Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

     

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

Background

Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

Results

In this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration.

Conclusion

We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

     

Mechanism of lignin inhibition of enzymatic biomass deconstruction

Background

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process.

Results

By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose binding of TrCel7A (Y466, Y492, and Y493).

Conclusions

Lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.

     

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Background

The non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.

Results

Eight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.

Conclusions

Our results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.

     

Strain improvement of <Emphasis Type="Italic">Trichoderma viride</Emphasis> for increased cellulase production by irradiation of electron and <Superscript>12</Superscript>C<Superscript>6+</Superscript>-ion beams

Objectives

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a ¹²C⁶⁺-ion beam.

Results

Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity.

Conclusions

Mutagenesis with electron and ¹²C⁶⁺-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

     

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.

     

Regular enzyme recovery enhances cellulase production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis> in fed-batch culture

Objective

To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals.

Results

Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l^?1.

Conclusion

Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

     

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

Background

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.

Results

CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.

Conclusion

We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

     

Heterologous expression of <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> cellobiohydrolase Cel7A in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> increases the efficiency of corncob residues saccharification

Objective

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii.

Results

Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction.

Conclusions

The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

     

A process for purifying xylosugars of pre-hydrolysis liquor from kraft-based dissolving pulp production process

Background

In the kraft-based dissolving pulp production process, pre-hydrolysis liquor (PHL) is produced, which contains hemicelluloses, lignin, furfural and acetic acid. PHL is currently burned in the recovery boiler of the kraft pulping process, but it can be utilized for the generation of high-valued products, such as xylitol and xylanase, via fermentation processes. However, some PHL constituents, e.g., furfural and lignin, are contaminants for fermentation processes and they must be eliminated for production of value-added products.

Results

In this work, a process is introduced for removing contaminants of PHL. Ca(OH)₂ treatment is the first step of this process, which removed 41.2% of lignin and negligible amount of sugars. In this step, a notable increase in the concentration of acetic acid was achieved (ranging from 6.2 to 11.7 g/L). In the second step, the implementation of adsorption using activated carbon (AC) at 1 wt% dosage led to additional 32% lignin and 5.9% xylosugar removals. In addition, laccase assisted activated carbon treatment led to further removal of lignin via accelerating lignin polymerization and adsorption on AC (i.e., removal from PHL). Overall, 90.7% of lignin, 100% of furfural, 5.7% of xylose, and 12% of xylan were removed from PHL, while the concentration of acetic acid became twofolds in the PHL.

Conclusions

This study reports an attractive process for purifying sugars and acetic acid of PHL. This process may be implemented for producing sugar-based value-added products from PHL. It also discusses the mechanism of Ca(OH)₂ treatment, AC adsorption and laccase assisted activated carbon treatment for lignin removal.

     

Expressing accessory proteins in cellulolytic <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to improve the conversion yield of recalcitrant cellulose

Background

A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate.

Results

The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively.

Conclusions

This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.

     

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches

Background

New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis.

Results

Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O-methyl-glucuronic acid and p-coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio.

Conclusions

Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation

Objectives

Efficient enzymatic saccharification of plant cell wall material is key to industrial processing of agricultural and forestry waste such as straw and wood chips into fuels and chemicals.

Results

Saccharification assays were performed on steam-pretreated wheat straw under ambient and O₂-deprived environments and in the absence and presence of a lytic polysaccharide monooxygenase (LPMO) and catalase. A kinetic model was used to calculate catalytic rate and first-order inactivation rate constants of the cellulases from reaction progress curves. The addition of a LPMO significantly (P < 0.01, Student’s T test) enhanced the rate of glucose release from 2.8 to 6.9 h^?1 under ambient O₂ conditions. However, this also significantly (P < 0.01, Student’s T test) increased the rate of inactivation of the enzyme mixture, thereby reducing the performance half-life from 65 to 35 h. Decreasing O₂ levels or, strikingly, the addition of catalase significantly reduced (P < 0.01, Student’s T test) enzyme inactivation and, as a consequence, higher efficiency of the cellulolytic enzyme cocktail was achieved.

