Overcoming extended lag phase on optically pure lactic acid production from pretreated softwood solids
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ABSTRACT:
SUBMITTER: Campos J
PROVIDER: S-EPMC10513496 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of L-lactic acid to D-lactic acid. Although the largest demand is for the L enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of D-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. D-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this D-lactic acid was approximately 98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of D-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.
| S-EPMC152430 | biostudies-literature
Project description:The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.
| S-EPMC7465028 | biostudies-literature
Project description:BackgroundOptically pure D-lactic acid (≥ 99%) is an important precursor of polylactic acid. However, there are relatively few studies on D-lactic acid fermentation compared with the extensive investigation of L-lactic acid production. Most lactic acid producers are mesophilic organisms. Optically pure D-lactic acid produced at high temperature not only could reduce the costs of sterilization but also could inhibit the growth of other bacteria, such as L-lactic acid producers.ResultsThermophilic Bacillus coagulans is an excellent producer of L-lactic acid with capable of growing at 50 °C. In our previous study, the roles of two L-lactic acid dehydrogenases have been demonstrated in B. coagulans DSM1. In this study, the function of another annotated possible L-lactate dehydrogenase gene (ldhL3) was verified to be leucine dehydrogenase with an activity of 0.16 units (μmol/min) per mg protein. Furthermore, the activity of native D-lactate dehydrogenase was too low to support efficient D-lactic acid production, even under the control of strong promoter. Finally, an engineered B. coagulans D-DSM1 strain with the capacity for efficient production of D-lactic acid was constructed by deletion of two L-lactate dehydrogenases genes (ldhL1 and ldhL2) and insertion of the D-lactate dehydrogenase gene (LdldhD) from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 at the position of ldhL1.ConclusionsThis genetically engineered strain produced only D-lactic acid under non-sterilized condition, and finally 145 g/L of D-lactic acid was produced with an optical purity of 99.9% and a high yield of 0.98 g/g. This is the highest optically pure D-lactic acid titer produced by a thermophilic strain.
| S-EPMC5702109 | biostudies-literature
Project description:Background2,3-butanediol is an important platform compound which has a wide range of applications, involving in medicine, chemical industry, food and other fields. Especially the optically pure (2R,3R)-2,3-butanediol can be employed as an antifreeze agent and as the precursor for producing chiral compounds. However, some (2R,3R)-2,3-butanediol overproducing strains are pathogenic such as Enterobacter cloacae and Klebsiella oxytoca.ResultsIn this study, a (3R)-acetoin overproducing C. glutamicum strain, CGS9, was engineered to produce optically pure (2R,3R)-2,3-butanediol efficiently. Firstly, the gene bdhA from B. subtilis 168 was integrated into strain CGS9 and its expression level was further enhanced by using a strong promoter Psod and ribosome binding site (RBS) with high translation initiation rate, and the (2R,3R)-2,3-butanediol titer of the resulting strain was increased by 33.9%. Then the transhydrogenase gene udhA from E. coli was expressed to provide more NADH for 2,3-butanediol synthesis, which reduced the accumulation of the main byproduct acetoin by 57.2%. Next, a mutant atpG was integrated into strain CGK3, which increased the glucose consumption rate by 10.5% and the 2,3-butanediol productivity by 10.9% in shake-flask fermentation. Through fermentation engineering, the most promising strain CGK4 produced a titer of 144.9 g/L (2R,3R)-2,3-butanediol with a yield of 0.429 g/g glucose and a productivity of 1.10 g/L/h in fed-batch fermentation. The optical purity of the resulting (2R,3R)-2,3-butanediol surpassed 98%.ConclusionsTo the best of our knowledge, this is the highest titer of optically pure (2R,3R)-2,3-butanediol achieved by GRAS strains, and the result has demonstrated that C. glutamicum is a competitive candidate for (2R,3R)-2,3-butanediol production.
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Project description:Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41 degrees C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.
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Project description:An inhibitor-tolerance strain, Bacillus coagulans GKN316, was developed through atmospheric and room temperature plasma (ARTP) mutation and evolution experiment in condensed dilute-acid hydrolysate (CDH) of corn stover. The fermentabilities of other hydrolysates with B. coagulans GKN316 and the parental strain B. coagulans NL01 were assessed. When using condensed acid-catalyzed steam-exploded hydrolysate (CASEH), condensed acid-catalyzed liquid hot water hydrolysate (CALH) and condensed acid-catalyzed sulfite hydrolysate (CASH) as substrates, the concentration of lactic acid reached 45.39, 16.83, and 18.71 g/L by B. coagulans GKN316, respectively. But for B. coagulans NL01, only CASEH could be directly fermented to produce 15.47 g/L lactic acid. The individual inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde and p-hydroxybenzaldehyde (pHBal) on xylose utilization by B. coagulans GKN316 was also studied. The strain B. coagulans GKN316 could effectively convert these toxic inhibitors to the less toxic corresponding alcohols in situ. These results suggested that B. coagulans GKN316 was well suited to production of lactic acid from undetoxified lignocellulosic hydrolysates.
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Project description:Softwood bark contains a large amounts of extractives-i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks-Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic digestibility of both steam-pretreated materials. This effect was more pronounced for the spruce than the pine bark, as evidenced by the 30 and 11% glucose yield improvement, respectively, in the enzymatic digestibility. Furthermore, analysis of the chemical composition showed that the acid-insoluble lignin content of the pretreated materials decreased when water-soluble extractives were removed prior to steam pretreatment. This can be explained by a decreased formation of water-insoluble "pseudo-lignin" from water-soluble bark phenolics during the acid-catalyzed pretreatment, which otherwise results in distorted lignin analysis and may also contribute to the impaired enzymatic digestibility of the barks. Thus, this study advocates the removal of extractives as the first step in the processing of bark or bark-rich materials in a sugar platform biorefinery.
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Project description:Dopamine is not only a widely used commodity pharmaceutical for treating neurological diseases but also a highly attractive base for advanced carbon materials. Lignin, the waste from the lignocellulosic biomass industry, is the richest source of renewable aromatics on earth. Efficient production of dopamine direct from lignin is a highly desirable target but extremely challenging. Here, we report an innovative strategy for the sustainable production of dopamine hydrochloride from softwood lignin with a mass yield of 6.4 wt.%. Significantly, the solid dopamine hydrochloride is obtained by a simple filtration process in purity of 98.0%, which avoids the tedious separation and purification steps. The approach begins with the acid-catalyzed depolymerization, followed by deprotection, hydrogen-borrowing amination, and hydrolysis of methoxy group, transforming lignin into dopamine hydrochloride. The technical economic analysis predicts that this process is an economically competitive production process. This study fulfills the unexplored potential of dopamine hydrochloride synthesis from lignin.
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Project description:The resolution of proton solid-state NMR spectra is usually limited by broadening arising from dipolar interactions between spins. Magic-angle spinning alleviates this broadening by inducing coherent averaging. However, even the highest spinning rates experimentally accessible today are not able to completely remove dipolar interactions. Here, we introduce a deep learning approach to determine pure isotropic proton spectra from a two-dimensional set of magic-angle spinning spectra acquired at different spinning rates. Applying the model to 8 organic solids yields high-resolution 1 H solid-state NMR spectra with isotropic linewidths in the 50-400 Hz range.
| S-EPMC10107932 | biostudies-literature