Biotechnology for Biofuels

Journal: Biotechnology for Biofuels

Abbreviation

Biotechnol. biofuels

Publisher

BioMed Central

ISSN

1754-6834

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Publications1 - 8 of 8

Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose
Bergdahl, Basti; Heer, Dominik; Sauer, Uwe; et al. (2012)
Biotechnology for Biofuels
Background:The concerted effects of changes in gene expression due to changes in the environment areultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition cantherefore give a description of the cellular state with a high degree of functional information. We used thispotential to evaluate the metabolic status of two recombinant strains ofSaccharomyces cerevisiaeduringanaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only inthe pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitoldehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationshipbetween the two strains ascertains that the observed responses are a result of the particular xylose pathwayand not due to unknown changes in regulatory systems. An increased understanding of the physiologicalstate of these strains is important for further development of efficient pentose-utilizing strains for bioethanolproduction.Results:Using LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites incentral carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The generalresponse to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycleintermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strainsinvestigated had significantly different uptake rates of xylose which led to an enhanced response in theXI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stablearound 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylatepool.Conclusions:The low uptake of xylose by the XI-strain led to several distinguished responses: depletion ofkey metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromaticamino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few suchtraits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy tothe cells but does not suppress a response similar to carbon starvation. Particular signals may play a role inthe latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.
CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars
Gabriel, Raphael; Mueller, Rebecca; Floerl, Lena; et al. (2021)
Biotechnology for Biofuels
Background Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results A fed-batch system with plant biomass-derived sugars d-xylose, l-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion This work has identified two additional CAZyme inducers for T. aurantiacus, l-arabinose and cellobiose, along with d-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.
Steam explosion pretreatment of softwood: the effect of the explosive decompression on enzymatic digestibility
Pielhop, Thomas; Amgarten, Janick; Rudolf von Rohr, Philipp; et al. (2016)
Biotechnology for Biofuels
Background Steam explosion pretreatment has been examined in many studies for enhancing the enzymatic digestibility of lignocellulosic biomass and is currently the most common pretreatment method in commercial biorefineries. The information available about the effect of the explosive decompression on the biochemical conversion is, however, very limited, and no studies prove that the latter is actually enhanced by the explosion. Hence, it is of great value to discern between the effect of the explosion on the one hand and the steaming on the other hand, to identify their particular influences on enzymatic digestibility. Results The effect of the explosive decompression in the steam explosion pretreatment of spruce wood chips on their enzymatic cellulose digestibility was studied systematically. The explosion had a high influence on digestibility, improving it by up to 90 % compared to a steam pretreatment without explosion. Two factors were identified to be essentially responsible for the effect of the explosion on enzymatic digestibility: pretreatment severity and pressure difference of the explosion. A higher pretreatment severity can soften up and weaken the lignocellulose structure more, so that the explosion can better break up the biomass and decrease its particle size, which enhances its digestibility. In particular, increasing the pressure difference of the explosion leads to more defibration, a smaller particle size and a better digestibility. Though differences were found in the micro- and nanostructure of exploded and non-exploded biomass, the only influence of the explosion on digestibility was found to be the macroscopic particle size reduction. Steam explosion treatments with a high severity and a high pressure difference of the explosion lead to a comparatively high cellulose digestibility of the—typically very recalcitrant—softwood biomass. Conclusions This is the first study to show that explosion can enhance the enzymatic digestibility of lignocellulosic biomass. If the enhancing effect of the explosion is thoroughly exploited, even very recalcitrant biomass like softwood can be made enzymatically digestible.
Pilot-scale steam explosion pretreatment with 2-naphthol to overcome high softwood recalcitrance
Pielhop, Thomas; Amgarten, Janick; Studer, Michael H.; et al. (2017)
Biotechnology for Biofuels
