Engineering 5-hydroxymethylfurfural (HMF) oxidation in Pseudomonas boosts tolerance and accelerates 2,5-furandicarboxylic acid (FDCA) production.

Due to its tolerance properties, Pseudomonas has gained particular interest as host for oxidative upgrading of the toxic aldehyde 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA), a promising biobased alternative to terephthalate in polyesters. However, until now, the native enzymes responsible for aldehyde oxidation are unknown. Here, we report the identification of the primary HMF-converting enzymes of P. taiwanensis VLB120 and P. putida KT2440 by extended gene deletions. The key players in HMF oxidation are a molybdenum-dependent periplasmic oxidoreductase and a cytoplasmic dehydrogenase. Deletion of the corresponding genes almost completely abolished HMF oxidation, leading instead to aldehyde reduction. In this context, two HMF-reducing dehydrogenases were also revealed. These discoveries enabled enhancement of Pseudomonas' furanic aldehyde oxidation machinery by genomic overexpression of the respective genes. The resulting BOX strains (Boosted OXidation) represent superior hosts for biotechnological synthesis of FDCA from HMF. The increased oxidation rates provide greatly elevated HMF tolerance, thus tackling one of the major drawbacks of whole-cell catalysis with this aldehyde. Furthermore, the ROX (Reduced OXidation) and ROAR (Reduced Oxidation And Reduction) deletion mutants offer a solid foundation for future development of Pseudomonads as biotechnological chassis notably for scenarios where rapid HMF conversion is undesirable.

One-pot Twostep Chemobiocatalytic Synthesis of a Furan Amino Acid from 5-Hydroxymethylfurfural.

Recently, catalytic valorization of biomass-derived furans has received growing interest. 5-Aminomethyl-2-furancarboxylic acid (AMFC), a furan amino acid, holds great promise in the aeras of polymer and pharmaceutical, but its synthesis remains limited. In this work, we report a chemobiocatalytic route toward AMFC by combining laccase-TEMPO system and recombinant Escherichia coli (named E. coli_TAF) harboring ω-transaminase (TA), L-alanine dehydrogenase (L-AlaDH) and formate dehydrogenase (FDH), starting from 5-hydroxymethylfurfural (HMF). In the cascade, HMF is oxidized into 5-formyl-2-furancarboxylic acid (FFCA) by laccase-TEMPO system, and then the resulting intermediate is converted into AMFC by E. coli_TAF via transamination with cheap ammonium formate instead of costly organic amine donors, theoretically generating H2O and CO2 as by-products. The tandem process was run in a one-pot twostep manner, affording AMFC with approximately 81 % yield, together with 10 % 2,5-furandicarboxylic acid (FDCA) as by-product. In addition, the scale-up production of AMFC was demonstrated, with 0.41 g/L h productivity and 8.6 g/L titer. This work may pave the way for green manufacturing of the furan-containing amino acid.

Human Keratinocyte Responses to Woodsmoke Chemicals.

Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1-2 via adaptive laboratory evolution.

BACKGROUND: Spathaspora passalidarum is a yeast with the highly effective capability of fermenting several monosaccharides in lignocellulosic hydrolysates, especially xylose. However, this yeast was shown to be sensitive to furfural released during pretreatment and hydrolysis processes of lignocellulose biomass. We aimed to improve furfural tolerance in a previously isolated S. passalidarum CMUWF1-2, which presented thermotolerance and no detectable glucose repression, via adaptive laboratory evolution (ALE). RESULTS: An adapted strain, AF2.5, was obtained from 17 sequential transfers of CMUWF1-2 in YPD broth with gradually increasing furfural concentration. Strain AF2.5 could tolerate higher concentrations of furfural, ethanol and 5-hydroxymethyl furfuraldehyde (HMF) compared with CMUWF1-2 while maintaining the ability to utilize glucose and other sugars simultaneously. Notably, the lag phase of AF2.5 was 2 times shorter than that of CMUWF1-2 in the presence of 2.0 g/l furfural, which allowed the highest ethanol titers to be reached in a shorter period. To investigate more in-depth effects of furfural, intracellular reactive oxygen species (ROS) accumulation was observed and, in the presence of 2.0 g/l furfural, AF2.5 exhibited 3.41 times less ROS accumulation than CMUWF1-2 consistent with the result from nuclear chromatins diffusion, which the cells number of AF2.5 with diffuse chromatins was also 1.41 and 1.24 times less than CMUWF1-2 at 24 and 36 h, respectively. CONCLUSIONS: An enhanced furfural tolerant strain of S. passalidarum was achieved via ALE techniques, which shows faster and higher ethanol productivity than that of the wild type. Not only furfural tolerance but also ethanol and HMF tolerances were improved.

