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  • 1. Alriksson, Björn
    et al.
    Cavka, Adnan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents2011In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 102, no 2, p. 1254-1263Article in journal (Refereed)
    Abstract [en]

    Inhibitory lignocellulose hydrolysates were treated with the reducing agents dithionite and sulfite to achieve improved fermentability. Addition of these reducing agents (in the concentration range 5.0-17.5mM) to enzymatic hydrolysates of spruce wood or sugarcane bagasse improved processes based on both SHF (simultaneous hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation). The approach was exemplified in ethanolic fermentations with Saccharomyces cerevisiae and by using hydrolysates with sugar concentrations >100g/L (for SHF) and with 10% dry-matter content (for SSF). In the SHF experiments, treatments with dithionite raised the ethanol productivities of the spruce hydrolysate from 0.2 to 2.5g×L(-1)×h(-1) and of the bagasse hydrolysate from 0.9 to 3.9g×L(-1)×h(-1), values even higher than those of fermentations with reference sugar solutions without inhibitors. Benefits of the approach include that the addition of the reducing agent can be made in-situ directly in the fermentation vessel, that the treatment can be performed at a temperature and pH suitable for fermentation, and that the treatment results in dramatically improved fermentability without degradation of fermentable sugars. The many benefits and the simplicity of the approach offer a new way to achieve more efficient manufacture of fermentation products from lignocellulose hydrolysates.

  • 2.
    Cavka, Adnan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Biorefining of lignocellulose: Detoxification of inhibitory hydrolysates and potential utilization of residual streams for production of enzymes2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Lignocellulosic biomass is a renewable resource that can be utilized for the production of biofuels, chemicals, and bio-based materials. Biochemical conversion of lignocellulose to advanced biofuels, such as cellulosic ethanol, is generally performed through microbial fermentation of sugars generated by thermochemical pretreatment of the biomass followed by an enzymatic hydrolysis of the cellulose. The aims of the research presented in this thesis were to address problems associated with pretreatment by-products that inhibit microbial and enzymatic biocatalysts, and to investigate the potential of utilizing residual streams from pulp mills and biorefineries to produce hydrolytic enzymes.

    A novel method to detoxify lignocellulosic hydrolysates to improve the fermentability was investigated in experiments with the yeast Saccharomyces cerevisiae. The method is based on treatment of lignocellulosic slurries and hydrolysates with reducing agents, such as sodium dithionite and sodium sulfite. The effects of treatment with sodium borohydride were also investigated. Treatment of a hydrolysate of Norway spruce by addition of 10 mM dithionite resulted in an increase of the balanced ethanol yield from 0.03 to 0.35 g/g. Similarly, the balanced ethanol yield of a hydrolysate of sugarcane bagasse increased from 0.06 to 0.28 g/g after treatment with 10 mM dithionite. In another study with a hydrolysate of Norway spruce, addition of 34 mM borohydride increased the balanced ethanol yield from 0.02 to 0.30 g/g, while the ethanol productivity increased from 0.05 to 0.57 g/(L×h). While treatment with sulfur oxyanions had a positive effect on microbial fermentation and enzymatic hydrolysis, treatment with borohydride resulted in an improvement only for the microbial fermentation. The chemical effects of treatments of hydrolysates with sodium dithionite, sodium sulfite, and sodium borohydride were investigated using liquid chromatography-mass spectrometry (LC-MS). Treatments with dithionite and sulfite were found to rapidly sulfonate inhibitors already at room temperature and at a pH that is compatible with enzymatic hydrolysis and microbial fermentation. Treatment with borohydride reduced inhibitory compounds, but the products were less hydrophilic than the products obtained in the reactions with the sulfur oxyanions.