Background Steam explosion pretreatment has been examined in many studies for enhancing the enzymatic digestibility of lignocellulosic biomass and is currently the most common pretreatment method in commercial biorefineries. It is however not effective for overcoming the extremely high recalcitrance of softwood to biochemical conversion. Recent fundamental research in small-scale liquid hot water pretreatment has shown, though, that the addition of a carbocation scavenger like 2-naphthol can prevent lignin repolymerization and thus enhance the enzymatic digestibility of softwood cellulose. This work studies the technical application potential of this approach in a larger steam explosion pilot plant for surmounting softwood recalcitrance. Results The addition of 35.36 g 2-naphthol to the steam explosion pretreatment of 1.5 kg spruce wood chips allowed to considerably enhance the enzymatic cellulose digestibility. Different ways of adding the solid 2-naphthol to steam pretreatment were tested. Mixing with the biomass before pretreatment could enhance digestibility by up to 55% compared to control experiments. Impregnation of the biomass with 2-naphthol was yet more effective. Acetone and ethanol were tested to dissolve 2-naphthol and impregnate the biomass. The solvents were then removed again by evaporation before the pretreatment. The impregnation allowed to enhance digestibility by up to 179 and 192%, respectively. A comparison to prevalent acid-catalyzed steam explosion pretreatments for softwood revealed that the scavenger approach allows for obtaining exceptionally high yields in enzymatic hydrolysis. The biomass impregnation with 2-naphthol even renders a complete enzymatic cellulose conversion possible, which is remarkable for a softwood pretreatment not removing lignin. Steam pretreatment experiments without explosive decompression revealed that the enhancing effects of the explosion and the scavenger complement each other well. The explosion enhances the accessibility of the cellulose while the use of the scavenger reduces particularly the deactivation of enzymes. Conclusions This is the first study to show that a carbocation scavenger in steam pretreatment can enhance the enzymatic digestibility of lignocellulosic biomass. The approach opens up a novel possibility for overcoming the high softwood recalcitrance in a process that does not require an acid catalyst or the removal of lignin from the biomass.
Improvement of corn stover fuel properties via hydrothermal carbonization combined with surfactant
Tu, Ren; Sun, Yan; Wu, Yujian; et al. (2019)
Biotechnology for Biofuels
Background Biomass fuel has been used to supply heat or crude materials in industry to replace the traditional fossil fuel which was one of the chief causes of climate warming. However, the large-scale utilization of biomass fuel was restricted due to the low density and high hydrophilicity of biomass, which causes the problem of transportation and storage. Therefore, pelletization of biomass was used to improve its fuel density. At present, the biomass pellet was widely used to supply heat, gas or electricity generation via gasification, which supplied clean and sustainable energy for industry. However, the energy consumption during pelletization and high hydrophilicity of pellets were still the problem for the large-scale application of biomass pellet. In this study, hydrothermal carbonization and surfactant played the role of permeation, adsorption and wetting in the solution, which was expected to improve the fuel properties and pelletization effectivity of corn stover. Results In the article, surfactant (PEG400, Span80, SDBS) was chosen to be combined with wet torrefaction to overcome the drawbacks and improve the pelletization and combustion properties of Corn stover (CS). Especially, hydrothermal carbonization (HTC) combined with surfactant improves the yield of solid products and reduces the ash content of solid product, which was beneficial for reducing the ashes of furnace during gasification. Meanwhile, surfactant promotes the formation of pseudo-lignin and the absorption for oil with low O and high C during HTC, which improves the energy density of solid product. Furthermore, the oil in solid product plays the role of lubricant and binder, which reduces the negative effect of high energy consumption, low bulk density and weak pellets strength caused by HTC during pelletization. HTC combined with surfactant improved the hydrophobicity of pellet as well as grindability due to the modification of solid product. Moreover, surfactant combined with HTC improved the combustion characteristic of solid product such as ignition and burning temperature as well as kinetic parameters due to the bio-oil absorbed and the improvement of surface and porosity. Conclusions The study supplied a new, less-energy intensive and effective method to improve the pelletization and combustion properties of corn stover via hydrothermal carbonization combined with surfactant, and provided a promising alternative fuel from corn stover
In muro deacetylation of xylan affects lignin properties and improves saccharification of aspen wood
Pawar, Prashant Mohan-Anupama; Derba-Maceluch, Marta; Chong, Sun-Li; et al. (2017)
Biotechnology for Biofuels
