Quality of life and socioeconomic and educational status in patients with congenital hypothyroidism.

BACKGROUND: The aim of this study was to investigate the influence of primary congenital hypothyroidism (CH) on quality of life, level of education and socioeconomic status (SES). METHODS: Two independent study cohorts, a national and a regional, were collected from Finnish national registers and patient records. Data on social security benefits, SES, marital status, and education were collected from Statistics Finland. Health-related quality of life (HRQoL) was studied in the regional patient cohort with the standardized 15D and 16D instruments. RESULTS: There were no statistically significant differences in education level, marital status, or SES between CH patients (n = 40) and their matched controls at the age of 25 years. The mean 15D score was both statistically significantly and clinically importantly lower in CH patients (n = 29) than controls (0.904 vs. 0.953, p = 0.008). CH patients reported significantly lower scores across various dimensions of physical and mental HRQoL, including breathing, sleeping, speech, excretion, mental function, distress, and vitality. The mean 16D score was lower in CH patients compared to controls (0.917, vs. 0.947), but without statistical significance. CONCLUSION: SES of CH patients did not differ from matched controls. Thus, most CH patients integrate well into society, but their HRQoL is impaired. IMPACT: Most patients with primary congenital hypothyroidism integrate well into society. In the current study, their socioeconomic and marital status did not differ from matched controls at the age of 25 years. However, health-related quality of life measured using 15D instrument was impaired. Every fourth patient reported that congenital hypothyroidism influenced everyday life.

Comorbidity in Congenital Hypothyroidism-A Nationwide, Population-based Cohort Study.

CONTEXT: Patients with congenital hypothyroidism (CH) are affected more often than the general population by other chronic diseases and neurological difficulties. OBJECTIVE: The aim of this nationwide population-based register study was to investigate the incidence of congenital malformations, comorbidities, and the use of prescribed drugs in patients with primary CH. METHODS: The study cohort and matched controls were identified from national population-based registers in Finland. All diagnoses from birth until the end of 2018 were collected from the Care Register, and subject-specific prescription drug purchases were identified from The Prescription Register from birth until the end of 2017. RESULTS: Diagnoses of neonatal and chronic diseases were collected for 438 full-term patients and 835 controls (median follow-up time 11.6 years; range, 0-23 years). Newborns with CH were more often found to have neonatal jaundice (11.2% and 2.0%; P < .001), hypoglycemia (8.9% and 2.8%; P < .001), metabolic acidemia (3.2% and 1.1%; P = .007), and respiratory distress (3.9% and 1.3%; P < .003) as compared to their matched controls.Congenital malformations were diagnosed in 66 of 438 (15.1%) CH patients and in 62 of 835 (7.4%) controls (P < .001). The most commonly affected extrathyroidal systems were the circulatory and musculoskeletal systems. The cumulative incidence of hearing loss and specific developmental disorders was higher among CH patients than controls. The use of antidepressant and antipsychotic drugs was similar in CH patients and their controls. CONCLUSION: CH patients have more neonatal morbidity and congenital malformations than their matched controls. The cumulative incidence of neurological disorders is higher in CH patients. However, our results do not support the existence of severe psychiatric comorbidity.

Incidence of primary congenital hypothyroidism over 24 years in Finland.

BACKGROUND: A rise in the incidence of congenital hypothyroidism (CH) has been reported worldwide. This nationwide study aimed to describe the secular trends and current incidence of CH in Finland. METHODS: Two independent study cohorts, a national and a regional, were collected from national registers and patient records. The national cohort represents all CH cases born in Finland between 1994 and 2017. Birth data, results of the screening test, and the incidence of CH were reviewed. RESULTS: Between 1994 and 2017, 1,400,028 children were born in Finland. Of these children, 503 were diagnosed with primary CH (incidence 1:2783). Male-to-female sex ratio was 1:2.0. The nationwide incidence was 33 cases per 100,000 live births between 1994 and 1999, 38 cases per 100,000 live births between 2000 and 2005, 40 cases per 100,000 live births between 2006 and 2011, and 33 cases per 100,000 live births between 2012 and 2017. In the regional cohort (n = 139), the incidence of transient CH was 3.6%. The incidence of mild, moderate, and severe CH remained constant. CONCLUSIONS: In Finland, the incidence of CH has not changed during the 24-year study period. IMPACT: As opposed to recent reports worldwide, the incidence of congenital hypothyroidism has not changed between 1994 and 2017 in Finland. The proportions of mild, moderate, and severe congenital hypothyroidism did not change significantly over the study period. Lowering the TSH cut-off limit or increasing immigration did not affect the incidence rate of primary congenital hypothyroidism in Finland.