    The potential of on-site enzyme production using low-value residual streams, such as stillage, was investigated utilizing recombinant Aspergillus niger producing xylanase and cellulase. A xylanase activity of 8,400 nkat/ml and a cellulase activity of 2,700 nkat/ml were reached using stillages from processes based on waste fiber sludge. The fungus consumed a large part of the xylose, the acetic acid, and the oligosaccharides that were left in the stillages after fermentation with S. cerevisiae. In another study, the capability of two filamentous fungi (A. niger and Trichoderma reesei) and three yeasts (S. cerevisiae, Pichia pastoris, and Yarrowia lipolytica) to grow on inhibitory lignocellulosic media were compared. The results indicate that the two filamentous fungi had the best capability to utilize different nutrients in the media, while the S. cerevisiae strain exhibited the best tolerance against the inhibitors. Utilization of different nutrients would be especially important in enzyme production using residual streams, while tolerance against inhibitors is desirable in a consolidated bio-process in which the fermenting microorganism also contributes by producing enzymes.

  • 3.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Björn
    Processum Biorefinery Initiative AB, SE-891 22 Örnsköldsvik, Sweden.
    Ahnlund, Maria
    Umeå Plant Science Center, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors2011In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 108, no 11, p. 2592-2599Article in journal (Refereed)
    Abstract [en]

    Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).

  • 4.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Björn
    Rose, Shaunita H
    van Zyl, Willem H
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge2011In: Journal of Industrial Microbiology & Biotechnology, ISSN 1367-5435, E-ISSN 1476-5535, Vol. 38, no 8, p. 891-899Article in journal (Refereed)
    Abstract [en]

    The possibility to utilize fiber sludge, waste fibers from pulp mills and lignocellulose-based biorefineries, for combined production of liquid biofuel and biocatalysts was investigated. Without pretreatment, fiber sludge was hydrolyzed enzymatically to monosaccharides, mainly glucose and xylose. In the first of two sequential fermentation steps, the fiber sludge hydrolysate was fermented to cellulosic ethanol with the yeast Saccharomyces cerevisiae. Although the final ethanol yields were similar, the ethanol productivity after 9.5 h was 3.3 g/l/h for the fiber sludge hydrolysate compared with only 2.2 g/l/h for a reference fermentation with similar sugar content. In the second fermentation step, the spent fiber sludge hydrolysate (the stillage obtained after distillation) was used as growth medium for recombinant Aspergillus niger expressing the xylanase-encoding Trichoderma reesei (Hypocrea jecorina) xyn2 gene. The xylanase activity obtained with the spent fiber sludge hydrolysate (8,500 nkat/ml) was higher than that obtained in a standard medium with similar monosaccharide content (1,400 nkat/ml). Analyses based on deglycosylation with N-glycosidase F suggest that the main part of the recombinant xylanase was unglycosylated and had molecular mass of 20.7 kDa, while a minor part had N-linked glycosylation and molecular mass of 23.6 kDa. Chemical analyses of the growth medium showed that important carbon sources in the spent fiber sludge hydrolysate included xylose, small aliphatic acids, and oligosaccharides. The results show the potential of converting waste fiber sludge to liquid biofuel and enzymes as coproducts in lignocellulose-based biorefineries.

  • 5.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Björn
    Processum Biorefinery Initiative AB, 891 22 Örnsköldsvik, Sweden.
    Rose, Shaunita H.
    Department of Microbiology, Stellenbosch University, Stellenbosch, 7602, South Africa.
    van Zyl, Willem H.
    Department of Microbiology, Stellenbosch University, Stellenbosch, 7602, South Africa.
    Jönsson, Leif J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger2014In: Journal of Industrial Microbiology & Biotechnology, ISSN 1367-5435, E-ISSN 1476-5535, Vol. 41, no 8, p. 1191-1200Article in journal (Refereed)
    Abstract [en]

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

  • 6.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Guo, Xiang
    Tang, Shui-Jia
    Winestrand, Sandra
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hong, Feng
    Production of bacterial cellulose and enzyme from waste fiber sludge2013In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 6, no 25Article in journal (Refereed)
    Abstract [en]

    Background: Bacterial cellulose (BC) is a highly crystalline and mechanically stable nanopolymer, which has excellent potential as a material in many novel applications, especially if it can be produced in large amounts from an inexpensive feedstock. Waste fiber sludge, a residue with little or no value, originates from pulp mills and lignocellulosic biorefineries. A high cellulose and low lignin content contributes to making the fiber sludge suitable for bioconversion, even without a thermochemical pretreatment step. In this study, the possibility to combine production of BC and hydrolytic enzymes from fiber sludge was investigated. The BC was characterized using field-emission scanning electron microscopy and X-ray diffraction analysis, and its mechanical properties were investigated.