Background Lignocellulose from fast growing hardwood species is a preferred source of polysaccharides for advanced biofuels and “green” chemicals. However, the extensive acetylation of hardwood xylan hinders lignocellulose saccharification by obstructing enzymatic xylan hydrolysis and causing inhibitory acetic acid concentrations during microbial sugar fermentation. To optimize lignocellulose for cost-effective saccharification and biofuel production, an acetyl xylan esterase AnAXE1 from Aspergillus niger was introduced into aspen and targeted to cell walls. Results AnAXE1-expressing plants exhibited reduced xylan acetylation and grew normally. Without pretreatment, their lignocellulose yielded over 25% more glucose per unit mass of wood (dry weight) than wild-type plants. Glucose yields were less improved (+7%) after acid pretreatment, which hydrolyses xylan. The results indicate that AnAXE1 expression also reduced the molecular weight of xylan, and xylan–lignin complexes and/or lignin co-extracted with xylan, increased cellulose crystallinity, altered the lignin composition, reducing its syringyl to guaiacyl ratio, and increased lignin solubility in dioxane and hot water. Lignin-associated carbohydrates became enriched in xylose residues, indicating a higher content of xylo-oligosaccharides. Conclusions This work revealed several changes in plant cell walls caused by deacetylation of xylan. We propose that deacetylated xylan is partially hydrolyzed in the cell walls, liberating xylo-oligosaccharides and their associated lignin oligomers from the cell wall network. Deacetylating xylan thus not only increases its susceptibility to hydrolytic enzymes during saccharification but also changes the cell wall architecture, increasing the extractability of lignin and xylan and facilitating saccharification.
Two-stage steam explosion pretreatment of softwood with 2-naphthol as carbocation scavenger
Seidel, Christoph-Maximilian; Brethauer, Simone; Gyenge, László; et al. (2019)
Biotechnology for Biofuels
Background Lignocellulosic biomass is considered as a potential source for sustainable biofuels. In the conversion process, a pretreatment step is necessary in order to overcome the biomass recalcitrance and allow for sufficient fermentable sugar yields in enzymatic hydrolysis. Steam explosion is a well known pretreatment method working without additional chemicals and allowing for efficient particle size reduction. However, it is not effective for the pretreatment of softwood and the harsh conditions necessary to achieve a highly digestible cellulose fraction lead to the partial degradation of the hemicellulosic sugars. Previous studies showed that the autohydrolysis pretreatreatment of softwood can benefit from the addition of 2-naphthol. This carbocation scavenger prevents lignin repolymerisation leading to an enhanced glucose yield in the subsequent enzymatic hydrolysis. Results In order to prevent the degradation of the hemicellulose, we investigated in this study a two-stage 2-naphthol steam explosion pretreatment. In the first stage, spruce wood is pretreated at a severity which is optimal for the autocatalytic hydrolysis of the hemicellulose. The hydrolyzate containing the solubilized sugars is withdrawn from the reactor and the remaining solids are pretreated with different amounts of 2-naphthol in a second stage at a severity that allows for high glucose yields in enzymatic hydrolysis. The pretreated spruce was subjected to enzymatic hydrolysis and to simultaneous saccharification and fermentation (SSF). In the first stage, the maximal yield of hemicellulosic sugars was 47.5% at a pretreatment severity of log R0 = 3.75 at 180 °C. In the second stage, a 2-naphthol dosage of 0.205 mol/mol lignin C9-unit increased the ethanol yield in SSF with a cellulose loading of 1% using the whole second stage pretreatment slurry by 17% from 73.6% for the control without 2-naphthol to 90.4%. At a higher solid loading corresponding to 5% w/w cellulose, the yields decreased due to higher concentrations of residual 2-naphthol in the biomass and the pretreatment liquor, but also due to higher concentrations of potential inhibitors like HMF, furfural and acetic acid. Experiments with washed solids, vacuum filtered solids and the whole slurry showed that residual 2-naphthol can inhibit the fermentation as a single inhibitor but also synergistically together with HMF, furfural and acetic acid. Conclusions This work shows that a two-stage pretreatment greatly enhances the recovery of hemicellulosic sugars from spruce wood. The presence of 2-naphthol in the second pretreatment stage can enhance the ethanol yield in SSF of steam explosion pretreated softwood at low cellulose concentrations of 1% w/w. However, with higher solid loadings of 5% w/w cellulose, the ethanol yields were in general lower due to the solid effect and a synergistic inhibition of HMF, furfural, acetic acid with residual 2-naphthol. The concentration of residual 2-naphthol tolerated by the yeast decreased with increasing concentrations of HMF, furfural, and acetic acid.
CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate
Gutmann, Friederike; Jann, Cosimo; Pereira, Filipa; et al. (2021)
Biotechnology for Biofuels
Background Baker’s yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity. Results Here, we employ CRISPR interference in S. cerevisiae to identify genes modulating fermentative growth in plant hydrolysate and in presence of lignocellulosic toxins. We find that at least one-third of hydrolysate-associated gene functions are explained by effects of known toxic compounds, such as the decreased growth of YAP1 or HAA1, or increased growth of DOT6 knock-down strains in hydrolysate. Conclusion Our study confirms previously known genetic elements and uncovers new targets towards designing more robust yeast strains for the utilization of lignocellulose hydrolysate as sustainable feedstock, and, more broadly, paves the way for applying CRISPRi screens to improve industrial fermentation processes.

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Journal: Biotechnology for Biofuels

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