Conclusion

Oxidative inactivation of commercial cellulase mixtures is a significant factor influencing the overall saccharification efficiency and the addition of catalase can be used to protect these mixtures from inactivation.

     

Identification and characterization of a novel bacterial pyranose 2-oxidase from the lignocellulolytic bacterium <Emphasis Type="Italic">Pantoea ananatis</Emphasis> Sd-1

Objective

To identify and characterize a novel bacterial pyranose 2-oxidase (P2Ox) and investigate its potential use in lignin degradation applications.

Results

A new bacterial P2Ox (PaP2Ox) enzyme was identified in the lignocellulolytic bacterium Pantoea ananatis Sd-1. The PaP2Ox open reading frame was cloned, and the encoded protein was heterologously expressed in an Escherichia coli expression system. Unlike another reported bacterial P2Ox enzyme, the purified PaP2Ox exhibits a homotetrameric spatial conformation that is similar to fungal P2Oxs, with each subunit having a molecular mass of 65 kDa. The recombinant PaP2Ox exhibits maximum activity at 50 °C and pH 6.5 with d-glucose as its preferred substrate. In addition, this enzyme was shown to work in combination with bacterial laccase in lignin degradation.

Conclusions

The bacterial enzyme PaP2Ox has potential use in ligninolytic systems and shows promising value in industrial biotechnological applications.

     

Consolidated bioprocessing performance of <Emphasis Type="Italic">Thermoanaerobacterium thermosaccharolyticum</Emphasis> M18 on fungal pretreated cornstalk for enhanced hydrogen production

Background

Biological hydrogen production from lignocellulosic biomass shows great potential as a promising alternative to conventional hydrogen production methods, such as electrolysis of water and coal gasification. Currently, most researches on biohydrogen production from lignocellulose concentrate on consolidated bioprocessing, which has the advantages of simpler operation and lower cost over processes featuring dedicated cellulase production. However, the recalcitrance of the lignin structure induces a low cellulase activity, making the carbohydrates in the hetero-matrix more unapproachable. Pretreatment of lignocellulosic biomass is consequently an extremely important step in the commercialization of biohydrogen, and for massive realization of lignocellulosic biomass as alternative fuel feedstock. Thus, development of a pretreatment method which is cost efficient, environmentally benign, and highly efficient for enhanced consolidated bioprocessing of lignocellulosic biomass to hydrogen is essential.

Results

In this research, fungal pretreatment was adopted for enhanced hydrogen production by consolidated bioprocessing performance. To confirm the fungal pretreatment efficiency, two typical thermochemical pretreatments were also compared side by side. Results showed that the fungal pretreatment was superior to the other pretreatments in terms of high lignin reduction of up to 35.3% with least holocellulose loss (the value was only 9.5%). Microscopic structure observation combined with Fourier transform infrared spectroscopy (FTIR) analysis further demonstrated that the lignin and crystallinity of lignocellulose were decreased with better holocellulose reservation. Upon fungal pretreatment, the hydrogen yield and hydrogen production rate were 6.8 mmol H₂ g^-1 pretreated substrate and 0.89 mmol L^-1 h^-1, respectively, which were 2.9 and 4 times higher than the values obtained for the untreated sample.

Conclusions

Results revealed that although all pretreatments could contribute to the enhancement of hydrogen production from cornstalk, fungal pretreatment proved to be the optimal method. It is apparent that besides high hydrogen production efficiency, fungal pretreatment also offered several advantages over other pretreatments such as being environmentally benign and energy efficient. This pretreatment method thus has great potential for application in consolidated bioprocessing performance of hydrogen production.

     

Lignocellulose conversion for biofuel: a new pretreatment greatly improves downstream biocatalytic hydrolysis of various lignocellulosic materials

Background

Lignocellulosic biomass is an attractive renewable resource for future liquid transport fuel. Efficient and cost-effective production of bioethanol from lignocellulosic biomass depends on the development of a suitable pretreatment system. The aim of this study is to investigate a new pretreatment method that is highly efficient and effective for downstream biocatalytic hydrolysis of various lignocellulosic biomass materials, which can accelerate bioethanol commercialization.

