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Laccase treatment of phenolic compounds found in lignocellulosic hydrolysates is an alternative to reduce the growth inhibitory effect of these compounds on fermenting microorganisms. In order to d...

Lignocellulosic biomass is the most abundant bio-resource on earth containing carbohydrates polymers cellulose, hemicellulose and lignin. With a number of methods including pretreatment and hydrolysis, lignocellulose-derived hydrolysates containing D-glucose, D-xylose, L-arabinose, and further sugars can be obtained. The hydrolysates consisting fermentable sugars typically display a high degree of variation depending on both the biomass source materials and process conditions, resulting in different compositions such as various concentrations of monosaccharide sugars and inhibitors. The fermentable sugars in lignocellulosic hydrolysates have attracted many people���s attention all over the world, because these fermentable sugars have a great potential to be used by microorganisms as a sole carbon source for the growth and production of bioproducts. To date, several bioproducts such as biofuels, chemicals and many high value-added products have been already investigated and produced, and the application field is still expanding. However, one of the key obstacles is that not all the microorganisms have the ability to metabolize all the different sugars in hydrolysates. Most of the known microorganisms only utilize D-glucose as the sole carbon source, which results in the energy loss of lignocellulosic biomass. Another obstacle is that the inhibitors formed during hydrolysis, limit the efficient usage of hydrolysates as a carbon source for biotechnological conversion. To further investigate and overcome these obstacles, in this Ph.D. thesis, the following research works have been examined and summarized: 1) Investigation the sugar utilization of Escherichia coli strains and the production of ��s1-casein proteins based on lignocellulosic hydrolysates. 2) Metabolic engineering of Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of D-xylose and L-arabinose. 3) Evaluation of different lignocellulosic hydrolysates as substrates for engineered P. putida KT2440 and the inhibitory effectiveness on P. putida KT2440.

The antifungal and growth-stimulating activity of biological detoxification waste products of hydrolysates of lignocellulosic raw materials in vitro and in vivo was evaluated in comparison with reference preparations (Alirin-B, Fitosporin-M) and control (sterile tap water). These wastes are specially adapted microbocenoses of activated sludge, worked out in the process of purification of hydrolyzates of lignocellulosic raw materials from inhibitors of acetone butyl fermentation. The agronomic value of biodetoxification waste was studied in three prototypes of different nature, using Iren spring wheat as an example. The results showed that detoxification bioagents, regardless of origin, showed fungistatic activity at the in vitro and in vivo study stages (biotest). In an in vitro experiment, all test samples showed significant antifungal activity against the fungus F. oxysporum. The most effective was the biodetoxification waste obtained on the basis of microbocenosis of activated sludge grown on a nutrient medium containing phenol, formic and acetic acid. As a result of exposure to this bioagent at the end of the experiment, the average diameter of the colonies of the fungus F. oxysporum was approximately 34 times less than in the control version. The detoxification bioagent, obtained on the basis of a specially adapted microbocenosis of activated sludge grown on a nutrient medium simulating wastewater, reduced the diameter of phytopathogenic fungus colonies by an average of 16 times. Specially adapted activated sludge from the sewage treatment facilities of the wood processing enterprise, worked out during the detoxification of hydrolysates of lignocellulosic raw materials, was also able to effectively suppress fungus growth, the average diameter of which was 19 times less than the control. The biotest results also confirmed the fungistatic activity of the test samples. The effectiveness of reducing the total infection with seminal infections in different experimental variants ranged from 52 to 84%. The growth-promoting ability of biodetoxification waste was weak.

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of “Design-Build-Test-Learn”, as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era.