Treatment of Congenital Hypothyroidism: Impact of Secular Changes in Levothyroxine Initial Dose on Early Growth.

INTRODUCTION: Newborn screening of congenital hypothyroidism (CH) has enabled early treatment with levothyroxine (LT4), ensuring normal growth and development. The initial LT4 dose recommendation has increased over decades. We evaluated whether the increased LT4 dosing influenced thyroid-stimulating hormone (TSH) and thyroxine (fT4) concentrations, growth, or treatment-related symptoms. METHODS: LT4 doses, TSH, fT4, anthropometrics, and treatment-related symptoms until age 2 years were evaluated in 172 Finnish CH patients born between 1980 and 2018. The patients were grouped according to birth decade: 1980s (n = 19, mean LT4 starting dose 6.8 µg/kg/day), 1990s (n = 50, 7.4 µg/kg/day), 2000s (n = 59, 9.7 µg/kg/day), and 2010s (n = 44, 10.8 µg/kg/day). RESULTS: TSH concentrations were higher during the first 2 years of life in children born in the 1980s compared to children born later. TSH concentrations were often subnormal in children receiving higher LT4 doses (children born in the 2000s and 2010s). However, symptoms of overtreatment were uncommon. Linear or head growth showed no differences between the groups during the first 2 years of life. Although growth was within the normal spectrum, children in all groups were shorter than their target length at 2 years and their weight-for-length was above the mean through the first 2 years of life. DISCUSSION: Current treatment practice with higher LT4 dose normalizes TSH rapidly without significant increase in side effects. However, irrespective of initial LT4 dose, children were shorter than expected at 2 years of age. Effects of different initial LT4 dose on cognitive development urges further investigation.

Screening for Mutations in Isolated Central Hypothyroidism Reveals a Novel Mutation in Insulin Receptor Substrate 4.

Background: Central hypothyroidism (CeH) is a rare condition affecting approximately 1:16 000- 100 000 individuals. Congenital forms can harm normal development if not detected and treated promptly. Clinical and biochemical diagnosis, especially of isolated CeH, can be challenging. Cases are not usually detected in neonatal screening, which, in most countries, is focused on detection of the more prevalent primary hypothyroidism. Until now, five genetic causes for isolated CeH have been identified. Here we aimed to identify the genetic cause in two brothers with impaired growth diagnosed with CeH at the age of 5 years. We further evaluated the candidate gene variants in a large genetic database. Methods: Clinical and biochemical characterization together with targeted next-generation sequencing (NGS) was used to identify the genetic cause in a family of two brothers presenting with CeH. Screening of insulin receptor substrate 4 (IRS4) variants was carried out in the FinnGen database. Results: A novel monoallelic frameshift mutation c.1712_1713insT, p.Gly572Trp fs*32 in the X-linked IRS4 gene was identified by NGS analysis in both affected males and confirmed using Sanger sequencing. Their mother was an unaffected carrier. In addition to the declined growth at presentation, central hypothyroidism and blunted TRH test, no other phenotypic alterations were found. Diagnostic tests included head MRI, thyroid imaging, bone age, and laboratory tests for thyroid autoantibodies, glucose, insulin and glycosylated hemoglobin levels. Examination of the IRS4 locus in FinnGen (R5) database revealed the strongest associations to a rare Finnish haplotype associated with thyroid disorders (p = 1.3e-7) and hypothyroidism (p = 8.3e-7). Conclusions: Here, we identified a novel frameshift mutation in an X-linked IRS4 gene in two brothers with isolated CeH. Furthermore, we demonstrate an association of IRS4 gene locus to a general thyroid disease risk in the FinnGen database. Our findings confirm the role of IRS4 in isolated central hypothyroidism.

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides.

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and ß-glucosidase played an important role in mannan hydrolysis. Addition of 10mg/ml mannan reduced the glucose yield of Avicel (at 20g/l) from 40.1 to 24.3%. No inhibition of ß-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on ß-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.

Impact of xylan on synergistic effects of xylanases and cellulases in enzymatic hydrolysis of lignocelluloses.

