Mechanistic understanding of the fermentative L‐glutamic acid overproduction by Corynebacterium glutamicum through combined metabolic flux profiling and transmembrane transport characteristics

Since the 1950s when Micrococcus glutamicus later renamed Corynebacterium glutamicum was discovered, the production of amino acids by fermentative methods has become an important aspect of industrial microbiology. Numerous studies to understand and improve the metabolic conditions leading to amino acid overproduction have been carried out. Most amino acids are currently produced by use of mutants that contain combinations of auxotrophic and regulatory mutations. L ‐Glutamic acid is the amino acid produced in the greatest quantities (10⁶ tonnes per year) and Corynebacteria are central to its industrial production. However, further improvements to strain performance are difficult to obtain by empirical optimization and a more rational approach is required. The use of metabolic flux analysis provides valuable information regarding bottlenecks in the formation of desired metabolites. Such techniques have found application in elucidating flux control, provided insight into metabolic network function and developed methods to amplify or redirect fluxes in engineered bioprocesses. Hence, branch points in biosynthesis, precursor supply in fuelling reactions and export of metabolites can be manipulated, resulting in high glutamic acid overproduction by Corynebacterium glutamicum fermentations. In this review, in addition to reviewing the state of play in metabolic flux analysis for glutamate overproduction, the metabolic pathways involved in the production of L ‐glutamic acid, the mechanisms mediating its efflux and secretion as well as their manipulation to achieve higher glutamate production, are discussed. The link between metabolic flux and transmembrane transport of glutamic acid are also considered. Copyright © 2004 Society of Chemical Industry     

Investigation of liquid–liquid phase equilibria for reactive extraction of lactic acid with organophosphorus solvents

BACKGROUND: Lactic acid is a versatile commodity chemical with a variety of applications. Synthesis of lactic acid either through fermentation of carbohydrates or through chemical synthesis is state of the art. Separation from dilute aqueous solution is complex and accounts for the major part of production costs. Reactive extraction based on reversible adduct formation is a promising alternative for the separation of lactic acid. RESULTS: Extraction was carried out with the organophosphorus solvents tri‐n‐butyl phosphate, tri‐n‐octylphosphine oxide and Cyanex 923. Shellsol T was used as diluent. Partition coefficients increase with increasing extractant content and decreasing temperature. Cyanex 923 has several advantages compared with tri‐n‐butyl phosphate and tri‐n‐octylphosphine oxide with respect to lactic acid distribution and hydrodynamic properties. Liquid‐liquid phase equilibria for lactic acid extraction with Cyanex 923 were modeled. Selectivity of lactic acid extraction with respect to glycolic acid and formic acid was discussed. CONCLUSION: The organophosphorus extractant Cyanex 923 was found to be an appropriate solvent for lactic acid extraction from aqueous solutions. Experimental data and model data based on the Law of Mass Action showed good agreement. Lactic acid extraction from multi‐acid solution showed good selectivity compared with glycolic acid. Lactic acid selectivity is low with respect to formic acid. © 2012 Society of Chemical Industry     

Design of reactive distillation processes for the production of butyl acrylate:Impact of bio-based raw materials☆

The chemical industry is nowadays predominantly using fossil raw materials, but the alternative use of bio-based resources is investigated to account for the foreseeable scarcity of fossil feedstocks. A main challenge of using bio-based feedstocks is the complexity of the impurity profile. For an economic production of bio-based chemicals, the use of intensified processes is inevitable and approaches are needed for the various process intensification techniques to identify their applicability to be used for the production of bio-based components. In the presented study, an approach is shown for the reactive distil ation (RD) technology to identify the most critical bio-based impurities and their impact on the reactive distillation process. The investigated case-study is the production of n-butyl acrylate from acrylic acid and n-butanol. Among al initially identified impurities, the key impurities, having the biggest impact on the product purity in the reactive distil ation process, are found. These impurities are then studied in more detail and an operating window depending on the impurity concentration is identified for the reactive distil ation column. Furthermore, an integrated design of upstream and downstream processes is facilitated, as the presented results can be used in the development of the fermentation processes for the produc-tion of the bio-based reactants by decreasing the concentration of the critical impurities.     