Furfural, an inhibitor of yeast growth, occurs in xylose-containing lignocellulosic hydrolysates that are potential substrates for the fermentative production of the natural sweetener xylitol from xylose. Effects of furfural on growth and xylitol production by the yeast Candida magnoliae TISTR 5663 are reported. Aerobic as well as oxygen limited conditions were used to assess the effects of furfural. A mathematical model was developed to predict the behavior of this fermentation. The model was validated using independent experimental data. The model and its kinetic parameters are reported. Furfural was found to be a competitive inhibitor of growth. Nevertheless, the presence of a certain amount of furfural (∼0.3 g L −1 ) in the production medium actually improved the productivity of xylitol and its yield from xylose under suitable oxygen limited conditions.

The paper deals with the investigation of the possible using of brewer’s grain as a source of growth substabces in acetone-butanol fermentation of lignocellulosic hydrolysates in order to reduce the cost of biobutanol production and to utilize the brewery waste. The fermentation of glucose was carried out at different concentrations of the brewer’s grain by Clostridium acetobutylicum ATCC 824. In the experiments on fermentation of the lignocellulosic hydrolysates an enzymatic hydrolysate of miscanthus cellulose containing 34.8 g/l glucose and 15.6 g/l xylose was used as a source of reducing substances. The sterilization of the medium was carried out at 0.5 KPa for 20 minutes. The sterilization of the growth and reducing substances sources was conducted separately to prevent caramelization of products and melanoidins. For inoculation the spores of 3% (vol/vol) C. acetobutylicum ATCC 824 were transferred to a fresh medium. The strain was grown at 37 °С under anaerobic conditions. In a series of experiments on the evaluation of the influence of the brewer’s grain on the fermentability of carbohydrates by the strain of C. acetobutylicum АТСС 824, limiting and inhibitive concentrations of brewer’s grain were determined in the medium, which were 2 and 20 % vol., respectively. The optimal amount of the brewer’s grain was about 6 % vol. At the optimal concentration of the brewer’s grain the fermentation of lignocellulosic hydrolysates occured in all replicates. It was characterized by intensive gas and foam formation that corresponds to the data in literature. After 79-88 h of fermentation of miscanthus cellulose hydrolysate the product yield amounted 10.14±0.87 g/L butanol, 02.48±0.53 acetone, 01.02±0.42 g/L ethanol. It was found that at an optimum concentration both the fresh and sour brewer’s grain can be used in the fermentation. After the acetone-butanol fermentation the brewer’s grain can be used as a food for farm animals. The obtained results indicate the possibility of using the brewer’s grain as a source of growth substances for the fermentation of lignocellulosic hydrolysates. The proposed method of using the brewer’s grains in acetone-butanol fermentation may reduce the cost of biobutanol and also solve the problem of disposing some waste from the brewing industry.

Background Lignocellulosic biomass is a promising resource of renewable biochemicals and biofuels. However, the presence of inhibitors existing in lignocellulosic hydrolysates (LCH) is a great challenge to acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. In particular, phenolic compounds (PCs) from LCH severely block ABE production even at low concentrations. Thus, it is urgent to gain insight into the intracellular metabolic disturbances caused by phenolic inhibitors and elucidate the underlying mechanisms to identify key industrial bottlenecks that undermine efficient ABE production. Results In this study, a time-course of ABE fermentation by C. acetobutylicum in the presence of four typical PCs (syringaldehyde, vanillin, ferulic acid, and p-coumaric acid) was characterized, respectively. Addition of PCs caused different irreversible effects on ABE production. Specifically, syringaldehyde showed the greatest inhibition to butanol production, followed by vanillin, ferulic acid, and p-coumaric acid. Subsequently, a weighted gene co-expression network analysis (WGCNA) based on RNA-sequencing data was applied to identify metabolic perturbations caused by four LCH-derived PCs, and extract the gene modules associated with extracellular fermentation traits. The hub genes in each module were subjected to protein–protein interaction analysis and enrichment analysis. The results showed that functional modules were PC-dependent and shared some unique features. Specifically, p-coumaric acid caused the most extensive transcriptomic disturbances, particularly affecting the gene expressions of ribosome proteins and the assembly of flagella, DNA replication, repair, and recombination; the addition of syringaldehyde caused significant metabolic disturbances on the gene expressions of ribosome proteins, starch and sucrose metabolism; vanillin mainly disturbed purine metabolism, sporulation and signal transduction; and ferulic acid caused a metabolic disturbance on glycosyl transferase-related gene expressions. Conclusion This study uncovers novel insights into the inhibitory mechanisms of PCs for the first time and provides guidance for future metabolic engineering efforts, which establishes a powerful foundation for the development of phenol-tolerant strains of C. acetobutylicum for economically sustainable ABE production with high productivity from lignocellulosic biomass.