Results

The optimal conditions for the hydrogen peroxide–acetic acid (HPAC) pretreatment were 80 °C, 2 h, and an equal volume mixture of H₂O₂ and CH₃COOH. Compared to organo-solvent pretreatment under the same conditions, the HPAC pretreatment was more effective at increasing enzymatic digestibility. After HPAC treatment, the composition of the recovered solid was 74.0 % cellulose, 20.0 % hemicelluloses, and 0.9 % lignin. Notably, 97.2 % of the lignin was removed with HPAC pretreatment. Fermentation of the hydrolyzates by S. cerevisiae resulted in 412 mL ethanol kg^?1 of biomass after 24 h, which was equivalent to 85.0 % of the maximum theoretical yield (based on the amount of glucose in the raw material).

Conclusion

The newly developed HPAC pretreatment was highly effective for removing lignin from lignocellulosic cell walls, resulting in enhanced enzymatic accessibility of the substrate and more efficient cellulose hydrolysis. This pretreatment produced less amounts of fermentative inhibitory compounds. In addition, HPAC pretreatment enables year-round operations, maximizing utilization of lignocellulosic biomass from various plant sources.

     

Simultaneous enzymatic saccharification and comminution for the valorization of lignocellulosic biomass toward natural products

Background

Large-scale processing of lignocellulosics for glucose production generally relies on high temperature and acidic or alkaline conditions. However, extreme conditions produce chemical contaminants that complicate downstream processing. A method that mainly rely on mechanical and enzymatic reaction completely averts such problem and generates unmodified lignin. Products from this process could find novel applications in the chemicals, feed and food industry. But a large-scale system suitable for this purpose is yet to be developed. In this study we applied simultaneous enzymatic saccharification and communition (SESC) for the pre-treatment of a representative lignocellulosic biomass, cedar softwood, under both laboratory and large-scale conditions.

Results

Laboratory-scale comminution achieved a maximum saccharification efficiency of 80% at the optimum pH of 6. It was possible to recycle the supernatant to concentrate the glucose without affecting the efficiency. During the direct alcohol fermentation of SESC slurry, a high yield of ethanol was attained. The mild reaction conditions prevented the generation of undesired chemical inhibitors. Large-scale SESC treatment using a commercial beads mill system achieved a saccharification efficiency of 60% at an energy consumption of 50?MJ/kg biomass.

Conclusion

SESC is very promising for the mild and clean processing of lignocellulose to generate glucose and unmodified lignin in a large scale. Economic feasibility is highly dependent on its potential to generate high value natural products for energy, specialty chemicals, feed and food application.

     

<Superscript>123</Superscript>I-ADAM SPET imaging of serotonin transporter in patients with epilepsy and comorbid depression

Background

Purpose of the study was to investigate alterations in midbrain serotonin transporter (SERT) binding in patients with epilepsy and symptoms of depression compared to patients with epilepsy with no symptoms of depression.

Methods

We studied 12 patients with epilepsy (7 patients had focal and 5 had generalized epilepsy syndromes). The presence of self-reported symptoms of depression was assessed using Beck Depression Inventory (BDI) and the Emotional State Questionnaire (EST-Q). The binding potential of the SERT was assessed by performing brain single photon emission tomography (SPET) using the SERT radioligand 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(123)iodophenylamine (¹²³I-ADAM).

Results

Seven patients had BDI and EST-Q subscale scores greater than 11 points, which was interpreted as the presence of symptoms of depression. We found that ¹²³I-ADAM binding was not significantly different between patients with epilepsy with and without symptoms of depression. In addition, ¹²³I-ADAM binding did not show a significant correlation to either BDI or EST-Q depression subscale scores and did not differ between patients with focal vs. generalized epilepsy.

Conclusion

The results of our study failed to demonstrate alterations of SERT binding properties in patients with epilepsy with or without symptoms of depression.