    Results: Bacterial cellulose and enzymes were produced through sequential fermentations with the bacterium Gluconacetobacter xylinus and the filamentous fungus Trichoderma reesei. Fiber sludges from sulfate (SAFS) and sulfite (SIFS) processes were hydrolyzed enzymatically without prior thermochemical pretreatment and the resulting hydrolysates were used for BC production. The highest volumetric yields of BC from SAFS and SIFS were 11 and 10 g/L (DW), respectively. The BC yield on initial sugar in hydrolysate-based medium reached 0.3 g/g after seven days of cultivation. The tensile strength of wet BC from hydrolysate medium was about 0.04 MPa compared to about 0.03 MPa for BC from a glucose-based reference medium, while the crystallinity was slightly lower for BC from hydrolysate cultures. The spent hydrolysates were used for production of cellulase with T. reesei. The cellulase activity (CMCase activity) in spent SAFS and SIFS hydrolysates reached 5.2 U/mL (87 nkat/mL), which was similar to the activity level obtained in a reference medium containing equal amounts of reducing sugar.

    Conclusions: It was shown that waste fiber sludge is a suitable raw material for production of bacterial cellulose and enzymes through sequential fermentation. The concept studied offers efficient utilization of the various components in fiber sludge hydrolysates and affords a possibility to combine production of two high value-added products using residual streams from pulp mills and biorefineries. Cellulase produced in this manner could tentatively be used to hydrolyze fresh fiber sludge to obtain medium suitable for production of BC in the same biorefinery.

  • 7.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jönsson, Leif J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Comparison of the growth of filamentous fungi and yeasts in lignocellulose-derived mediaManuscript (preprint) (Other academic)
  • 8.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Detoxification of lignocellulosic hydrolysates using sodium borohydride2013In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 136, p. 368-376Article in journal (Refereed)
    Abstract [en]

    Addition of sodium borohydride to a lignocellulose hydrolysate of Norway spruce affected the fermentability when cellulosic ethanol was produced using Saccharomyces cerevisiae. Treatment of the hydrolysate with borohydride improved the ethanol yield on consumed sugar from 0.09 to 0.31 g/g, the balanced ethanol yield from 0.02 to 0.30 g/g, and the ethanol productivity from 0.05 to 0.57 g/(L×h). Treatment of a sugarcane bagasse hydrolysate gave similar results, and the experiments indicate that sodium borohydride is suitable for chemical in-situ detoxification. The model inhibitors coniferyl aldehyde, p-benzoquinone, 2,6-dimethoxybenzoquinone, and furfural were efficiently reduced by treatment with sodium borohydride, even under mild reaction conditions (20°C and pH 6.0). While addition of sodium dithionite to pretreatment liquid from spruce improved enzymatic hydrolysis of cellulose, addition of sodium borohydride did not. This result indicates that the strong hydrophilicity resulting from sulfonation of inhibitors by dithionite treatment was particularly important for alleviating enzyme inhibition.

  • 9.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Martin, Carlos
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Bjorn
    Mortsell, Marlene
    Jönsson, Leif J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Techno-economic evaluation of conditioning with sodium sulfite for bioethanol production from softwood2015In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 196, p. 129-135Article in journal (Refereed)
    Abstract [en]

    Conditioning with reducing agents allows alleviation of inhibition of biocatalytic processes by toxic by-products generated during biomass pretreatment, without necessitating the introduction of a separate process step. In this work, conditioning of steam-pretreated spruce with sodium sulfite made it possible to lower the yeast and enzyme dosages in simultaneous saccharification and fermentation (SSF) to 1 g/L and 5 FPU/g WIS, respectively. Techno-economic evaluation indicates that the cost of sodium sulfite can be offset by benefits resulting from a reduction of either the yeast load by 0.68 g/L or the enzyme load by 1 FPU/g WIS. As those thresholds were surpassed, inclusion of conditioning can be justified. Another potential benefit results from shortening the SSF time, which would allow reducing the bioreactor volume and result in capital savings. Sodium sulfite conditioning emerges as an opportunity to lower the financial uncertainty and compensate the overall investment risk for commercializing a softwood-to-ethanol process. (C) 2015 The Authors. Published by Elsevier Ltd.