To avoid complex procedures in measurement of lipid content of oleaginous yeast especially for that can accumulate microbial lipid in lignocellulosic hydrolysates, fluorescent method using Nile Red as fluorescent dye was applied to measure lipid content of oleaginous yeast Trichosporon dermatis. The fluorescent method was built by fitting of lipid content identified by both conventional gravimetric method and fluorescence intensity of oleaginous yeast. Within the range of lipid content measured, the fitting curves showed linear relationship with good correlation coefficient (R2=0.95), showing this method is suitable for measuring lipid content of T. dermatis in the simulated medium. To evaluate the applicability of this method for lipid fermentation using lignocellulosic acid hydrolysates as substrate, T. dermatis was cultured in corncob acid hydrolysate and rice straw acid hydrolysate and then its lipid content measured by both fluorescent method and gravimetric method were compared. The results showed that the lipid content measured by these two methods were close, therefore, this method was promising for the application in lipid fermentation in lignocellulosic acid hydrolysates.

The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl)furfural (HMF) and furfural are specifically well known. This study investigated the furfural and 5-HMF tolerance in Candida strains, which could be used for the development of advanced generation bioethanol processes. The 10 isolates of Candida were selected based on the cell growth and bioethanol production on YPD medium containing several concentrations of furfural and 5-HMF by using spectrophotometer and HPLC. Candida parapsilosis Y80 could grow and produce bioethanol in the medium that contains furfural and 5-HMF with a concentration of 60 mM and 40 mM, respectively. Based on the results, C. parapsilosis Y80 has potential activity in the development of bioethanol fermentation.

Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains., This study was supported by the Portuguese Foundation for Science and Technology (FCT, Portugal) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to Joana T. Cunha), the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte, the MultiBiorefinery project (POCI-01–0145FEDER-016403) and the Biomass and Bioenergy Research Infrastructure (PINFRA/22059/2016)., info:eu-repo/semantics/publishedVersion

The aims of this work were to improve cell tolerance towards high concentrations of furfural and 5-hydroxymethylfurfural (HMF) of an osmotolerant strain of Wickerhamomyces anomalus by means of evolutionary engineering, and to determine its ethanol production under stress conditions. Cells were grown in the presence of furfural, HMF, either isolated or in combination, and under high osmotic pressure conditions. The most toxic condition for the parental strain was the combination of both furans, under which it was unable to grow and to produce ethanol. However, the tolerant adapted strain achieved a yield of ethanol of 0.43 g g-1glucose in the presence of furfural and HMF, showing an alcohol dehydrogenase activity of 0.68 mU mg protein-1. For this strain, osmotic pressure, did not affect its growth rate. These results suggest that W. anomalus WA-HF5.5strain shows potential to be used in second-generation ethanol production systems.