Plant-based meat substitute analysis using microextraction with deep eutectic solvent followed by LC-MS/MS to determine acrylamide, 5-hydroxymethylfurfural and furaneol.

For the analysis of plant-based meat substitutes and the determination of Maillard reaction products such as acrylamide, 5-hydroxymethylfurfural and furaneol, a novel and effective procedure based on hydrophobic natural deep eutectic solvent and liquid chromatography coupled with tandem mass spectrometry was developed for the first time. The 49 compositions of the deep eutectic solvents were designed and screened to select the most suitable option. The terpenoids eugenol and thymol in a molar ratio of 2:1 were selected as precursors for solvent formation, allowing effective extraction of the target analytes. The developed procedure comprised two main steps: extraction - in which the analytes are isolated from the solid sample due to the salting-out effect and pre-concentrated in the deep eutectic solvent, and back-extraction - in which the analytes are re-extracted into the formic acid solution for subsequent mass spectrometric detection. As the density of the aqueous phases changed during the extraction and back-extraction steps, the phenomenon of inversion of the coalesced organic phase was observed, which simplified the withdrawing of the phases. The linear range was 1-50 ng/mL for acrylamide, 10-1000 ng/mL for 5-hydroxymethylfurfural and 200-1000 ng/mL for furaneol with coefficients of determination above 0.9952. The developed method was fully validated and found recoveries were in the range 83-120%, with CVs not exceeding 4.9%. The method was applied to real sample analysis of pea-based meat substitutes.

Green Synthesis of UV-Reactive Polycarbonates from Levoglucosenone and 5-Hydroxymethyl Furfural.

This study focuses on the synthesis of fully renewable polycarbonates (PCs) starting from cellulose-based platform molecules levoglucosenone (LGO) and 2,5-bis(hydroxymethyl)furan (BHMF). These unique bio-based PCs are obtained through the reaction of a citronellol-containing triol (Triol-citro) derived from LGO, with a dimethyl carbonate derivative of BHMF (BHMF-DC). Solvent-free polymerizations are targeted to minimize waste generation and promote an eco-friendly approach with a favorable environmental factor (E-factor). The choice of metal catalyst during polymerization significantly influences the polymer properties, resulting in high molecular weight (up to 755 kDa) when Na2 CO3 is employed as an inexpensive catalyst. Characterization using nuclear magnetic resonance confirms the successful incorporation of the furan ring and the retention of the terminal double bond of the citronellol pendant chain. Furthermore, under UV irradiation, the presence of both citronellol and furanic moieties induces singular structural changes, triggering the formation of three distinct structures within the polymer network, a phenomenon herein occurs for the first time in this type of polymer. These findings pave the way to new functional materials prepared from renewable monomers with tunable properties.

Acid insoluble lignin material production by chemical activation of olive endocarps for an efficient furfural adsorption-removal from aqueous solutions.

