Efficient simultaneous saccharification and fermentation of inulin to 2,3-butanediol by thermophilic Bacillus licheniformis ATCC 14580.
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SUBMITTER: Li L
PROVIDER: S-EPMC4178658 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Efficient simultaneous saccharification and fermentation of inulin to 2,3-butanediol by thermophilic Bacillus licheniformis ATCC 14580.
Li Lixiang L Chen Chao C Li Kun K Wang Yu Y Gao Chao C Ma Cuiqing C Xu Ping P
Applied and environmental microbiology 20140808 20
2,3-Butanediol (2,3-BD) is an important starting material for the manufacture of bulk chemicals. For efficient and large-scale production of 2,3-BD through fermentation, low-cost substrates are required. One such substrate, inulin, is a polydisperse fructan found in a wide variety of plants. In this study, a levanase with high inulinase activity and high pH and temperature stability was identified in Bacillus licheniformis strain ATCC 14580. B. licheniformis strain ATCC 14580 was found to effici ...[more]
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Project description:Background2,3-Butanediol (2,3-BD) can be used as a liquid fuel additive to replace petroleum oil, and as an important platform chemical in the pharmaceutical and plastic industries. Microbial production of 2,3-BD by Bacillus licheniformis presents potential advantages due to its GRAS status, but previous attempts to use this microorganism as a chassis strain resulted in the production of a mix of D-2,3-BD and meso-2,3-BD isomers.ResultsThe aim of this work was to develop an engineered strain of B. licheniformis suited to produce the high titers of the pure meso-2,3-BD isomer. Glycerol dehydrogenase (Gdh) was identified as the catalyst for D-2,3-BD biosynthesis from its precursor acetoin in B. licheniformis. The gdh gene was, therefore, deleted from the wild-type strain WX-02 to inhibit the flux of acetoin to D-2,3-BD biosynthesis. The acoR gene involved in acetoin degradation through AoDH ES was also deleted to provide adequate flux from acetoin towards meso-2,3-BD. By re-directing the carbon flux distribution, the double-deletion mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD isomer with >99 % purity. The titer was 50 % higher than that of the wide type. A bench-scale fermentation by the double-deletion mutant was developed to further improve meso-2,3-BD production. In a fed-batch fermentation, meso-2,3-BD titer reached 98.0 g/L with a purity of >99.0 % and a productivity of 0.94 g/L-h.ConclusionsThis work demonstrates the potential of producing meso-2,3-BD with high titer and purity through metabolic engineering of B. licheniformis.
| S-EPMC4890260 | biostudies-literature
Project description:BACKGROUND:D-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis. RESULTS:We developed a genetically modified strain of B. licheniformis (WX-02 ?budC) as a D-2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the budC gene in B. licheniformis WX-02. The budC knock-out strain successfully abolished meso-2,3-butanediol production with enhanced D-2,3-butanediol production. No meso-BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced meso-2,3-butanediol and possessed meso-BDH activity. All of these data suggested that budC encoded the major meso-BDH catalyzing the reversible reaction from acetoin to meso-2,3-butanediol in B. licheniformis. The budC knock-out strain produced D-2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h. CONCLUSIONS:We confirmed the hypothesis that budC gene is responsible to reversibly transfer acetoin to meso-2,3-butanediol in B. licheniformis. A mutant strain of B. licheniformis with depleted budC gene was successfully developed and produced high level of the D-2,3-butanediol with high optimal purity.
| S-EPMC3909405 | biostudies-literature
Project description:UNLABELLED: BACKGROUND:Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination. RESULTS:This work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7?g/L of acetoin and 14.5?g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography-mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. ?-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C. CONCLUSIONS:Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work.
| S-EPMC3538569 | biostudies-literature
Project description:Background2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical, can be efficiently produced by Klebsiella pneumonia, K. oxytoca, and Serratia marcescens. However, these strains are opportunistic pathogens and not favorable for industrial application. Although some generally regarded as safe (GRAS) microorganisms have been isolated in recent years, there is still a demand for safe 2,3-BD producing strains with high productivity and yield under thermophilic fermentation.ResultsBacillus licheniformis strain 10-1-A was newly isolated for 2,3-BD production. The optimum temperature and medium pH were 50°C and pH 7.0 for 2,3-BD production by strain 10-1-A. The medium composition was optimized through Plackett-Burman design and response surface methodology techniques. With a two-stage agitation speed control strategy, 115.7 g/L of 2,3-BD was obtained from glucose by fed-batch fermentation in a 5-L bioreactor with a high productivity (2.4 g/L·h) and yield (94% of its theoretical value). The 2,3-BD produced by strain 10-1-A comprises (2R,3R)-2,3-BD and meso-2,3-BD with a ratio of nearly 1:1. The bdh and gdh genes encoding meso-2,3-butanediol dehydrogenase (meso-BDH) and glycerol dehydrogenase (GDH) of strain 10-1-A were expressed in Escherichia coli and the proteins were purified. meso-2,3-BD and (2R,3R)-2,3-BD were transformed from racemic acetoin by meso-BDH and GDH with NADH, respectively.ConclusionsCompared with the reported GRAS 2,3-BD producers, B. licheniformis 10-1-A could thermophilically produce 2,3-BD with a high concentration, productivity and yield. Thus, the newly isolated GRAS strain 10-1-A might be a promising strain for industrial production of 2,3-BD. Two key enzymes for meso-2,3-BD and (2R,3R)-2,3-BD production were purified and further studied, and this might be helpful to understand the mechanism for 2,3-BD stereoisomers forming in B. licheniformis.
