.

BACKGROUND: Inhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation. RESULTS: The presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield. CONCLUSIONS: Short-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.

Treatment of acute Achilles tendon rupture in Scandinavia does not adhere to evidence-based guidelines: a cross-sectional questionnaire-based study of 138 departments.

The best treatment of acute Achilles tendon rupture has been discussed for decades. During the past half decade, evidence has increased in favor of nonoperative treatment and dynamic and weightbearing rehabilitation. We hypothesized that the treatment strategies would show great variation and that adherence to evidence-based recommendations would not be as good as desired. The purpose of the present study was to investigate how acute Achilles tendon rupture is treated in Scandinavia. A questionnaire was distributed to all orthopedic departments treating acute Achilles tendon ruptures in Denmark, Sweden, Norway, and Finland. The questionnaire was returned by 138 of 148 departments (response rate 93%). Two-way tables with Fisher's exact test were used for statistical analysis. In Denmark, Norway, Sweden, and Finland, 19 of 23 (83%), 44 of 48 (92%), 26 of 40 (65%), and 8 of 27 (30%) departments recommended surgical treatment (p < .001). Dynamic rehabilitation was used significantly less often in Denmark (5 of 23 [22%]), Norway (17 of 45 [38%]), and Sweden (11 of 40 [28%]) than in Finland (15 of 26 [58%]; p = .015). A significant difference was found among the countries in the educational level of the performing surgeons (p < .001). Surgical treatment was the treatment of choice in Danish, Norwegian, and Swedish hospitals regardless of the increasing evidence favoring nonoperative treatment. Although increasing evidence has favored dynamic rehabilitation, it has gained limited use across Scandinavia. Weightbearing was used in most hospitals. Surgery was performed by junior surgeons in most hospitals across Scandinavia. Treatment algorithms showed considerable variation and often did not adhere to the clinical evidence.

Simultaneous saccharification and co-fermentation for bioethanol production using corncobs at lab, PDU and demo scales.

BACKGROUND: While simultaneous saccharification and co-fermentation (SSCF) is considered to be a promising process for bioconversion of lignocellulosic materials to ethanol, there are still relatively little demo-plant data and operating experiences reported in the literature. In the current work, we designed a SSCF process and scaled up from lab to demo scale reaching 4% (w/v) ethanol using xylose rich corncobs. RESULTS: Seven different recombinant xylose utilizing Saccharomyces cerevisiae strains were evaluated for their fermentation performance in hydrolysates of steam pretreated corncobs. Two strains, RHD-15 and KE6-12 with highest ethanol yield and lowest xylitol yield, respectively were further screened in SSCF using the whole slurry from pretreatment. Similar ethanol yields were reached with both strains, however, KE6-12 was chosen as the preferred strain since it produced 26% lower xylitol from consumed xylose compared to RHD-15. Model SSCF experiments with glucose or hydrolysate feed in combination with prefermentation resulted in 79% of xylose consumption and more than 75% of the theoretical ethanol yield on available glucose and xylose in lab and PDU scales. The results suggest that for an efficient xylose conversion to ethanol controlled release of glucose from enzymatic hydrolysis and low levels of glucose concentration must be maintained throughout the SSCF. Fed-batch SSCF in PDU with addition of enzymes at three different time points facilitated controlled release of glucose and hence co-consumption of glucose and xylose was observed yielding 76% of the theoretical ethanol yield on available glucose and xylose at 7.9% water insoluble solids (WIS). With a fed-batch SSCF in combination with prefermentation and a feed of substrate and enzymes 47 and 40 g l-1 of ethanol corresponding to 68% and 58% of the theoretical ethanol yield on available glucose and xylose were produced at 10.5% WIS in PDU and demo scale, respectively. The strain KE6-12 was able to completely consume xylose within 76 h during the fermentation of hydrolysate in a 10 m3 demo scale bioreactor. CONCLUSIONS: The potential of SSCF is improved in combination with prefermentation and a feed of substrate and enzymes. It was possible to successfully reproduce the fed-batch SSCF at demo scale producing 4% (w/v) ethanol which is the minimum economical requirement for efficient lignocellulosic bioethanol production process.

Radiation induced spent nuclear fuel dissolution under deep repository conditions.

The dynamics of spent nuclear fuel dissolution in groundwater is an important part of the safety assessment of a deep geological repository for high level nuclear waste. In this paperwe discussthe most important elementary processes and parameters involved in radiation induced oxidative dissolution of spent nuclear fuel. Based on these processes, we also present a new approach for simulation of spent nuclear fuel dissolution under deep repository conditions. This approach accounts for the effects of fuel age, burn up, noble metal nanoparticle contents, aqueous H2 and HCO3- concentration, water chemistry, and combinations thereof. The results clearly indicate that solutes consuming H202 and combined effects of noble metal nanoparticles and H2 have significant impact on the rate of spent nuclear fuel dissolution. Using data from the two possible repository sites in Sweden, we have employed the new approach to estimate the maximum rate of spent nuclear fuel dissolution. This estimate indicates that H2 produced from radiolysis of groundwater alone will be sufficient to inhibit the dissolution completely for spent nuclear fuel older than 100 years.