The present work describes a protocol of chemical activation, with acid catalyst, of olive endocarps to obtain acid insoluble lignin-rich materials with high capacities for the adsorption of furfural present in aqueous media. During biomass activation, factors such as acid concentration, reaction time and temperature, solid/liquid ratio and the presence of water extractives strongly affected both the surface characteristics of the treated bioadsorbents and their capacities for furfural retention (percentage increase close to 600% with respect to the crude biomass). Once a treated solid with good adsorbent properties was obtained, the optimal conditions for adsorption were found: stirring speed 80 rpm, temperature 303 K and adsorbent load 7.5 g solid/50 cm3. Kinetic study indicated the pseudo-second order model provided the best fit of the experimental data. At 303 K, the equilibrium adsorption capacities values ranged from 2.27 mg g-1 to 29.29 mg g-1, for initial furfural concentrations between 0.49 g dm-3 and 12.88 g dm-3. Freundlich model presented the best isotherm (R2 = 0.996 and SE = 4.7%) providing KF and n values of 0.115 (mg g-1) (mg dm-3)-n and 0.610, respectively. Since physical interactions predominate in the adsorption of furfural on chemically activated olive endocarps, the furfural removal process could have occurred reversibly on the heterogeneous surface of the bioadsorbents.

A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors.

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.

Nb-Based Catalysts for the Valorization of Furfural into Valuable Product through in One-Pot Reaction.

Nb-based catalysts supported on porous silica with different textural properties have been synthesized, characterized, and tested in the one-pot reaction of furfural to obtain valuable chemicals. The catalytic results reveal that the presence of fluoride in the synthesis, which limits the growing of the porous silica, limits diffusional problems of the porous silica, obtaining higher conversion values at shorter reaction times. On the other hand, the incorporation of NbOx species in the porous silica provides Lewis acid sites and a small proportion of Brönsted acid sites, in such a way that the main products are alkyl furfuryl ethers, which can be used as fuel additives.

The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC.

OBJECTIVE: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. METHODS: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. RESULTS: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. CONCLUSIONS: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Electric Field Promoted Click Surface-Enhanced Raman Spectroscopy for Rapid and Specific Detection of DNA 2-Deoxyribose 5'-Aldehyde Oxidation Products in Plasma.

Rapid identification of DNA oxidative damage sites is of great significance for disease diagnosis. In this work, electric field-regulated click reaction surface-enhanced Raman spectroscopy (e-Click-SERS) was developed aiming at the rapid and specific analysis of furfural, the biomarker of oxidative damage to the 5-carbon site of DNA deoxyribose. In e-Click-SERS, cysteamine-modified porous Ag filaments (cys@p-Ag) were prepared and used as electrodes, amine-aldehyde click reaction sites, and SERS substrates. Cysteamine was controlled as an "end-on" conformation by setting the voltage of cys@p-Ag at -0.1 V, which ensures its activity in participating in the amine-aldehyde click reaction during the detection of furfural. Benefiting from this, the proposed e-Click-SERS method was found to be sensitive, rapid-responding, and interference-resistant in analyzing furfural from plasma. The method detection limits of furfural were 5 ng mL-1 in plasma, and the whole "extraction and detection" procedure was completed within 30 min with satisfactory recovery. Interference from 13 kinds of common plasma metabolites was investigated and found to not interfere with the analysis, according to the exclusive adaptation of the amine-aldehyde click reaction. Notably, the e-Click-SERS technique allows in situ analysis of biological samples, which offers great potential to be a point-of-care testing tool for detecting DNA oxidative damage.

Efficient Oxidation of 5-Hydroxymethylfurfural Using a Flavoprotein Oxidase from the Honeybee Apis mellifera.

The chemical 5-hydroxymethylfurfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only a few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemoselective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source of novel biocatalysts.

Unveiling bacterial consortium for xenobiotic biodegradation from Pichavaram mangrove forest soil: a metagenomic approach.

