Valorization Of Glycerol Research Articles

Electro-oxidation of waste glycerol to tartronic acid over Pt/CNT nanocatalyst: study of effect of reaction time on product distribution

ABSTRACT In this work, glycerol valorization into tartronic acid – one of the most valuable derivatives of glycerol – was reported over platinum supported on multi-walled carbon nanotubes (Pt/CNT) electrocatalyst. The nanocatalyst was synthesized using a chemical reduction method with hydrazine as a reducing agent. The physical analyses of catalyst were performed by BET, XRD, FESEM, EDX, and TEM. The XRD spectroscopy indicated a face-centered cubic crystalline structure with an average crystallite size of 4.2 nm. The field emission scanning electron microscopy analysis revealed that the particles were uniformly distributed over multi-walled carbon nanotubes, while EDX showed the elemental composition of Pt (17.52%) and C (82.48%). Electrochemical activity and stability of the catalyst were measured using cyclic voltammetry and chronoamperometry techniques. Electrochemical surface area (ECSA, 226.52 m2/g) and mass activity (MA, 5.74 mA/mg) were calculated in 0.5 M H2SO4 and 0.5 M glycerol/0.5 M KOH solution, respectively. The catalytic activity of Pt/CNT nanoparticles was analyzed from glycerol oxidation reaction (GOR), while the products distribution and selectivity were measured using high-performance liquid chromatography (HPLC). It was found that the tartronic acid was produced as a major product with a selectivity of 95.3% while small quantities (<1%) of mesoxalic acid, dihydroxyacetone, and glyceric acid were also produced. The higher activity and selectivity of Pt/CNT can be attributed to high electrochemical surface area and uniform distribution over support.

Cost, energy and GHG emission assessment for microbial biodiesel production through valorization of municipal sludge and crude glycerol.

In this study, cost simulations were made based on 20 million L blended biodiesel B-10 production per year using INRS and conventional process. In case of INRS process, microbial lipid was produced by T. oleaginosus using washed municipal secondary sludge fortified with crude glycerol while lipid was extracted from wet biomass using biodegradable surfactant and petroleum-diesel (PD). The conventional process uses commercial substrates for lipid production and organic solvents for lipid extraction from dry biomass. The unit B-10 production cost of INRS process was estimated to be $ 0.72/L for an annual capacity of 20 million L, which is 9.5 times more economical than conventional biodiesel production process. For INRS process, the unit B-10 biodiesel production cost was sensitive to plant capacity and lipid productivity during the fermentation. INRS process exhibited positive net energy gain and positive GHG capture, which proves to be energetically and environmentally viable.

Glycerol valorization: conversion to lactic acid by heterogeneous catalysis and separation by ion exchange chromatography

AbstractThe consumption and production of biodiesel has grown dramatically in recent decades, motivated by a strong interest in renewable energy. This has generated a surplus of its main by‐product, glycerol. Several catalytic processes have been proposed to generate alternatives for using this excess glycerol, mainly focusing on its transformation into high value‐added chemical products. Various products, ranging from syngas to organic acids, such as acrylic or lactic acid, along with 1,2‐propanediol, 1,3‐propanediol, glycerol ethers or even polymers, have been produced starting from glycerol. Among this multitude of possibilities, the production of lactic acid is one of the most interesting alternatives to valorize glycerol, because of the wide range of applications described for this organic acid. This review focuses on recent studies dealing with the catalytic conversion of glycerol to lactic acid, using different heterogeneous catalysts, and evaluates the factors influencing the oxidation catalytic process and the proposed reaction mechanisms. Finally, recent studies on the separation and purification of lactic acid using ion exchange chromatography resins are also reported. © 2019 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Electrochemical valorization of crude glycerol in alkaline medium for energy conversion using Pd, Au and PdAu nanomaterials

Crude glycerol is a byproduct from biodiesel production, and due to overproduction, new applications requiring the minimum purification steps are needed. The electrochemical valorization of crude glycerol is one of the most attractive applications. In this work, the use of crude glycerol as fuel for energy conversion applications was studied on noble materials such as Pd/C, Au/C and PdAu/C nanoparticles with different Pd/Au atomic composition (1:1, 3:1 and 5:1) with the aim of identifying the main barriers to improve the crude glycerol electro–oxidation reaction (crude GOR). High–resolution transmission electron micrographs revealed that Pd and PdAu nanoparticles had average sizes below 10 nm, while Au nanoparticles had particle sizes of 16.2 ± 1.4 nm. Besides, crude glycerol was obtained from a biodiesel prepared by a transesterification reaction assisted by sonication, resulting in a crude glycerol with a high glycerol content (>95 V/V %). The electrocatalytic evaluation indicated that Pd5Au1/C was the material with the highest current density for crude GOR, displaying 140.1 mA mgPdAu−1, which was 1.8, 2.73, 2.79, 4.14 and 2.2 times higher to that obtained for Pd3Au1/C, Pd/C, Au/C, Pd1Au1/C, and commercial Pd/C, respectively. Moreover, this current density was 20% higher to that obtained for analytical grade glycerol, which was attributed to the bimetallic capability of Pd5Au1 to oxidize both, glycerol and methanol presented in crude glycerol. In summary, it was found that the competition between the adsorption of methanol and glycerol on active sites plays a major role on the activity for crude GOR.

