| 1. |
- Arnling Bååth, Jenny, 1987, et al.
(författare)
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Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Lignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration. Results Ten CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass. Conclusions Bacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin–carbohydrate disassembly.
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| 2. |
- Arnling Bååth, Jenny, 1987, et al.
(författare)
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Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo-β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus (CjMan5A and CjMan26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions. Results: CjMan5A and CjMan26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. CjMan5A action resulted in higher amounts of mannotriose and mannotetraose than that of CjMan26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by CjMan5A, whereas CjMan26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity. Conclusions: Our findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important resource spruce GGM. CjMan26A released higher amounts of fermentable sugars suitable for biofuel production, while CjMan5A, producing higher amounts of oligosaccharides, could be a good candidate for the production of oligomeric platform chemicals and food additives. Furthermore, chemical acetylation of mannan polymers was found to be a potential strategy for limiting the biodegradation of mannan-containing materials.
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| 3. |
- Aulitto, Martina, 1991, et al.
(författare)
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Bacillus coagulans MA-13: a promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:210
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Tidskriftsartikel (refereegranskat)abstract
- Background: The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed. Results: A thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans, named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 degrees C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-) hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 degrees C), still retaining an optimal operational activity at 50 degrees C. Conclusions: The above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto employed for the depolymerisation of lignocellulosic biomasses to fermentable sugars. Moreover, the robustness of B. coagulans MA-13 is a desirable trait, given the presence of microbial growth inhibitors in the pre-treated biomass hydrolysate.
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| 4. |
- Aulitto, Martina, 1991, et al.
(författare)
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Seed culture pre-adaptation of Bacillus coagulans MA-13 improves lactic acid production in simultaneous saccharification and fermentation
- 2019
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Lignocellulosic biomass is an abundant and sustainable feedstock, which represents a promising raw material for the production of lactic acid via microbial fermentation. However, toxic compounds that affect microbial growth and metabolism are released from the biomass upon thermochemical pre-treatment. So far, susceptibility of bacterial strains to biomass-derived inhibitors still represents a major barrier to lactic acid production from lignocellulose. Detoxification of the pre-treated lignocellulosic material by water washing is commonly performed to alleviate growth inhibition of the production microorganism and achieve higher production rates. Results In this study, we assessed the feasibility of replacing the washing step with integrated cellular adaptation during pre-culture of Bacillus coagulans MA-13 prior to simultaneous saccharification and lactic acid fermentation of steam exploded wheat straw. Using a seed culture pre-exposed to 30% hydrolysate led to 50% shorter process time, 50% higher average volumetric and 115% higher average specific productivity than when using cells from a hydrolysate-free seed culture. Conclusions Pre-exposure of B. coagulans MA-13 to hydrolysate supports adaptation to the actual production medium. This strategy leads to lower process water requirements and combines cost-effective seed cultivation with physiological pre-adaptation of the production strain, resulting in reduced lactic acid production costs.
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| 5. |
- Bhalla, Aditya, et al.
(författare)
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Effective alkaline metal-catalyzed oxidative delignification of hybrid poplar
- 2016
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundStrategies to improve copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment of hybrid poplar were investigated. These improvements included a combination of increasing hydrolysis yields, while simultaneously decreasing process inputs through (i) more efficient utilization of H2O2 and (ii) the addition of an alkaline extraction step prior to the metal-catalyzed AHP pretreatment. We hypothesized that utilizing this improved process could substantially lower the chemical inputs needed during pretreatment.ResultsHybrid poplar was pretreated utilizing a modified process in which an alkaline extraction step was incorporated prior to the Cu-AHP treatment step and H2O2 was added batch-wise over the course of 10 h. Our results revealed that the alkaline pre-extraction step improved both lignin and xylan solubilization, which ultimately led to improved glucose (86 %) and xylose (95 %) yields following enzymatic hydrolysis. An increase in the lignin solubilization was also observed with fed-batch H2O2 addition relative to batch-only addition, which again resulted in increased glucose and xylose yields (77 and 93 % versus 63 and 74 %, respectively). Importantly, combining these strategies led to significantly improved sugar yields (96 % glucose and 94 % xylose) following enzymatic hydrolysis. In addition, we found that we could substantially lower the chemical inputs (enzyme, H2O2, and catalyst), while still maintaining high product yields utilizing the improved Cu-AHP process. This pretreatment also provided a relatively pure lignin stream consisting of ≥90 % Klason lignin and only 3 % xylan and 2 % ash following precipitation. Two-dimensional heteronuclear single-quantum coherence (2D HSQC) NMR and size-exclusion chromatography demonstrated that the solubilized lignin was high molecular weight (Mw ≈ 22,000 Da) and only slightly oxidized relative to lignin from untreated poplar.ConclusionsThis study demonstrated that the fed-batch, two-stage Cu-AHP pretreatment process was effective in pretreating hybrid poplar for its conversion into fermentable sugars. Results showed sugar yields near the theoretical maximum were achieved from enzymatically hydrolyzed hybrid poplar by incorporating an alkaline extraction step prior to pretreatment and by efficiently utilizing H2O2 during the Cu-AHP process. Significantly, this study reports high sugar yields from woody biomass treated with an AHP pretreatment under mild reaction conditions.
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| 6. |
- Bhalla, Aditya, et al.
(författare)
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Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline–oxidative pretreatment of hybrid poplar
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer. - 1754-6834 .- 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H2O2) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss in sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified.
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| 7. |
- Bhalla, Aditya, et al.