The growing concerns regarding climate change, population growth, depletion of fossil fuel, and pollution arising from the combustion of petroleum-based fuel can be identified as the most important factors driving the urgent need for environmentally friendly renewable energy. Among all the recognized alternatives to gasoline-based fuel, hydrogen is not only considered as a clean energy but also it has a high energy content of 142 kJ/g which is almost three times higher compared to other fossil fuels. Only water and heat are the by-products of hydrogen combustion. Dark fermentative hydrogen production is a feasible option in which inexpensive, low-grade, carbohydrate-rich, and renewable lignocellulosic biomass can be used as a substrate and anaerobic digester sludge (ADS) as a seed for biohydrogen production. Lignocellulosic substances are abundant in nature and are suitable for dark fermentative hydrogen production. Pretreatment of these carbohydrate-rich materials is required to get rid of lignin and increase the readily biodegradable sugars required for fermentation. There are several methods to break down the rigid structure of lignin and increase the fermentable sugar content. Although chemical treatment may be appropriate, it produces not only readily biodegradable sugars but also other by-products which inhibit microbial growth. The main purpose of this study was to assess the significance of acclimatization and the impact of furfural inhibition in both batches and continuous-flow systems for biohydrogen production from synthetic lignocellulosic hydrolysates. First, acclimatization of ADS was tested for biohydrogen production in a patented continuous-flow system known as integrated biohydrogen reactor clarifier systems (IBRCS), and in batches. IBRCS, R1, was fed initially with glucose at a concentration of 10 g/L (phase 1) and then the feed was switched to a mixture of C6 and C5 sugars: glucose, cellobiose, xylose, arabinose at a concentration of 2.5 g/L each (phase 2) and then the feed reverted to glucose at the same concentration of 10 g/L (phase 3). The results showed that hydrogen production yields were negatively affected by changing the feed substrates, despite their biodegradability. Additionally, propionate, which is not favorable for both biohydrogen and biomethane production, was predominant as a result of feed changes. This was evident by microbial community analysis which revealed that the propionate-producing Megasphaera were predominant while the hydrogen and acetate-producing bacteria i.e. Clostridium were washed out after switching substrates in phases 2 and 3. On the other hand, neither hydrogen yields nor volatile fatty acids (VFAs) distribution was negatively affected in the batch study, but rather changing the feed from mono substrate to co-substrate enhanced the hydrogen production yields. A confirmation experiment has been conducted in IBRCS, R2, to investigate the effect of feed changes on the acclimatized anaerobic hydrogen-producing mesophilic mixed cultures where the system was initially fed with a mixture of C5 and C6 sugars similar in concentration and composition to R1 in the second phase of this project. The results showed a significantly higher hydrogen production yields in R2 compared to R1 phase 2 (1.9 mol H2/mol sugar vs 1.1 mol H2/mol sugar) verifying that the reduction in hydrogen yields resulted from feed changes. Second, the impact of furfural inhibition on biohydrogen production was investigated in both continuous-flow systems and batch studies. In the continuous-flow systems, IBRCS were used to test glucose and xylose individually in presence of gradual increase of furfural concentrations from 0-4 g/L for mesophilic biohydrogen production. The results of this study showed that the biohydrogen-producing microorganisms in both glucose-fed reactor and xylose-fed reactor behaved similarly towards furfural inhibition. The acclimatized anaerobic mesophilic hydrogen-producing cultures could tolerate furfural inhibition up to 2 g/L with 29% percent reduction of the hydrogen yields compared to the control phase with 0 g/L furfural in the feed. However, the furfural inhibition threshold level ranged from 2-4 g/L. The revivability of the inhibited cultures from the glucose-fed reactor at 4 g/L furfural was assessed by removing furfural from the feed. The revivability of the inhibited cultures was proven as evident by the 95% recovery of the specific hydrogen production rate. On the other hand, synthetic lignocellulosic hydrolysate comprised mainly of 76% (by weight) xylose, 10% glucose, 9% arabinose, and the rest a mixture of other sugars i.e. galactose and mannose was investigated as a substrate at concentrations of 2-32 g/L in the presence of furfural at concentrations of 0, 1, and 2 g/L. The results showed that furfural completely inhibits the biohydrogen producers at 2 g/L and the optimum substrate concentration tested was 16 g/L.