  • 10.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Stagge, Stefan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jönsson, Leif J.
    Identification of Small Aliphatic Aldehydes in Pretreated Lignocellulosic Feedstocks and Evaluation of Their Inhibitory Effects on Yeast2015In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 63, no 44, p. 9747-9754Article in journal (Refereed)
    Abstract [en]

    Six lignocellulosic hydrolysates produced through acid pretreatment were analyzed for the occurrence of formaldehyde, acetaldehyde, and glycolaldehyde. Acetaldehyde was found in all six (0.3-1.6 mM) and formaldehyde in four (<= 4.4 mM), whereas glycolaldehyde was not detected. To assess the relevance of these findings, fermentations with yeast and formaldehyde or acetaldehyde were performed in the concentration interval 0.5-10 mM. Formaldehyde already inhibited at 1.0 mM, whereas 5.0 mM acetaldehyde was needed to obtain a clear inhibitory effect. After 24 h of fermentation, 1.5 mM formaldehyde reduced the glucose consumption by 85%, the balanced ethanol yield by 92%, and the volumetric productivity by 91%. The results show that formaldehyde and acetaldehyde are prevalent in pretreated lignocellulose and that formaldehyde in some cases could explain a large part of the inhibitory effects on yeast by lignocellulosic hydrolysates, as three of six hydrolysates contained >= 1.9 mM formaldehyde, which was shown to be strongly inhibitory.

  • 11.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallenius, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Björn
    Nilvebrant, Nils-Olof
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ozone detoxification of steam-pretreated Norway spruce2015In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 8, article id 196Article in journal (Refereed)
    Abstract [en]

    Background: Pretreatment of lignocellulose for biochemical conversion commonly results in formation of by-products that inhibit microorganisms and cellulolytic enzymes. To make bioconversion processes more efficient, inhibition problems can be alleviated through conditioning. Ozone is currently commercially employed in pulp and paper production for bleaching, as it offers the desirable capability to disrupt unsaturated bonds in lignin through an ionic reaction known as ozonolysis. Ozonolysis is more selective towards lignin than cellulose, for instance, when compared to other oxidative treatment methods, such as Fenton's reagent. Ozone may thus have desirable properties for conditioning of pretreated lignocellulose without concomitant degradation of cellulose or sugars. Ozone treatment of SO2- impregnated steam-pretreated Norway spruce was explored as a potential approach to decrease inhibition of yeast and cellulolytic enzymes. This novel approach was furthermore compared to some of the most effective methods for conditioning of pretreated lignocellulose, i.e., treatment with alkali and sodium dithionite. Results: Low dosages of ozone decreased the total contents of phenolics to about half of the initial value and improved the fermentability. Increasing ozone dosages led to almost proportional increase in the contents of total acids, including formic acid, which ultimately led to poor fermentability at higher ozone dosages. The decrease of the contents of furfural and 5-hydroxymethylfurfural was inversely proportional (R-2 > 0.99) to the duration of the ozone treatment, but exhibited no connection with the fermentability. Ozone detoxification was compared with other detoxification methods and was superior to treatment with Fenton's reagent, which exhibited no positive effect on fermentability. However, ozone detoxification was less efficient than treatment with alkali or sodium dithionite. High ozone dosages decreased the inhibition of cellulolytic enzymes as the glucose yield was improved with 13 % compared to that of an untreated control. Conclusions: Low dosages of ozone were beneficial for the fermentation of steam-pretreated Norway spruce, while high dosages decreased the inhibition of cellulolytic enzymes by soluble components in the pretreatment liquid. While clearly of interest for conditioning of lignocellulosic hydrolysates, future challenges include finding conditions that provide beneficial effects both with regard to enzymatic saccharification and microbial fermentation.