(författare)
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Performance of three delignifying pretreatments on hardwoods: hydrolysis yields, comprehensive mass balances, and lignin properties
- 2019
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Ingår i: Biotechnology for Biofuels. - : BioMed Central (BMC). - 1754-6834 .- 1754-6834. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundIn this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading. Lignins recovered from the pretreatments were characterized for polysaccharide content, molar mass distributions, β-aryl ether content, and response to depolymerization by thioacidolysis.ResultsAll three pretreatments resulted in significant solubilization of lignin and xylan, with the CELF pretreatment solubilizing the majority of both biopolymer categories. Enzymatic hydrolysis yields were shown to exhibit a strong, positive correlation with the lignin solubilized for the low enzyme loadings. The pretreatment-derived solubles in the [Ch][Lys]-pretreated biomass were presumed to contribute to inhibition of enzymatic hydrolysis in the eucalyptus as a substantial fraction of the pretreatment liquor was carried forward into hydrolysis for this pretreatment. The pretreatment-solubilized lignins exhibited significant differences in polysaccharide content, molar mass distributions, aromatic monomer yield by thioacidolysis, and β-aryl ether content. Key trends include a substantially higher polysaccharide content in the lignins recovered from the [Ch][Lys] pretreatment and high β-aryl ether contents and aromatic monomer yields from the Cu-AHP pretreatment. For all lignins, the 13C NMR-determined β-aryl ether content was shown to be correlated with the monomer yield with a second-order functionality.ConclusionsOverall, it was demonstrated that the three pretreatments highlighted in this study demonstrated uniquely different functionalities in reducing biomass recalcitrance and achieving higher enzymatic hydrolysis yields for the hybrid poplar while yielding a lignin-rich stream that may be suitable for valorization. Furthermore, modification of lignin during pretreatment, particularly cleavage of β-aryl ether bonds, is shown to be detrimental to subsequent depolymerization.
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| 8. |
- Björkmalm, Johanna, et al.
(författare)
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A non-linear model of hydrogen production by Caldicellulosiruptor saccharolyticus for diauxic-like consumption of lignocellulosic sugar mixtures
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 11:1, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Caldicellulosiruptor saccharolyticus is an attractive hydrogen producer suitable for growth on various lignocellulosic substrates. The aim of this study was to quantify uptake of pentose and hexose monosaccharides in anindustrial substrate and to present a kinetic growth model of C. saccharolyticus that includes sugar uptake on defined and industrial media. The model is based on Monod and Hill kinetics extended with gas-to-liquid mass transfer and acybernetic approach to describe diauxic-like growth.Results: Mathematical expressions were developed to describe hydrogen production by C. saccharolyticus consuming glucose, xylose, and arabinose. The model parameters were calibrated against batch fermentation data. Theexperimental data included four different cases: glucose, xylose, sugar mixture, and wheat straw hydrolysate (WSH) fermentations. The fermentations were performed without yeast extract. The substrate uptake rate of C. saccharolyticuson single sugar-defined media was higher on glucose compared to xylose. In contrast, in the defined sugar mixture and WSH, the pentoses were consumed faster than glucose. Subsequently, the cultures entered a lag phasewhen all pentoses were consumed after which glucose uptake rate increased. This phenomenon suggested a diauxic-like behavior as was deduced from the successive appearance of two peaks in the hydrogen and carbon dioxideproductivity. The observation could be described with a modified diauxic model including a second enzyme system with a higher affinity for glucose being expressed when pentose saccharides are consumed. This behavior was morepronounced when WSH was used as substrate.Conclusions: The previously observed co-consumption of glucose and pentoses with a preference for the latter was herein confirmed. However, once all pentoses were consumed, C. saccharolyticus most probably expressed anotheruptake system to account for the observed increased glucose uptake rate. This phenomenon could be quantitatively captured in a kinetic model of the entire diauxic-like growth process. Moreover, the observation indicates a regulationsystem that has fundamental research relevance, since pentose and glucose uptake in C. saccharolyticus has only been described with ABC transporters, whereas previously reported diauxic growth phenomena have been correlatedmainly to PTS systems for sugar uptake.
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| 9. |
- Bondesson, Pia-Maria, et al.
(författare)
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Process design of SSCF for ethanol production from steam-pretreated, acetic-acid-impregnated wheat straw
- 2016
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPretreatment is an important step in the production of ethanol from lignocellulosic material. Using acetic acid together with steam pretreatment allows the positive effects of an acid catalyst to be retained, while avoiding the negative environmental effects associated with sulphuric acid. Acetic acid is also formed during the pretreatment and hydrolysis of hemicellulose, and is a known inhibitor that may impair fermentation at high concentrations. The purpose of this study was to improve ethanol production from glucose and xylose in steam-pretreated, acetic-acid-impregnated wheat straw by process design of simultaneous saccharification and co-fermentation (SSCF), using a genetically modified pentose fermenting yeast strain Saccharomyces cerevisiae.ResultsEthanol was produced from glucose and xylose using both the liquid fraction and the whole slurry from pretreated materials. The highest ethanol concentration achieved was 37.5 g/L, corresponding to an overall ethanol yield of 0.32 g/g based on the glucose and xylose available in the pretreated material. To obtain this concentration, a slurry with a water-insoluble solids (WIS) content of 11.7 % was used, using a fed-batch SSCF strategy. A higher overall ethanol yield (0.36 g/g) was obtained at 10 % WIS.ConclusionsEthanol production from steam-pretreated, acetic-acid-impregnated wheat straw through SSCF with a pentose fermenting S. cerevisiae strain was successfully demonstrated. However, the ethanol concentration was too low and the residence time too long to be suitable for large-scale applications. It is hoped that further process design focusing on the enzymatic conversion of cellulose to glucose will allow the combination of acetic acid pretreatment and co-fermentation of glucose and xylose.
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| 10. |
- Borisova, Anna, et al.
(författare)
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Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, similar to 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs.Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.
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