Acetic acid, a potential growth inhibitor, commonly occurs in lignocellulosic hydrolysates. The growth of Cupriavidus necator DSM 545 and production of poly(3-hydroxybutyrate) (PHB) by this bacterium in a glucose-based medium supplemented with various initial concentrations of acetic acid are reported. The bacterium could use both glucose and acetic acid to grow and produce PHB, but acetic acid inhibited growth once its initial concentration exceeded 0.5 g/L. As acetic acid is an unavoidable contaminant in hydrolysates used as sugar sources in commercial fermentations, a mathematical model was developed to describe its impact on growth and the production of PHB. The model was shown to satisfactorily apply to growth and PHB production data obtained in media made with acetic-acid-containing hydrolysates of Napier grass and oil palm trunk as carbon substrates.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122054., This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main 9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks., This work was supported by: the Spanish Ministerio de Economía y Competitividad (grant numbers BIO2014-56930-PandBIO2017-88435R) and the Junta de Castilla y León (grant number SA016P17); the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019, PhD grant PD/ BD/113812/2015 to R. Silva (Doctoral Program in Applied and Environmental Microbiology), the MultiBiorefinery project (POCI-010145-FEDER-016403) and the BioTecNorte operation (NORTE-010145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. DDF was recipient of USAL predoctoral fellowship from the University of Salamanca., info:eu-repo/semantics/publishedVersion

In the present study we investigated the possibility of using the waste of the lignocellulosic hydrolysates biodetoxification as a seed treater for wheat varieties zoned in the West Siberian region including the Tomsk region. The wastes are microbiocenoses of activated sludge, depleted during the detoxification process and capable of growing on toxic substrates. The study was conducted on three experimental objects and on wheat varieties Iren, Novosibirskaya 3, Novosibirskaya 51. For comparison, reference objects of biological preparations for plant protection Alirin-B, Fitosporin-M were used. The study showed that the waste of lignocellulosic hydrolysates biodetoxification had antifungal activity. The effectiveness of reducing the total contamination of seed infection in different experiments with experimental samples ranged from 52 to 82%. In some cases, the reference variants proved to be ineffective; the maximum effect on reducing the total seed contamination was achieved by Alirin-B and amounted about 30%. Seed treatment of wheat by the waste biodetoxification of different origins reduced the prevalence pathogens of seed wheat. At the same time the treatment did not significantly affect the increase in the parameters of plant growth and development (germination, length and weight of sprouts).

Microbial chemicals and fuels production from renewable resources requires the development of biocatalysts that can tolerate toxic chemicals produced during the lignocellulosic hydrolyzation process and also tolerate the end product toxicity. Evolutionary engineering makes use of adaptive strategies and selection procedures to generate and study mutations that will increase tolerance to harmful chemicals and therefore increase productivity and titer in such processes. In this chapter, we will review recent advances in evolutionary engineering strategies, their results and challenges to generate better microorganisms for the production of chemicals and fuels from lignocellulosic hydrolysates.

The main purpose of this study was to investigate the impact of furfural on mixed cultures during fermentative hydrogen production from lignocellulosic biomass. Small batch studies using synthetic lignocellulosic hydrolysate grown on mesophilic mixed cultures, revealed a threshold furfural concentration of greater than 1 g/L with enhancement to the yields (from the control) observed at 0.5 g/L furfural (at initial substrate-to–biomass (S°/X°) ratios of 0.5 and 1 gCOD/gVSS) and at both 0.5 g/L and 1 g/L furfural (at S°/X° of 2 and 4 gCOD/gVSS). This study was scaled-up from 200 mL to 11 L working volume batches, using half the substrate concentration of the small batch studies, at an S°/X° of 4 gCOD/gVSS in order to determine the Monod microbial kinetics of mixed cultures in the presence of furfural at both mesophilic and thermophilic temperatures. A 45 % enhancement at 1 g/L furfural was observed in the mesophilic experiment but a 50 % reduction at the same furfural concentration was observed at thermophilic conditions both relative to the yields from their respective controls. Enhanced kinetics observed in the control without furfural at both temperatures emphasized that although furfural is indeed an inhibitor, it can be broken down at low concentrations by mesophilic hydrogen-producers to increase hydrogen yields. Liquid and solid real waste hydrolysates obtained from poplar wood biomass treated using twin-screw extrusion technology were evaluated for their biohydrogen potential and the feasibility of a two-stage anaerobic digestion process. This study proved that acidification of the first-stage biohydrogen production process brought about a 50 % increase (on average) in TVFA/SCOD initial which enhanced methane yields in the second-stage. In the two-stage anaerobic digestion process, energy yields were 33 % and 18 % higher, while feedstock COD removal efficiencies were 16 % and 14 % higher than the single-stage BMP tests for the liquid and solid samples respectively.