Supplementation of xylanase (XYL) has been found to synergistically improve the performance of cellulases (CEL) in the hydrolysis of lignocelluloses. However, the effect of xylan on the synergistic effects of XYL and CEL is still unclear. In this work, the effect of xylan on the synergy between CEL and XYL was investigated. Xylan content in corn stover was generally a good indicator of the degree of the synergism between CEL and XYL. Strongest synergism was observed in the hydrolysis of cellulose in corn stover with the highest xylan contents. A more evident synergistic effect of CEL in xylan hydrolysis was observed in the substrates with low original xylan content. It was also found that the ratio of cellulose to xylan in substrates correlated to the synergism between the two types of enzymes. The results indicated that supplementation of XYL with CEL was most effective in the hydrolysis of corn stover with the highest xylan content. For efficient enzymatic hydrolysis of lignocelluloses, both cellulases and xylanase were important because cellulose and xylan coved each other and these enzymes could improve their performance each other in the hydrolysis of cellulose and xylan in lignocelluloses.

Carbohydrate-binding modules of fungal cellulases: occurrence in nature, function, and relevance in industrial biomass conversion.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.

Cellulases without carbohydrate-binding modules in high consistency ethanol production process.

BACKGROUND: Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. RESULTS: High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. CONCLUSIONS: The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce.

BACKGROUND: The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs). RESULTS: LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis. CONCLUSIONS: LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.

Effect of temperature on lignin-derived inhibition studied with three structurally different cellobiohydrolases.

Non-productive enzyme adsorption onto lignin inhibits enzymatic hydrolysis of lignocellulosic biomass. Three cellobiohydrolases, Trichoderma reesei Cel7A (TrCel7A) and two engineered fusion enzymes, with distinctive modular structures and temperature stabilities were employed to study the effect of temperature on inhibition arising from non-productive cellulase adsorption. The fusion enzymes, TeCel7A-CBM1 and TeCel7A-CBM3, were composed of a thermostable Talaromyces emersonii Cel7A (TeCel7A) catalytic domain fused to a carbohydrate-binding module (CBM) either from family 1 or from family 3. With all studied enzymes, increase in temperature was found to increase the inhibitory effect of supplemented lignin in the enzymatic hydrolysis of microcrystalline cellulose. However, for the different enzymes, lignin-derived inhibition emerged at different temperatures. Low binding onto lignin and thermostable structure were characteristic for the most lignin-tolerant enzyme, TeCel7A-CBM1, whereas TrCel7A was most susceptible to lignin especially at elevated temperature (55 °C).

The role of carbohydrate binding module (CBM) at high substrate consistency: comparison of Trichoderma reesei and Thermoascus aurantiacus Cel7A (CBHI) and Cel5A (EGII).

The role of CBM in two fungal model cellulase systems, consisting of Cel7A and Cel5A, from Trichoderma reesei and Thermoascus aurantiacus, were compared in the hydrolysis of various substrates. For comparison, family-1 CBM's were introduced to the T. aurantiacus and removed from the T. reesei enzymes. Especially at high dry matter consistencies of lignocellulosic substrates, pretreated wheat straw and spruce, the T. aurantiacus enzymes lacking CBM outperformed the enzymes carrying the CBM. In these conditions, the CBM-less enzymes from both organisms obviously recognized and bound to the substrate at higher probability than in dilute systems. Avoiding the unproductive binding to lignin caused by the CBMs obviously enhanced the hydrolytic performance. The lignin binding effect was, however, not entirely caused by the CBM, but also by the different structures and affinities of the core enzymes to lignin. Due to decreased binding, the CBM-less enzymes would allow reuse, potentially decreasing hydrolysis costs.

Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs.