Vegetable oil-based epoxy resins and their composites with bio-based hardener: a short review

ABSTRACT

Vegetable oil (VO) is the latest alternative found to be one of the ideal candidates for production of bio-based polymeric units. There are two main methods for producing bio-based epoxy resins and composites: the direct synthesis of VO and the blends of epoxy with VO incorporated with natural or synthetic fibers. However, it is more challenging to produce a fully green bio-based epoxy composite, as they have relatively low strength that limits their applications. Thus, this article is designed to review the present research advances on VO-based epoxy resins and their composites with bio-based hardener incorporated with fibers or fillers.     

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

A new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L (+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L^?1) was reached under semi‐batch conditions. Potato starch (5–70 g L^?1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry     

Metabolic strategies for microbial glycerol overproduction

Glycerol as a platform chemical has wide applications in chemical, pharmaceutical, and food industries and is a useful feedstock for the production of bio‐based chemicals. There is an increasing demand for sustainable glycerol production and microbial glycerol production is a promising alternative. The paper highlighted this new trend and summarized recent research progress on microbial glycerol production, and focused mainly on the efforts achieved in rational design of S. cerevisiae, as well as Escherichia coli, to overcome the limitations of glycerol production in these two microorganisms. More efforts are required for overcoming hurdles in yield and productivity enhancement for large scale production. © 2017 Society of Chemical Industry     

High cell density fermentation via a metabolically engineered Escherichia coli for the enhanced production of succinic acid

BACKGROUND: Succinic acid is a valuable four‐carbon organic chemical with applications in many fields. It was found that cell mass was an important factor in succinic acid production by metabolically engineered Escherichia coli strains. In this work, high cell density fermentation was investigated for succinic acid production by a metabolically engineered strain SD121 with ldhA, pflB, ptsG mutation and heterogenous cyanobacterial ppc overexpression. RESULTS: Under two‐stage cultivation, the controlled DO feeding strategy during the aerobic growth phase facilitated biomass up to a dry cell weight of 19.6 g L^?1, and enhanced succinic acid production in the following anaerobic fermentation phase to a concentration of 116.2 g L^?1. A near theoretical maximum succinic acid yield of 1.73 mol mol^?1 glucose was achieved with an average productivity of 1.55 g L^?1 h^?1. CONCLUSION: The results indicated the potential advantage of high cell density fermentation for improvement of succinic acid production by E. coli. Copyright © 2010 Society of Chemical Industry     

Semicontinuous distillation of impurities for the production of butyl acrylate from bio-butanol and bio-acrylic acid

Chemical production processes which use biosynthetic pathways typically experience variations in the concentration and composition of impurities contained in the products, due to the natural variability of biological behaviour. As a result, downstream processes which use these biosynthetic chemical products need to be quite flexible with respect to the impurity concentrations. In this study, a ternary semicontinuous distillation process is investigated, separating a mixture into three fractions, as a potential candidate for the purification of three different bio-based chemicals (bio-acrylic acid, bio-n-butanol, and bio-n-butyl acrylate) which are generated at various steps in the process to produce bio-n-butyl acrylate from bio-based feedstocks. In this model-based investigation, the performance of this approach is discussed in terms of the energy demand, product loss, and the cycle time. It is shown, that a single ternary semicontinuous system can be applied to purify several bio-based chemicals, allowing a simple increase or decrease of the desired final purity, while being able to handle natural variations of the initial compositions of the feed.     