| S-EPMC3766113 | biostudies-literature
Project description:BACKGROUND:Acetoin (3-hydroxy-2-butanone), the precursor of biofuel 2,3-butanediol, is an important bio-based platform chemical with wide applications. Fermenting the low-cost and renewable plant biomass is undoubtedly a promising strategy for acetoin production. Isothermal simultaneous saccharification and fermentation (SSF) is regarded as an efficient method for bioconversion of lignocellulosic biomass, in which the temperature optima fitting for both lignocellulose-degrading enzymes and microbial strains. RESULTS:A thermotolerant (up to 52 °C) acetoin producer Bacillus subtilis IPE5-4 which simultaneously consumed glucose and xylose was isolated and identified. By compound mutagenesis, the mutant IPE5-4-UD-4 with higher acetoin productivity was selected. When fermenting at 50 °C in a 5-L bioreactor using glucose as the feedstock by strain IPE5-4-UD-4, the acetoin concentration reached 28.83 ± 0.91 g L-1 with the acetoin yield and productivity of 0.34 g g-1 glucose and 0.60 g L-1 h-1, respectively. Furthermore, an optimized and thermophilic SSF process operating at 50 °C was conducted for acetoin production from alkali-pretreated corncob (APC). An acetoin concentration of 12.55 ± 0.28 g L-1 was achieved by strain IPE5-4-UD-4 in shake flask SSF, with the acetoin yield and productivity of 0.25 g g-1 APC and 0.17 g L-1 h-1. Meanwhile, the utilization of cellulose and hemicellulose in the SSF approach reached 96.34 and 93.29%, respectively. When further fermented at 50 °C in a 5-L bioreactor, the concentration of acetoin reached the maximum of 22.76 ± 1.16 g L-1, with the acetoin yield and productivity reaching, respectively, 0.46 g g-1 APC and 0.38 g L-1 h-1. This was by far the highest acetoin yield in SSF from lignocellulosic biomass. CONCLUSIONS:This thermophilic SSF process provided an efficient and economical route for acetoin production from lignocellulosic biomass at ideal temperature for both enzymatic hydrolysis and microbial fermentation.
| S-EPMC5635544 | biostudies-literature
Project description:Microaerobic cultivation conditions are often beneficial for the biotechnological production of reduced metabolites like 2,3-butanediol. However, due to oxygen limitation, process monitoring based on oxygen transfer rate, or dissolved oxygen measurement provides only limited information. In this study, online monitoring of the respiratory quotient is used to investigate the metabolic activity of Bacillus licheniformis DSM 8785 during mixed acid-2,3-butanediol production under microaerobic conditions. Thereby, the respiratory quotient provides valuable information about different metabolic phases. Based on partial reaction stoichiometries, the metabolic activity in each phase of the cultivation was revealed, explaining the course of the respiratory quotient. This provides profound information on the formation or consumption of glucose, 2,3-butanediol, ethanol and lactate, both, in shake flasks and stirred tank reactor cultivations. Furthermore, the average respiratory quotient correlates with the oxygen availability during the cultivation. Carbon mass balancing revealed that this reflects the increased formation of reduced metabolites with increasing oxygen limitation. The results clearly demonstrate that the respiratory quotient is a valuable online signal to reveal and understand the metabolic activity during microaerobic cultivations. The approach of combining respiratory quotient monitoring with stoichiometric considerations can be applied to other organisms and processes to define suitable cultivation conditions to produce the desired product spectrum.
