•Low-cost alkaline neutralizer could replace MgCO3 to produce succinic acid.•Key enzymes’ activities in the succinic acid synthesis pathway were improved.•The inhibitory effects by lignocellulosic ...hydrolysate was relieved.•In a 3-L bioreactor, the overall productivity was 2.15gL−1h−1.
In this study, a novel engineered Escherichia coli strain KMG111 was constructed by overexpression of mgtA in E. coli mutant DC1515. By adopting KMG111, nearly a concentration of succinic acid (32.41gL−1) with a yield of 0.81gg−1 glucose, could be obtained in a batch fermentation by using the low-cost mixture of Mg(OH)2 and NH3·H2O to replace MgCO3 as the alkaline neutralizer. Moreover, the effect of the inhibitory compounds in lignocellulosic hydrolyzates on cell growth and succinic acid production could be relieved. In a 3-L bioreactor, the overall productivity and yield of succinic acid in the whole anaerobic stage were 2.15gL−1h−1 and 0.86gg−1 total sugar, respectively. This study was the first to report decreased alkaline neutralizer cost via genetic manipulation for succinic acid production, which contributed to the industrialization of this microbial synthesis process.
We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this ...approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility. Biotechnol. Bioeng. 2009; 102: 822-827.
Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting ...Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.
Abstract
To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host ...should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for their tolerance to hydrolysate inhibitors. A unique resistant strain was selected and identified as Pichia anomala (Wickerhamomyces anomalus), deposited as CBS 132101. The strain is able to produce ethanol in various biomass hydrolysates, both with and without oxygen. Besides, the strain could assimilate xylose and use nitrate as N source. These physiological characteristics make P. anomala an interesting strain for bioethanol production from lignocellulosic biomass hydrolysates.
Corynebacterium glutamicum strains NC-2 were able to grow on xylose as sole carbon sources in our previous work. Nevertheless, it exhibited the major shortcoming that the xylose consumption was ...repressed in the presence of glucose. So far, regarding C. glutamicum, there are a number of reports on ptsG gene, the glucose-specific transporter, involved in glucose metabolism. Recently, we found ptsG had influence on xylose utilization and investigated the ptsG gene in response to xylose utilization in C. glutamicum with the aim to improve xylose consumption and simultaneously utilized glucose and xylose. The ptsG-deficient mutant could grow on xylose, while exhibiting noticeably reduced growth on xylose as sole carbon source. A mutant deficient in ptsH, a general PTS gene, exhibited a similar phenomenon. When complementing ptsG gene, the mutant ΔptsG-ptsG restored the ability to grow on xylose similarly to NC-2. These indicate that ptsG gene is not only essential for metabolism on glucose but also important in xylose utilization. A ptsG-overexpressing recombinant strain could not accelerate glucose or xylose metabolism. When strains were aerobically cultured in a sugar mixture of glucose and xylose, glucose and xylose could not be utilized simultaneously. Interestingly, the ΔptsG strain could co-utilize glucose and xylose under oxygen-deprived conditions, though the consumption rate of glucose and xylose dramatically declined. It was the first report of ptsG gene in response to xylose utilization in C. glutamicum.
We report here the production of pure (R,R)-2,3-butanediol (2,3-BDO) isomer by the non-pathogenic
Paenibacillus polymyxa
ICGEB2008 using lignocellulosic hydrolysate as substrate. Experimental design ...based on Plackett-Burman resulted in identification of Mn and K as most crucial salt elements along with the yeast extract for 2,3-BDO production. Further experiments using Box-Behnken design indicated that both KCl and yeast extract together had major impact on 2,3-BDO production. Optimized medium resulted in 2,3-BDO production with 2.3-fold higher maximum volumetric productivity (2.01 g/L/h) and similar yield (0.33 g/g sugar) as compared to rich yeast extract-peptone-dextrose medium in the bioreactor studies. Considering that the balance substrate was channeled towards ethanol, carbon recovery was close to theoretical yield between the two solvents, i.e., 2,3-BDO and ethanol. Biomass hydrolysate and corn-steep liquor was used further to produce 2,3-BDO without impacting its yield. In addition, 2,3-BDO was also produced via simultaneous saccharification and fermentation, signifying robustness of the strain.
Yeasts are truly fascinating microorganisms. Due to their diverse and dynamic activities, they have been used for the production of many interesting products, such as beer, wine, bread, biofuels, and ...biopharmaceuticals. Saccharomyces cerevisiae (brewers’ or bakers’ yeast) is the yeast species that is surely the most exploited by humans. Saccharomyces is a top-choice organism for industrial applications, although its use for producing beer dates back to at least the 6th millennium BC. Bakers’ yeast has been a cornerstone of modern biotechnology, enabling the development of efficient production processes. Today, diverse yeast species are explored for industrial applications. This Special Issue “Yeast Biotechnology 2.0” is a continuation of the first Special Issue, “Yeast Biotechnology” (https://www.mdpi.com/books/pdfview/book/324). It compiles the current state-of-the-art of research and technology in the area of “yeast biotechnology” and highlights prominent current research directions in the fields of yeast synthetic biology and strain engineering, new developments in efficient biomolecule production, fermented beverages (beer, wine, and honey fermentation), and yeast nanobiotechnology.
Acetic acid is an unavoidable constituent of the biomass hydrolysates generated from acetylated hemicellulose and lignin, and acetate affects the performance of microbes used to convert these ...hydrolysates into biofuels or other biochemicals. In this study, acetate was selectively removed from synthetic mixtures of glucose and xylose using metabolically engineered Escherichia coli strains having mutations in the glucose phosphotransferase system (PTS) genes (ptsG, manZ, crr), glucokinase (glk), and xylose (xylA). In batch culture, ALS1060 (ptsG manZ glk xylA) consumed exclusively acetate to depletion, and then consumed the two sugars only at a very slow rate (a growth rate of about 0.01 h−1). We also examined the effects of an additional knockout of either malX, fruA, fruB, bglF, or crr, genes that are involved in other PTSs, and a batch process using KD840 (ptsG manZ glk crr xylA) demonstrated a further reduction in glucose or xylose consumption by E. coli. These results demonstrate the feasibility of using a substrate-selective approach for the pre-treatment of biomass hydrolysate for microbial processes.
Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d ...biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The "Iogen Process" for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.
Bio-conversion of lignocellulosic biomass to high-value products offers numerous benefits; however, its development is hampered by chemical inhibitors generated during the pretreatment process. A ...better understanding of how microbes naturally respond to those inhibitors is valuable in the process of designing microorganisms with improved tolerance.
VLB120 is a natively tolerant strain that utilizes a wide range of carbon sources including pentose and hexose sugars. To this end, we investigated the tolerance and metabolic response of
VLB120 towards biomass hydrolysate-derived inhibitors including organic acids (acetic acid, formic acid, and levulinic acid), furans (furfural, 5-hydroxymethylfurfural), and phenols (vanillin).
The inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However,
was able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when
VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of
in the presence of biomass-derived inhibitors.
In this study, the tolerance and metabolic response of
VLB120 to biomass hydrolysate-derived inhibitors was investigated.
VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors compared to most wild-type microbes reported in the literature. It adopts different resistance mechanisms, including detoxification, efflux, and repair, which require additional energy and resources. Thus, targeting redox and energy metabolism in strain engineering may be a successful strategy to overcome inhibition during biomass hydrolysate conversion and lead to development of more robust strains.
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