Synthesis of a Sustainable and Bisphenol A-Free Epoxy Resin Based on Sorbic Acid and Characterization of the Cured Thermoset

In the present study, an epoxy compound, 1,2-epoxy-6-methyl-triglycidyl-3,4,5-cyclohexanetricarboxylate (EGCHC) synthesized from sorbic acid, maleic anhydride, and allyl alcohol is proposed. Using commodity chemicals, a bio-based carbon content of 68.4 % for the EGCHC resin is achieved. When cured with amine hardeners, the high oxirane content of EGCHC forms stiff cross-linked networks with strong mechanical and thermal properties. The characterization of the epoxy specimens showed that EGCHC can compete with conventional epoxy resins such as DGEBA. A maximum stiffness of 3965 MPa, tensile strength of 76 MPa, and T_g of 130 °C can be obtained by curing EGCHC with isophorone diamine (IPD). The cured resin showed to be decomposable under mild conditions due to the ester bonds. The solid material properties of EGCHC expose its potential as a promising bisphenol A, and epichlorohydrine free alternative to conventional petroleum-based epoxies with an overall high bio-based carbon content.     

Melt compounded polylactic acid-hexagonal boron nitride-aluminum oxide hybrid composites for electronic applications: impact of hybrid fillers on thermophysical,dielectric, optical,and hardness properties

ABSTRACT

The dielectric, thermophysical, optical, and hardness of pure polylactic acid (PLA) and hBN micropowder and Al₂O₃ nanopowder (1% to 30%) reinforced PLA hybrid composites were investigated. Hybrid composites exhibit improved thermal conductivity (k – 0.54 W/mK), permittivity (?? – 4.6720 @ 1 MHz to 1 GHz) with very low loss tangent (tan δ < 0.02). High absorption in UV region was observed for all hybrid composites. Overall, the prepared hybrid composites can be used as a bio-based dielectric substrates, enclosures, thermal interface material for low temperature applications and UV-absorbable coating materials for fabric, packaging, and storage applications.     

Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(��-L-Malic Acid)

Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(β-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.     

Use of sweet sorghum juice for lactic acid fermentation: preliminary steps in a process optimization

BACKGROUND: Lactic acid has many applications in the chemical industries and it can be produced economically by microorganisms using biomass raw materials of different origins. Sweet sorghum juice is a high sugar content raw material with potential for lactic acid production because after hydrolysis of its sucrose content the remaining glucose and fructose can supply the carbon demand of most lactic acid bacteria. However, satisfying the nitrogen and B‐vitamin needs of the bacteria by supplementation with yeast extract and/or other alternative nitrogen‐containing supplements can make the process too expensive. RESULTS: Using a statistical optimization process much of the yeast extract can be replaced by a cheaper alternative nitrogen source, namely wheat gluten. This resulted in a fermentation with 99% lactic acid yield and 3.04 g L^?1 h^?1 volumetric productivity. CONCLUSION: Using response surface methodology (RSM) media optimization was performed for lactic acid fermentation with an industrially acceptable result, reducing the costs of raw materials by half, replacing yeast extract by an alternative nitrogen source and applying yeast extract only as a source of micro‐elements (vitamins, salts, etc.) Copyright © 2010 Society of Chemical Industry     

Effective microbial production of poly(4‐hydroxybutyrate) homopolymer by Ralstonia eutropha H16

