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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.

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.

Background Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. Results In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. Conclusion The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.

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

Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication.

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.

Background Formic acid is an inhibitory compound present in lignocellulosic hydrolysates. Understanding the complex molecular mechanisms underlying Saccharomyces cerevisiae tolerance to this weak acid at the system level is instrumental to guide synthetic pathway engineering for robustness improvement of industrial strains envisaging their use in lignocellulosic biorefineries. Results This study was performed to identify, at a genome-wide scale, genes whose expression confers protection or susceptibility to formic acid, based on the screening of a haploid deletion mutant collection to search for these phenotypes in the presence of 60, 70 and 80 mM of this acid, at pH 4.5. This chemogenomic analysis allowed the identification of 172 determinants of tolerance and 41 determinants of susceptibility to formic acid. Clustering of genes required for maximal tolerance to this weak acid, based on their biological function, indicates an enrichment of those involved in intracellular trafficking and protein synthesis, cell wall and cytoskeleton organization, carbohydrate metabolism, lipid, amino acid and vitamin metabolism, response to stress, chromatin remodelling, transcription and internal pH homeostasis. Among these genes is HAA1 encoding the main transcriptional regulator of yeast transcriptome reprograming in response to acetic acid and genes of the Haa1-regulon; all demonstrated determinants of acetic acid tolerance. Among the genes that when deleted lead to increased tolerance to formic acid, TRK1, encoding the high-affinity potassium transporter and a determinant of resistance to acetic acid, was surprisingly found. Consistently, genes encoding positive regulators of Trk1 activity were also identified as formic acid susceptibility determinants, while a negative regulator confers protection. At a saturating K+ concentration of 20 mM, the deletion mutant trk1Δ was found to exhibit a much higher tolerance compared with the parental strain. Given that trk1Δ accumulates lower levels of radiolabelled formic acid, compared to the parental strain, it is hypothesized that Trk1 facilitates formic acid uptake into the yeast cell. Conclusions The list of genes resulting from this study shows a few marked differences from the list of genes conferring protection to acetic acid and provides potentially valuable information to guide improvement programmes for the development of more robust strains against formic acid. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0781-5) contains supplementary material, which is available to authorized users.

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 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.

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.

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.

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!

Two mutants of Pachysolen tannophilus were isolated which produced considerably more acetic acid from several sugars than a wild type strain. Such mutants are of potential interest for the production of acetic acid rather than ethanol from lignocellulosic hydrolysates.

Background Industrial biotechnology that is able to provide environmentally friendly bio-based products has attracted more attention in replacing petroleum-based industries. Currently, most of the carbon sources used for fermentation-based bioprocesses are obtained from agricultural commodities that are used as foodstuff for human beings. Lignocellulose-derived sugars as the non-food, green, and sustainable alternative carbon sources have great potential to avoid this dilemma for producing the renewable, bio-based hydrocarbon fuel precursors, such as microbial lipid. Efficient bioconversion of lignocellulose-based sugars into lipids is one of the critical parameters for industrial application. Therefore, the fed-batch cultivation, which is a common method used in industrial applications, was investigated to achieve a high cell density culture along with high lipid yield and productivity. Results In this study, several fed-batch strategies were explored to improve lipid production using lignocellulosic hydrolysates derived from corn stover. Compared to the batch culture giving a lipid yield of 0.19 g/g, the dissolved-oxygen-stat feeding mode increased the lipid yield to 0.23 g/g and the lipid productivity to 0.33 g/L/h. The pulse feeding mode further improved lipid productivity to 0.35 g/L/h and the yield to 0.24 g/g. However, the highest lipid yield (0.29 g/g) and productivity (0.4 g/L/h) were achieved using an automated online sugar control feeding mode, which gave a dry cell weight of 54 g/L and lipid content of 59 % (w/w). The major fatty acids of the lipid derived from lignocellulosic hydrolysates were predominately palmitic acid and oleic acid, which are similar to those of conventional oilseed plants. Conclusions Our results suggest that the fed-batch feeding strategy can strongly influence the lipid production. The online sugar control feeding mode was the most appealing strategy for high cell density, lipid yield, and lipid productivity using lignocellulosic hydrolysates as the sole carbon source. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0542-x) contains supplementary material, which is available to authorized users.