The complete bioconversion of the carbohydrate fraction is of great importance for a lignocellulosic-based biorefinery. However, due to the structure of the lignocellulosic materials, and depending basically on the main parameters within the pretreatment steps, numerous byproducts are generated and they act as inhibitors in the fermentation operations. In this sense, the impact of inhibitory compounds derived from lignocellulosic materials is one of the major challenges for a sustainable biomass-to-biofuel and -bioproduct industry. In order to minimise the negative effects of these compounds, numerous methodologies have been tested including physical, chemical, and biological processes. The main physical and chemical treatments have been studied in this work in relation to the lignocellulosic material and the inhibitor in order to point out the best mechanisms for fermenting purposes. In addition, special attention has been made in the case of lignocellulosic hydrolysates obtained by chemical processes with SO2, due to the complex matrix of these materials and the increase in these methodologies in future biorefinery markets. Recommendations of different detoxification methods have been given. The authors gratefully acknowledge the financial support by KBBE-2012-6-311935 BRIGIT research project www.brigit-project.eu and the COST FP1306 and COST FP1205 actions. Alberto Coz acknowledges the COST FP1205 Action for his research stay at Aalto University under a Sort Term Scientific Mission.

The concentration of polymerised sugars in enzymic hydrolysates from lignocellulosic materials was determined by high pressure liquid chromatography (h.p.l.c.) of hydrolysates before and after dilute acid hydrolysis: significant quantities of polymerised glucose and xylose were found in lignocellulosic hydrolysates, and these accounted for the discrepancy between the dinitrosalicyclic acid (DNS) and h.p.l.c. assay methods.

The dinitrosalicylic acid (DNS) method gives a rapid and simple estimation of the extent of saccharification by measuring the total amount of reducing sugars in the hydrolysate. However, it is subject to interference by citrate buffer and other substances and by the differing reactivities of the various reducing sugars. These interferences become more apparent when complex substrates such as sugar cane bagasse are employed. The paper also shows how the DNS method can be adapted for use on a Technicon Autoanalyser.

Aims: To show that the ethanol-induced lag phase in yeast can be almost eliminated by combining pre-adaptation with acetaldehyde supplementation. Methods and Results: Pre-adaptation to noninhibitory concentrations of ethanol and supplementation of unadapted cultures with acetaldehyde each separately reduced the lag phase of ethanol-inhibited cultures by c. 70%. By combining the two methods the ethanol-induced lag phase was virtually eliminated (90% reduction in lag time). Conclusions: Pre-adaptation to ethanol and acetaldehyde supplementation appear to promote yeast growth through different mechanisms, which are additive when combined. Significance and Impact of the Study: The combination of the above procedures is a potentially powerful tool for reducing the lag of stressed cultures, which may have practical applications: e.g. in reducing the lag of yeasts inoculated into lignocellulosic hydrolysates employed in fuel ethanol production.

Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors

Most of the current processes for bioethanol production are based on the use of Very-High-Gravity (VHG) technology and the processing of lignocellulosic biomass, limited by the high osmotic pressure and ethanol concentration in the fermentation medium, and by inhibitors resulting from biomass pre-treatments, respectively. Aiming the optimization of strains for industrial bioethanol production an integrated approach was undertaken to identify genes required for simultaneous yeast resistance to different fermentation-related stresses. The integration of previous chemogenomics data was used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to different inhibitors present during lignocellulosic fermentations. The expression of BUD31 and HPR1 lead to the increase of both ethanol yield and fermentation rate, while PHO85, VRP1 and YGL024w expression is required for maximal ethanol production in VHG fermentations. Five genes, ERG2, PRS3, RAV1, RPB4 and VMA8 were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or for maximal fermentation rate of this substrate [1]. Moreover, the yeast disruptome was screened for strains with increased susceptibility to inhibitory compounds present in an industrial lignocellulosic hydrolysate obtained from wheat straw. With this genome-wide analysis, 42 determinants of resistance to inhibitors were identified showing a high susceptibility phenotype compared to the parental strain. The identified genes stand as preferential targets for genetic engineering manipulation to generate more robust and efficient industrial strains.

Background The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. Results We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 μM) compared to that of NADPH (39 μM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. Conclusions In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde.

Mestrado em Engenharia de Sistemas Bioenergéticos - Instituto Superior de Agronomia Bioethanol production from lignocellulosic materials is only viable if the process of converting the biomass to ethanol is efficient. One of the conditions that affect this efficiency is the resistance of the fermentative microorganisms to the toxic compounds that are formed during the necessary lignocellulosic materials pre-treatment. The current work analyzed the behavior of Saccharomyces cerevisiae growing on a medium containing inhibitory compounds to the fermentation, compounds that are present on an industrial lignocellulosic hydrolysate. The effect of the elimination of non-essential genes on the cells fermentative metabolism was studied. The non-essential genes eliminated were genes involved in the regulation and metabolism of nitrogen (TOR1, GLN3, PRO1, PUT4, ARO4), in the cell’s response to oxidative stress (SOD2, ZWF1, ADH6), osmotic stress (HOG1, TPS1, NTH1, HSP12) and general stress (HSP26), on the regulation of the intracellular pH (RIM101, VMA1), and in apoptosis (AIF1, FIS1). The results obtained from these procedures were indicative that some of these genes mutations are relevant for determining the fermentation efficiency. The behavior of four selected mutant strains was further characterized. Besides the determination of glucose concentration, extracellular pH and CFU at the beginning of stationary phase and glucose exhaustion, for these four chosen strains, the ethanol concentration and the influx and efflux of protons through the plasmatic membrane were also collected.

The production of chemicals from lignocellulosics continues to be an active area of research. Improved economics for lignocellulosic-based chemical production processes can be realized through the integration of these processes into existing bioprocessing facilities that are often described as biorefineries. The focus of some of the most recent work in this area is the topic of session 5. Research described by the speakers in this session illustrates the capturing of additional value from low value coproducts and alternative products from several industries. Examples from the talks include the production of fuels such as ethanol and biodiesel from industrial processing co-products; increased value from feed fibers as provided in the talk on the corn processing industry; multiple products and co-products from woody biomass and sugarcane fiber lignins; and syngas from glycerol obtained as a byproduct from vegetable oil processing to biodiesel. While many of the presentations provided an overview of the employment of separation technologies to biomass feedstocks, one presentation directly highlighted the impediments to the production of polyols by chemical catalysis of lignocellulosic feedstocks. Since a similar problem exists in microbial biocatalyst inhibition by lignocellulosic hydrolysates, the development of innovative pretreatment, hydrolysis and separation technologies to overcome microbial inhibition and catalyst poisoning will be essential for progress in this area.

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates.