The effective microbial production of copolyesters of 3‐hydroxybutyrate (3HB) and 4‐hydroxybutyrate (4HB) with high mole fractions of 4HB units by a wild‐type strain of Ralstonia eutropha H16 was investigated in culture solutions containing 4‐hydroxybutyric acid (4HBA) and various carbon substrates in the presence of a nitrogen source such as ammonium sulfate. The addition of glucose or acetic acid to the culture solution containing 4HBA in the presence of ammonium sulfate resulted in the production of random copolymers of P(3HB‐co‐4HB) with compositions of up to 82 mol% 4HB, but the yield of copolymers was less than 7 wt% of dried cell weights. In contrast, when n‐alkanoic acids such as propionic acid, butyric acid, valeric acid and hexanoic acid, being subject to β‐oxidation metabolism in the cell, were used as the co‐substrates of 4HBA in the presence of ammonium sulfate, a mixture of copolymers with two different 4HB compositions was produced, and copolyesters with compositions of 93–100 mol% 4HB were isolated from chloroform–n‐hexane insoluble fractions in the mixture of copolymers. Especially, when this wild‐type Ralstonia eutropha H16 was cultivated in a medium containing 4HBA (15 g litre⁻¹), propionic acid (5 g litre⁻¹) and ammonium sulfate (5 g litre⁻¹), namely C/N (mol/mol) = 10, the P(4HB) homopolymer was produced at maximally 34 wt% of dry cell weight (7.8 g litre⁻¹), and the conversion yield of 4HBA to P(4HB) homopolymer resulted in values as high as 21 mol%. © 1999 Society of Chemical Industry     

Selective Hydrogenation of Biomass-Derived Succinic Acid: Reaction Network and Kinetics

The conversion of bio-based succinic acid (SA) to the value-added chemicals 1,4-butanediol (BDO), γ-butyrolactone (GBL), and tetrahydrofuran (THF) can replace the corresponding petrochemical production routes to achieve a sustainable process. The reaction network for aqueous-phase catalytic hydrogenation of succinic acid over a supported Re-Pd catalyst was identified and the reaction kinetics was determined. With the developed kinetic model, the composition of the product mixture regarding the desired products (BDO, GBL, THF) can be described as a function of educt concentration, temperature, and pressure. The maximum BDO yield was achieved at high pressure and low temperature, while low pressure and high temperature favored GBL and THF production.     

The properties and suitability of commercial bio-based epoxies for use in fiber-reinforced composites

Environmental concerns about fiber composites are leading manufacturers to consider bio-based alternatives to petroleum-derived epoxies. Such a substitution is hindered by a lack of information, so commercially available bio-based epoxy systems have been compared, their mechanical properties measured, and fiber composites produced by vacuum infusion. Most high bio-based content resins for infusion use conventional curing agents. Bio-based content is generally added using Epicerol, but also other bio-based precursors. A diglycidyl ether of bisphenol A system produced using Epicerol achieves 20 % bio-based content, but achieves higher contents when Epicerol is used in diluents. Fully bio-based monomers can be deleterious to the mechanical properties and glass transition temperature (T_g), so are used sparingly. The most-promising systems (28 % to 43 % bio-based) compare well to conventional epoxies, possessing good strength, stiffness, toughness, and a reasonable T_g. These partially bio-based epoxies offer an immediate lower-carbon alternative for vacuum-infused composites in marine, sports equipment, and wind energy.     

Synthesis of bio-based unsaturated polyester resins and their application in waterborne UV-curable coatings

In this paper, three bio-based unsaturated polyesters were synthesized from itaconic acid and different diols which could be derived from renewable resources. Their chemical structures were confirmed by FT-IR, ¹H NMR and acid value as well as hydroxyl value. Waterborne UV curable networks based on these polyesters were manufactured and their mechanical properties, thermal stability and coating properties including pencil hardness, flexibility, adhesion, water resistance and solvent resistance were investigated. Results showed that the UV-cured polyester coatings exhibited high hardness, good water resistance and solvent resistance. The coatings reported in this paper combined the merits of bio-based materials, UV-curing process and water distribution.     

The Peculiar Structure of Acetobacter pasteurianus CIP103108 LPS Core Oligosaccharide

Acetobacter pasteurianus, a member of the Alphaproteobacteria, is an acetic acid-producing bacterium present on sugar-rich substrates such as such as fruits, flowers and vegetables and traditionally used in the production of fermented food. The preferred living habitat associated with acid conditions makes the structure of the bacterial cell wall interesting to study, due to expected uncommon features. We have used a combination of chemical, analytical and NMR spectroscopy approaches to define the complete structure of the core oligosaccharide from A. pasteurianus CIP103108 LPS. Interestingly, the core oligosaccharide displays a high concentration of negatively charged groups, structural features that might contribute to reinforcing the bacterial membrane.     