BACKGROUND: A vast number of organisms are known to produce structurally diversified cellulases capable of degrading cellulose, the most abundant biopolymer on earth. The generally accepted paradigm is that the carbohydrate-binding modules (CBMs) of cellulases are required for efficient saccharification of insoluble substrates. Based on sequence data, surprisingly more than 60% of the cellulases identified lack carbohydrate-binding modules or alternative protein structures linked to cellulases (dockerins). This finding poses the question about the role of the CBMs: why would most cellulases lack CBMs, if they are necessary for the efficient hydrolysis of cellulose? RESULTS: The advantage of CBMs, which increase the affinity of cellulases to substrates, was found to be diminished by reducing the amount of water in the hydrolytic system, which increases the probability of enzyme-substrate interaction. At low substrate concentration (1% w/w), CBMs were found to be more important in the catalytic performance of the cellobiohydrolases TrCel7A and TrCel6A of Trichoderma reesei as compared to that of the endoglucanases TrCel5A and TrCel7B. Increasing the substrate concentration while maintaining the enzyme-to-substrate ratio enhanced adsorption of TrCel7A, independent of the presence of the CBM. At 20% (w/w) substrate concentration, the hydrolytic performance of cellulases without CBMs caught up with that of cellulases with CBMs. This phenomenon was more noticeable on the lignin-containing pretreated wheat straw as compared to the cellulosic Avicel, presumably due to unproductive adsorption of enzymes to lignin. CONCLUSIONS: Here we propose that the water content in the natural environments of carbohydrate-degrading organisms might have led to the evolution of various substrate-binding structures. In addition, some well recognized problems of economical saccharification such as unproductive binding of cellulases, which reduces the hydrolysis rate and prevents recycling of enzymes, could be partially overcome by omitting CBMs. This finding could help solve bottlenecks of enzymatic hydrolysis of lignocelluloses and speed up commercialization of second generation bioethanol.

The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin.

BACKGROUND: The enzymatic hydrolysis step converting lignocellulosic materials into fermentable sugars is recognized as one of the major limiting steps in biomass-to-ethanol process due to the low efficiency of enzymes and their cost. Xylanases have been found to be important in the improvement of the hydrolysis of cellulose due to the close interaction of cellulose and xylan. In this work, the effects of carbohydrate-binding module (CBM family II) of the xylanase 11 from Nonomuraea flexuosa (Nf Xyn11) on the adsorption and hydrolytic efficiency toward isolated xylan and lignocellulosic materials were investigated. RESULTS: The intact family 11 xylanase of N. flexuosa clearly adsorbed on wheat straw and lignin, following the Langmuir-type isotherm. The presence of the CBM in the xylanase increased the adsorption and hydrolytic efficiency on insoluble oat spelt xylan. But the presence of the CBM did not increase adsorption on pretreated wheat straw or isolated lignin. On the contrary, the CBM decreased the adsorption of the core protein to lignin containing substrates, indicating that the CBM of N. flexuosa xylanase did not contribute to the non-productive adsorption. CONCLUSION: The CBM of the N. flexuosa xylanase was shown to be a xylan-binding module, which had low affinity on cellulose. The CBM of the N. flexuosa xylanase reduced the non-specific adsorption of the core protein to lignin and showed potential for improving the hydrolysis of lignocellulosic materials to platform sugars.

Synergy between cellulases and pectinases in the hydrolysis of hemp.

The impact of pectinases in the hydrolysis of fresh, steam-exploded and ensiled hemp was investigated and the synergy between cellulases, pectinases and xylanase in the hydrolysis was evaluated. About half; 59.3% and 46.1% of pectin in the steam-exploded and ensiled hemp, respectively, could be removed by a low dosage of pectinases used. Pectinases were more efficient than xylanase in the hydrolysis of fresh and ensiled hemp whereas xylanase showed higher hydrolytic efficiency than the pectinase preparation used in the hydrolysis of steam-exploded hemp. Clear synergistic action between cellulases and xylanase could be observed in the hydrolysis of steam-exploded hemp. Supplementation of pectinase resulted in clear synergism with cellulases in the hydrolysis of all hemp substrates. Highest hydrolysis yield of steam-exploded hemp was obtained in the hydrolysis with cellulases and xylanase. In the hydrolysis of ensiled hemp, the synergistic action between cellulases and pectinases was more obvious for efficient hydrolysis.

Xylan as limiting factor in enzymatic hydrolysis of nanocellulose.

The role of xylan as a limiting factor in the enzymatic hydrolysis of cellulose was studied by hydrolysing nanocellulose samples prepared by mechanical fibrillation of birch pulp with varying xylan content. Analyzing the nanocelluloses and their hydrolysis residues with dynamic FT-IR spectroscopy revealed that a certain fraction of xylan remained tightly attached to cellulose fibrils despite partial hydrolysis of xylan with xylanase prior to pulp fibrillation and that this fraction remained in the structure during the hydrolysis of nanocellulose with cellulase mixture as well. Thus, a loosely bound fraction of xylan was predicted to have been more likely removed by purified xylanase. The presence of loosely bound xylan seemed to limit the hydrolysis of crystalline cellulose, indicated by an increase in cellulose crystallinity and by preserved crystal width measured with wide-angle X-ray scattering. Removing loosely bound xylan led to a proportional hydrolysis of xylan and cellulose with the cellulase mixture.