| S-EPMC7447875 | biostudies-literature
Project description:ObjectiveA primary drawback to simultaneous saccharification and fermentation (SSF) processes is the incompatibility of the temperature and pH optima for the hydrolysis and fermentation steps-with the former working best at 50-55 °C and pH 4.5-5.5. Here, nine thermophilic Bacillus and Parageobacillus spp. were evaluated for growth and lactic acid fermentation at high temperature and low pH. The most promising candidate was then carried forward to demonstrate SSF using the cellulosic fraction from municipal solid waste (MSW) as a feedstock.ResultsB. smithii SA8Eth was identified as the most promising candidate and in a batch SSF maintained at 55 °C and pH 5.0, using a cellulase dose of 5 FPU/g glucan, it produced 5.1 g/L lactic acid from 2% (w/v) MSW cellulosic pulp in TSB media.ConclusionThis work has both scientific and industrial relevance, as it evaluates a number of previously untrialled bacterial hosts for their compatibility with lignocellulosic SSF for lactic acid production and successfully identifies B. smithii as a potential candidate for such a process.
| S-EPMC7873104 | biostudies-literature
Project description:Background:2,3-Butanediol (BD) is a largely used fossil-based platform chemical. The yield and productivity of bio-based BD fermentative production must be increased and cheaper substrates need to be identified, to make bio-based BD production more competitive. As BD bioproduction occurs under microaerobic conditions, a fine tuning and control of the oxygen transfer rate (OTR) is crucial to maximize BD yield and productivity. Very few studies on BD bioproduction focused on the use of non-pathogenic microorganisms and of byproducts as substrate. The goal of this work was to optimize BD bioproduction by the non-pathogenic strain Bacillus licheniformis ATCC9789 by (i) identifying the ranges of volumetric and biomass-specific OTR that maximize BD yield and productivity using standard sugar and protein sources, and (ii) performing a preliminary evaluation of the variation in process performances and cost resulting from the replacement of glucose with molasses, and beef extract/peptone with chicken meat and bone meal, a byproduct of the meat production industry. Results:OTR optimization with an expensive, standard medium containing glucose, beef extract and peptone revealed that OTRs in the 7-15 mmol/L/h range lead to an optimal BD yield (0.43?±?0.03 g/g) and productivity (0.91?±?0.05 g/L/h). The corresponding optimal range of biomass-specific OTR was equal to 1.4-7.9 [Formula: see text], whereas the respiratory quotient ranged from 1.8 to 2.5. The switch to an agro-industrial byproduct-based medium containing chicken meat and bone meal and molasses led to a 50% decrease in both BD yield and productivity. A preliminary economic analysis indicated that the use of the byproduct-based medium can reduce by about 45% the BD production cost. Conclusions:A procedure for OTR optimization was developed and implemented, leading to the identification of a range of biomass-specific OTR and respiratory quotient to be used for the scale-up and control of BD bioproduction by Bacillus licheniformis. The switch to a byproduct-based medium led to a relevant decrease in BD production cost. Further research is needed to optimize the process of BD bioproduction from the tested byproduct-based medium.
| S-EPMC5964669 | biostudies-literature
Project description:BackgroundPreviously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.Methodology/principal findingsIn the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.Conclusions/significanceCo-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.
| S-EPMC3788785 | biostudies-literature
Project description:BackgroundIndustrial fermentations can generally be described as dynamic biotransformation processes in which microorganisms convert energy rich substrates into a desired product. The knowledge of active physiological pathways, reflected by corresponding gene activities, allows the identification of beneficial or disadvantageous performances of the microbial host. Whole transcriptome RNA-Seq is a powerful tool to accomplish in-depth quantification of these gene activities, since the low background noise and the absence of an upper limit of quantification allow the detection of transcripts with high dynamic ranges. Such data enable the identification of potential bottlenecks and futile energetic cycles, which in turn can lead to targets for rational approaches to productivity improvement. Here we present an overview of the dynamics of gene activity during an industrial-oriented fermentation process with Bacillus licheniformis, an important industrial enzyme producer. Thereby, valuable insights which help to understand the complex interactions during such processes are provided.ResultsWhole transcriptome RNA-Seq has been performed to study the gene expression at five selected growth stages of an industrial-oriented protease production process employing a germination deficient derivative of B. licheniformis DSM13. Since a significant amount of genes in Bacillus strains are regulated posttranscriptionally, the generated data have been confirmed by 2D gel-based proteomics. Regulatory events affecting the coordinated activity of hundreds of genes have been analyzed. The data enabled the identification of genes involved in the adaptations to changing environmental conditions during the fermentation process. A special focus of the analyses was on genes contributing to central carbon metabolism, amino acid transport and metabolism, starvation and stress responses and protein secretion. Genes contributing to lantibiotics production and Tat-dependent protein secretion have been pointed out as potential optimization targets.ConclusionsThe presented data give unprecedented insights into the complex adaptations of bacterial production strains to the changing physiological demands during an industrial-oriented fermentation. These are, to our knowledge, the first publicly available data that document quantifiable transcriptional responses of the commonly employed production strain B. licheniformis to changing conditions over the course of a typical fermentation process in such extensive depth.
| S-EPMC3878961 | biostudies-literature