Copolyesters developed from bio-based 2,5-furandicarboxylic acid: Synthesis,sequence distribution,mechanical, and barrier properties of poly(propylene-co-1,4-cyclohexanedimethylene 2,5-furandicarboxylate)s

A series of poly(propylene-co-1,4-cyclohexanedimethylene 2,5-furandicarboxylate)s (PPCFs) with different compositions were synthesized from bio-based aromatic monomer 2,5-furandicarboxylic acid and 1,3-propanediol (PDO), along with 1,4-cyclohexanedimethylene (CHDM). The chemical structure, composition, and sequence distribution of PPCFs were determined by nuclear magnetic resonance (¹H NMR and ¹³C NMR). Results showed that the compositions of copolyesters depended on the feed molar ratio of PDO and CHDM, and all the obtained PPCFs copolyesters had triad component in a random sequential structure. With the addition of CHDM, the gas barrier properties of poly(propylene 2,5-furandicarboxylate) (PPF) deteriorated. However, when the content of CHDM reached 79%, PPCF79 still showed the CO₂ and O₂ barrier improvement factor of 4.5 and 3.25, respectively, which were better than those of poly(ethylene naphthalate), a well-known polymer with barrier property. Besides, PPCF79 showed good performance on tensile modulus, tensile strength, elongation at break, and the crystallization enthalpy was improved from 3.2 J g⁻¹ for PPF to 30.7 J g⁻¹ for PPCF79. It has the potential to serve as a promising bio-based polymer with excellent comprehensive performance. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47291.     

Emerging tools,enabling technologies,and future opportunities for the bioproduction of aromatic chemicals

Aromatic compounds, which are traditionally derived from petroleum feedstocks, represent a diverse class of molecules with a wide range of industrial and commercial applications. Significant progress has been made to alternatively and sustainably produce many aromatics from renewable substrates using microbial biocatalysts. While the construction of both natural and non-natural pathways has expanded the number and diversity of aromatic bioproducts, pathway modularization in both single- and multi-strain systems continues to support the enhancement of key production metrics towards economically-viable levels. Emerging tools for implementing more precise metabolic control (e.g. CRISPRi, sRNA) as well as the engineering of novel high-throughput screening platforms utilizing in vivo aromatic biosensors, meanwhile, continue to facilitate further optimization of both pathways and hosts. While product toxicity persists as a key challenge limiting the production of many aromatics, various successful strategies have been demonstrated towards improving tolerance, including via membrane and efflux pump engineering as well as by exploiting alternative production hosts. Finally, as a further step towards sustainable and economical aromatic bioproduction, non-model substrates including lignin-derived compounds continue to emerge as viable feedstocks. This review highlights recent and notable achievements related to such efforts while offering future outlooks towards engineering microbial cell factories for aromatic production. © 2018 Society of Chemical Industry     

Differentiation of pH and free lactic acid effects on the various growth and production phases of lactobacillus helveticus

Growth and acid production coupling for Lactobacillus helveticus was found to be mainly controlled by cultivation pH. The kinetics of the two processes were uncoupled at acidic pH. This conclusion has been established by means of a simple plot, under conditions where the nitrogen source was not limiting. During the deceleration phase, the acid production rate remained close to its maximal value at a given cultivation pH, while growth rate decreased notably. Both growth and acid production were inhibited by undissociated lactic acid accumulated in the medium, but the growth rate was shown to be more affected than acid production. For the strain of L helveticus used throughout this work, irrespective of cultivation pH, concentrations of free lactic acid of 8.5 and 13.4 g dm⁻³ resulted in a full inhibition of growth (leading to a stationary phase) and acid production respectively. These findings will be useful to optimize the mean volumetric acid productivity of batch fermentations, both at constant and programmed pH values. © 1999 Society of Chemical Industry