Pichavaram mangrove forest was established as a wetland of International Importance by Article 2.1 in April 2022 by the Ministry of Environment, Forest and Climate Change, India. Even though it is a conserved site, xenobiotic agrochemical leaching on the forest land during monsoon is inevitable. These threaten the microbial diversity in the environment. Xenobiotic degradation is achieved using bacterial consortia already acclimatised to this environment. This study aims to identify the indigenous microbial consortia able to degrade xenobiotic compounds such as fluorobenzoate, furfural, and steroids. Pichavaram mangrove metagenomic dataset was obtained by shotgun sequencing of soil DNA and processed using the automated tool SqueezeMeta. Further, the DIAMOND database provided the taxonomical classification of the microbes in each contig. With reference to the KEGG database, the selected xenobiotic degradation pathways were confirmed in the dataset. Of 1,253,029 total contigs, 1332, 72 and 1262 were involved in fluorobenzoate, furfural and steroid degradation, respectively. This study identified that microbial consortia comprising Marinobacter, Methyloceanibacter and Vibrio natriegens/Gramella sp. can degrade fluorobenzoate. While Afipia, Nitrosopumilus sp., and Phototrophicus methaneseepsis favour the degradation of furfural compound. The steroid degradation pathway possessed a plethora of bacteria belonging to the phylum Proteobacteria.

Mechanism of furfural toxicity and metabolic strategies to engineer tolerance in microbial strains.

Lignocellulosic biomass represents a carbon neutral cheap and versatile source of carbon which can be converted to biofuels. A pretreatment step is frequently used to make the lignocellulosic carbon bioavailable for microbial metabolism. Dilute acid pretreatment at high temperature and pressure is commonly utilized to efficiently solubilize the pentose fraction by hydrolyzing the hemicellulose fibers and the process results in formation of furans-furfural and 5-hydroxymethyl furfural-and other inhibitors which are detrimental to metabolism. The presence of inhibitors in the medium reduce productivity of microbial biocatalysts and result in increased production costs. Furfural is the key furan inhibitor which acts synergistically along with other inhibitors present in the hydrolysate. In this review, the mode of furfural toxicity on microbial metabolism and metabolic strategies to increase tolerance is discussed. Shared cellular targets between furfural and acetic acid are compared followed by discussing further strategies to engineer tolerance. Finally, the possibility to use furfural as a model inhibitor of dilute acid pretreated lignocellulosic hydrolysate is discussed. The furfural tolerant strains will harbor an efficient lignocellulosic carbon to pyruvate conversion mechanism in presence of stressors in the medium. The pyruvate can be channeled to any metabolite of interest by appropriate modulation of downstream pathway of interest. The aim of this review is to emphasize the use of hydrolysate as a carbon source for bioproduction of biofuels and other compounds of industrial importance.

Molecular mechanism of engineered Zymomonas mobilis to furfural and acetic acid stress.

Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

Yarrowia lipolytica produces lipid-rich biomass in medium mimicking lignocellulosic biomass hydrolysate.

In recent years, lignocellulosic biomass has become an attractive low-cost raw material for microbial bioprocesses aiming the production of biofuels and other valuable chemicals. However, these feedstocks require preliminary pretreatments to increase their utilization by microorganisms, which may lead to the formation of various compounds (acetic acid, formic acid, furfural, 5-hydroxymethylfurfural, p-coumaric acid, vanillin, or benzoic acid) with antimicrobial activity. Batch cultures in microplate wells demonstrated the ability of Yarrowia strains (three of Y. lipolytica and one of Y. divulgata) to grow in media containing each one of these compounds. Cellular growth of Yarrowia lipolytica W29 and NCYC 2904 (chosen strains) was proven in Erlenmeyer flasks and bioreactor experiments where an accumulation of intracellular lipids was also observed in culture medium mimicking lignocellulosic biomass hydrolysate containing glucose, xylose, acetic acid, formic acid, furfural, and 5-HMF. Lipid contents of 35% (w/w) and 42% (w/w) were obtained in bioreactor batch cultures with Y. lipolytica W29 and NCYC 2904, respectively, showing the potential of this oleaginous yeast to use lignocellulosic biomass hydrolysates as feedstock for obtaining valuable compounds, such as microbial lipids that have many industrial applications. KEY POINTS: • Yarrowia strains tolerate compounds found in lignocellulosic biomass hydrolysate • Y. lipolytica consumed compounds found in lignocellulosic biomass hydrolysate • 42% (w/w) of microbial lipids was attained in bioreactor batch cultures.

Physiological and transcriptome analyses of Kluyveromyces marxianus reveal adaptive traits in stress response.