Dear Readers, Microbial Biotechnology (MBT) is undergoing a number of interesting changes that we would like to report. The first is its transition to an author-pays open access model from 2013. We believe open access will offer the journal a number of advantages, including the flexibility to grow without the constraints of a page budget, and substantial increases in exposure, with all articles being made freely available via Wiley Online Library and PubMed Central. MBT's transition to open access will significantly benefit both authors and readers, and hence the field of biotechnology as a whole. The Journal will continue to be editorially independent and its editorial team will continue to apply ever increasing standards of peer review, thus ensuring that the journal continues to be the high quality publication we know and value. Another is that MBT was assigned its first Impact Factor at the end of June and, since that time, has experienced a significant increase in submissions. As has been the case with its sister journals, Environmental Microbiology and Environmental Microbiology Reports, this rise in submissions will translate into an Editor-implemented rise in quality–originality threshold for acceptance, which in turn will positively influence subsequent IFs. Given the steady increase in exciting science we receive, we are confident that MBT will experience healthy increases in IF over the coming years. An important characteristic of MBT is the strength of its Editorial team – its Editors, Editorial Board and army of ad hoc reviewers, composed of leaders in the different sectors of microbial biotechnology, leaders both in the sense of representing the field and in pioneering the way forward. It is these dedicated scientists who set the benchmark for the field and for the papers that are published in MBT. They do this by providing constructive, critical reviews on submissions and by submitting their own work, and thereby ensure that MBT publishes some of the best research from high profile groups. The Editors are deeply indebted to the dedicated selfless support of its Editorial Board and ad hoc reviewers: thank you all for your tremendous support! The declared goal of MBT is to promote the field of applied microbiology, inter alia by stimulating young researchers to carry out original research on novel topics at the interfaces of the various disciplines that impact on biotechnology. It does so not only by showcasing important developments by leading groups, but also by profiling topics and new developments the Editors consider will lead to ground-breaking discoveries. One means it employs to do this is to publish Special and Themed Issues on such topics, edited by top researchers in the field. Exciting recent ones have been on Microbial Vaccines and Immunomodulators (http://onlinelibrary.wiley.com/doi/10.1111/mbt.2012.5.issue-2/issuetoc) edited by Carlos Guzman, Ennio de Gregorio, Jan ter Meulen and Martin Friede, and Microbial Resource Mangement (http://onlinelibrary.wiley.com/doi/10.1111/mbt.2012.5.issue-3/issuetoc), edited by Nico Boon and Willy Verstraete. Another means is the Web Alert of Larry Wackett which assembles the best websites for relevant diverse aspects of applied microbiology. A third is the biannual original, sometimes provocative, Crystal Ball series, in which international experts speculate on the key new developments/theories/paradigms that will drive discoveries in the field over the following years. This feature, which appears in the first issue of the year, always creates considerable interest and, in some cases, amusement. Take a look at the 2013 CB in this issue. And finally: check out below the most downloaded and most cited papers in MBT (see the MBT website for latest updates) to see who is publishing what in hot interest-generating biotechnology. Top downloaded articles Marine genomics: at the interface of marine microbial ecology and biodiscovery Karla B. Heidelberg, Jack A. Gilbert, Ian Joint Bacterial persistence increases as environmental fitness decreases Seok Hoon Hong, Xiaoxue Wang, Hazel F. O'Connor, Michael J. Benedik, Thomas K. Wood Strategies for discovery and improvement of enzyme function: state of the art and opportunities Praveen Kaul, Yasuhisa Asano Natural products for cancer chemotherapy Arnold L. Demain and Preeti Vaishnav Anaerobic benzene degradation by bacteria Carsten Vogt, Sabine Kleinsteuber, Hans-Hermann Richnow Top cited articles Metabolic engineering to enhance bacterial hydrogen production Maeda, T; Sanchez-Torres, V; Wood, TK Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain Heer, D; Sauer, U Microbial degradation of lignin: how a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this Ruiz-Duenas, FJ; Martinez, AT Microbial reporters of metal bioavailability Magrisso, S; Erel, Y; Belkin, S Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence Lee, J; Attila, C; Cirillo, SLG; Cirillo, JD; Wood, TK So, submit your best work to MBT, participate in its upward march to become the flagship of microbial biotechnology research, and your paper may be in one of these lists in future!