  • 12. Guo, Xiang
    et al.
    Cavka, Adnan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Industrial Graduate School.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hong, Feng
    Comparison of methods for detoxification of spruce hydrolysate for bacterial cellulose production2013In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 12, article id 93Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Bacterial cellulose (BC) is a nanostructured material with unique properties and wide applicability. In order to decrease the production cost of bacterial cellulose, lignocellulose-based media have considerable potential as alternative cost-effective feedstocks. However, pretreatment and enzymatic hydrolysis of lignocellulose to sugars also generate fermentation inhibitors. Detoxification of lignocellulosic hydrolysates is needed to achieve efficient production of BC. In this investigation, different methods for detoxification of spruce hydrolysate prior to production of BC were compared with respect to effects on potential inhibitors and fermentable sugars, sugar consumption, BC yield, and cell viability. The objectives were to identify efficient detoxification methods and to achieve a better understanding of the role played by different inhibitors in lignocellulosic hydrolysates.

    RESULTS: In a first series of experiments, the detoxification methods investigated included treatments with activated charcoal, alkali [sodium hydroxide, calcium hydroxide (overliming), and ammonium hydroxide], anion and cation ion-exchange resins, and reducing agents (sodium sulfite and sodium dithionite). A second series of detoxification experiments included enzymatic treatments (laccase and peroxidase). The potential inhibitors studied included aliphatic acids, furan aldehydes, and phenolic compounds. The best effects in the first series of detoxification experiments were achieved with activated charcoal and anion exchanger. After detoxification with activated charcoal the BC yield was 8.2 g/L, while, it was 7.5 g/L in a reference medium without inhibitors. Treatments with anion exchanger at pH 10 and pH 5.5 gave a BC yield of 7.9 g/L and 6.3 g/L, respectively. The first series of experiments suggested that there was a relationship between the BC yield and phenolic inhibitors. Therefore, the second series of detoxification experiments focused on treatments with phenol-oxidizing enzymes. The BC yield in the laccase-detoxified hydrolysate reached 5.0-5.5 g/L after 14 days cultivation, which demonstrated the important inhibitory role played by phenolic compounds.

    CONCLUSIONS: The investigation shows that detoxification methods that efficiently remove phenolics benefit bacterial growth and BC production. Negative effects of salts could not be excluded and the osmotolerance of Gluconacetobacter xylinus needs to be further investigated in the future. Combinations of detoxification methods that efficiently decrease the concentration of inhibitors remain as an interesting option.

  • 13.
    Stagge, Stefan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cavka, Adnan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Identification of benzoquinones in pretreated lignocellulosic feedstocks and inhibitory effects on yeast2015In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 5, article id 62Article in journal (Refereed)
    Abstract [en]

    Pretreatment of lignocellulosic biomass under acidic conditions gives rise to by-products that inhibit fermenting microorganisms. An analytical procedure for identification of p-benzoquinone (BQ) and 2,6-dimethoxybenzoquinone (DMBQ) in pretreated biomass was developed, and the inhibitory effects of BQ and DMBQ on the yeast Saccharomyces cerevisiae were assessed. The benzoquinones were analyzed using ultra-high performance liquid chromatographyelectrospray ionization-triple quadrupole-mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Pretreatment liquids examined with regard to the presence of BQ and DMBQ originated from six different lignocellulosic feedstocks covering agricultural residues, hardwood, and softwood, and were produced through impregnation with sulfuric acid or sulfur dioxide at varying pretreatment temperature (165-204 degrees C) and residence time (6-20 min). BQ was detected in all six pretreatment liquids in concentrations ranging up to 6 mg/l, while DMBQ was detected in four pretreatment liquids in concentrations ranging up to 0.5 mg/l. The result indicates that benzoquinones are ubiquitous as by-products of acid pretreatment of lignocellulose, regardless of feedstock and pretreatment conditions. Fermentation experiments with BQ and DMBQ covered the concentration ranges 2 mg/l to 1 g/l and 20 mg/l to 1 g/l, respectively. Even the lowest BQ concentration tested (2 mg/l) was strongly inhibitory to yeast, while 20 mg/l DMBQ gave a slight negative effect on ethanol formation. This work shows that benzoquinones should be regarded as potent and widespread inhibitors in lignocellulosic hydrolysates, and that they warrant attention besides more well-studied inhibitory substances, such as aliphatic carboxylic acids, phenols, and furan aldehydes.

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