Kluyveromyces marxianus is a non-conventional yeast with outstanding physiological characteristics and a high potential for lignocellulosic ethanol production. However, achieving high ethanol productivity requires overcoming several biotechnological challenges due to the cellular inhibition caused by the inhibitors present in the medium. In this work, K. marxianus SLP1 was adapted to increase its tolerance to a mix of inhibitory compounds using the adaptive laboratory evolution strategy to study the adaptation and stress response mechanisms used by this non-Saccharomyces yeast. The fermentative and physiological parameters demonstrated that the adapted K. marxianus P8 had a better response against the synergistic effects of multiple inhibitors because it reduced the lag phase from 12 to 4 h, increasing the biomass by 40% and improving the volumetric ethanol productivity 16-fold than the parental K. marxianus SLP1. To reveal the effect of adaptation process in P8, transcriptome analysis was carried out; the result showed that the basal gene expression in P8 changed, suggesting the biological capability of K. marxianus to activate the adaptative prediction mechanism. Similarly, we carried out physiologic and transcriptome analyses to reveal the mechanisms involved in the stress response triggered by furfural, the most potent inhibitor in K. marxianus. Stress response studies demonstrated that P8 had a better physiologic response than SLP1, since key genes related to furfural transformation (ALD4 and ALD6) and stress response (STL1) were upregulated. Our study demonstrates the rapid adaptability of K. marxianus to stressful environments, making this yeast a promising candidate to produce lignocellulosic ethanol. KEY POINTS: • K. marxianus was adapted to increase its tolerance to a mix of inhibitory compounds • The basal gene expression of K. marxianus changed after the adaptation process • Adapted K. marxianus showed a better physiological response to stress by inhibitors • Transcriptome analyses revealed key genes involved in the stress response.

New concept for conversion of lignocellulose to ethanol and furfural.

Studies on the chemical mechanisms of furfural formation showed the possibility to apply a new differential catalysis of hemicellulose - its depolymerisation and pentose dehydration to furfural. This change led to the increase in furfural yield and essential decrease of cellulose destruction. The lignocellulose residue that remains after the production of furfural may be subjected to enzymatic hydrolysis to glucose and the subsequent fermentation to ethanol. The remaining lignin appeared to be suitable for the production of additional various value-added products including medicinal mushrooms and laccase-containing enzyme complexes. Based on these developments, an innovative concept is proposed for the waste-free use of lignocellulose to obtain various valuable products. KEY POINTS: • New chemical mechanism of furfural production. • New lignocellulose pretreatment does not damage cellulose and lignin. • Lignocellulose may be processed using waste-free technology.

Production of Extracellular Enzymes by Lentinula edodes Strains in Solid-State Fermentation on Lignocellulosic Biomass Sterilized by Physical and Chemical Methods.

Two methods of sterilization of lignocellulosic biomass were performed in this study. Eucalypt waste (EW) supplemented with rice bran (RB) was added in the proportions 80:20 and 90:10 in dry weight. The compositions were sterilized by physical method (autoclaving) and by chemical method (H2O2). The production of extracellular enzymes by Lentinula edodes strains was compared within the two methods. Inactivation of catalase present in RB was achieved with 250 mM H2O2. The use of H2O2, when compared by physical method, favored high production of hydrolytic enzymes such as endoglucanase (1,600 IU/kg), twofold higher, ß-glucosidase (1,000 IU/kg), fivefold higher, xylanase (55,000 IU/kg), threefold higher and ß-xylosidase (225 IU/kg), similar results. Oxidative enzymes, MnP and laccase, were produced within a different profile between strains, with shorter times for laccase (2,200 IU/kg) by SJC in 45 days and MnP (2,000 IU/kg) by CCB-514 in 30 days. High production of extracellular enzymes is achieved by the use of the chemical method of sterilization of lignocellulosic biomass; in addition to no energy consumption, this process is carried out in a shorter execution time when compared to the physical process. The use of H2O2 in sterilization does not produce toxic compounds from the degradation of hemicellulose and cellulose such as furfural and hydroxy-methyl-furfural that cause inhibition of microorganisms and enzymes.