Enzymatic modification of flaxseed fibers.

Flaxseed (Linum usitatissimum L.) fibers were modified by oxidoreductive and cellulolytic enzymes. The lignin amount and intrinsic plant peroxidase activity was evaluated by histochemical and spectrophotometric assays. Peroxidase activity was not found from bast fibers. The flaxseed fibers were further separated and treated with laccase to conjugate the model compounds, that is, the hydrophobic gallate molecules on fiber surfaces. Laccase was able to slowly oxidize fiber-conjugated phenolics, but no fundamental changes in fiber cell surface structure or notable coupling of the applied hydrophobic gallate molecules onto the fibers occurred, as revealed by Fourier transform infrared spectroscopy. The reactivity of the mature fibers was further investigated using cellulolytic enzymes. Cellobiohydrolase (CBH) and endoglucanase (EG)-rich enzyme preparations were applied to reach a hydrolysis degree of 1-6% (of the fiber dry matter) using a standard enzyme dosage. The CBH mixture altered the fiber surface morphology distinctly, and SEM images illustrated fibers in which the cellulose fibrils seemed to be loosened and partially hydrolyzed. In contrast, the effect of the EG-rich preparation without CBH activity was notable on the fiber surface, polishing the surfaces. The cellulolytic treatments were potentially interesting for specific enzymatic modifications of flax fiber surfaces, whereas the approach to use oxidoreductive enzyme treatments on mature linseed fibers offered little potential, obviously due to the low lignin content of the fibers.

Reed canary grass as a feedstock for 2nd generation bioethanol production.

The enzymatic hydrolysis and fermentation of reed canary grass, harvested in the spring or autumn, and barley straw were studied. Steam pretreated materials were efficiently hydrolysed by commercial enzymes with a dosage of 10-20FPU/g d.m. Reed canary grass harvested in the spring was hydrolysed more efficiently than the autumn-harvested reed canary grass. Additional ß-glucosidase improved the release of glucose and xylose during the hydrolysis reaction. The hydrolysis rate and level of reed canary grass with a commercial Trichoderma reesei cellulase could be improved by supplementation of purified enzymes. The addition of CBH II improved the hydrolysis level by 10% in 48hours' hydrolysis. Efficient mixing was shown to be important for hydrolysis already at 10% dry matter consistency. The highest ethanol concentration (20g/l) and yield (82%) was obtained with reed canary grass at 10% d.m. consistency.

Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases.

Hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose, and prevent the access of enzymes to cellulose surface. In addition, soluble hemicelluloses may strongly inhibit the cellulase activity. In this work, birchwood xylan clearly inhibited the enzymatic hydrolysis of wheat straw, Avicel and nanocellulose by cellulases. Hydrolysis efficiencies of cellobiohydrolase I (CBHI, from Thermoascus aurantiacus), cellobiohydrolase II (CBHII, from Trichoderma reesei) and endoglucanase II (from T. aurantiacus) were clearly inhibited by birchwood xylan, respectively. The strongest inhibitory effect of birchwood xylan was observed on the hydrolysis of Avicel by CBHI and CBHII, as a dramatically decreased formation of the main product, cellobiose. After additions of soluble and insoluble oat spelt xylan, cleaved cellobiose units by CBHI from cellulose chain decreased from 8 to 4 and 6, respectively. The results in this work demonstrated that xylans clearly inhibited the hydrolysis efficiencies of both endoglucanase and cellobiohydrolase.

Conversion of carbohydrates in herbaceous crops during anaerobic digestion.

The methane yields and conversion of pentoses (xylose) and hexoses (cellulose) in hemp, maize, and white lupin were studied over 30 days of anaerobic digestion. Preservation of hemp increased the methane yield by 23% compared with the fresh hemp. The increased methane yield of hemp was verified by the enhanced conversion of C6 sugars, increasing from 48% to about 70%, whereas the conversion of C5 sugars increased from only 9% to nearly 50%. The consumption of all carbohydrates in fresh maize was almost complete in the 30 days of anaerobic digestion. Hence, there was no major difference in carbohydrate consumption between fresh and preserved maize during biogas production. Fresh white lupin produced the highest methane yield (343 ± 33 dm(3) kg(-1) TS) in this work, mainly due to its highest amount of proteins. Conversion of C6 sugars was 80%, but that of C5 sugars was notably less at 46%.