Anaerobic production of valeric acid from crude glycerol via chain elongation

Glycerol from biodiesel production was used as a substrate for valerate production in a 13.1-L anaerobic filter with an open microbiome. Ethanol at 15% of the influent chemical oxygen demand was supplemented as an additional electron donor source. The 114-day experimental period was divided in three phases which included initial adaptation phase, external sludge addition from a caproic acid producing reactor and valerate extraction using a pertraction system. Propionate (0.63–1.75 g COD L−1 day−1) and valerate (0.86–1.81 g COD L−1 day−1) were the main carboxylates formed throughout all operational phases. An increase in production rates of butyrate (0.26–0.31 g COD L−1 day−1), caproate (0.01–0.08 g COD L−1 day−1), and 1,3-propanediol (0.43–0.52 g COD L−1 day−1) was observed with addition of external caproic acid producing sludge rich in Clostridium members. In the operational phase with pertraction, a sudden decrease in 1,3-propanediol and concomitant increase in acid production were verified. Propionate and valerate reached higher production rates compared with the other two phases, suggesting that the pertraction system favored the oxidative pathway of glycerol fermentation. Valerate extraction reached a maximum of 30 g COD m−2 day−1. The filter microbiome was highly diverse with Simpson index values close to 0.1 for the four sludge samples collected, and a concomitant increase in Megasphaera elsdenii and an increase in valerate production rates were observed. Hence, high valerate production and extraction rates through the carboxylate platform are a feasible alternative for crude glycerol valorization with a great potential for improvement in future research.

A review on glycerol reforming processes over Ni-based catalyst for hydrogen and syngas productions

The rapid increase in energy demand coupled with the depletion of fossil-based resources has elevated the need for cleaner, renewable and sustainable fuels. Amongst alternative energies, hydrogen-based energy solution has become a promising replacement candidate due to its clean emission, high efficiency and it is considered to be a perfect substitute to reduce the dependency on non-renewable sources. Recently, the valorization biomass has become one of the attractive routes for hydrogen production and it has received growing attentions from worldwide researchers. Glycerol, the by-product from the biodiesel production faced oversupply crisis due to the high refining cost and this has affected the economics and sustainability of biodiesel industry. Hence, the most attractive way to boost the economic value of biodiesel is through the valorization of crude glycerol into value-added products, i.e., H2 and syngas. Previously, the production of H2 from glycerol has been carried out using various reforming processes such as aqueous phase reforming, pyrolysis, steam reforming, partial oxidation and dry reforming reactions. In the large scale industrial applications, Ni-based catalyst has been reported as the most common catalyst used in reforming reactions since this type of catalyst is readily available, inexpensive and possesses high catalytic activity. Ni was also found to have a good intrinsic activity and easily dispersed over the support materials. Throughout the years, various production routes and catalyst design have been reported in literature; however, none of the literatures are specifically focusing on benefits, constraints, limitation and challenges faced by glycerol reforming reactions catalyzed by Ni-based catalysts. Therefore, the focus of this review is to highlight the recent findings on Ni-based thermochemical processes of glycerol reforming reactions and emphasis will be given on the recent advances in catalyst and reactor designs as well as discovering the main routes of catalyst deactivation.

A Kinetic Study of Glycerol Esterification with Acetic Acid Over a Commercial Amberlyst-35 Ion Exchange Resin

The increased quantities of glycerol available on the market initiated research efforts oriented to new valorization technologies, particularly by its conversion into medium tonnage chemicals, replacing petroleum derivatives. In this work it was investigated the valorization of glycerol by its transformation in glycerol acetates, by direct esterification with acetic acid, over a commercial Amberlyst-35 resin. Experiments were carried out batch-wise, in an autoclave reactor under controlled working conditions, at temperatures between 95 and 112 oC and initial acetic acid to glycerol molar ratios between 4 and 9. The experimental data evidenced that the glycerol conversion to monoacetate is faster than the next esterification steps. A relatively simple kinetic model was proposed and its parameters were evaluated from the experimental measurements. It proved reasonable predicting capacity for products distribution dependencies on the reactants molar ratio and reaction temperature.

Effective Hydrogenolysis of Glycerol to 1,3‐Propanediol over Metal‐Acid Concerted Pt/WOx/Al2O3 Catalysts

AbstractSelective cleavage of secondary C−O bond is an important yet challenging strategy in glycerol valorization, and the product 1,3‐propanediol (1,3‐PDO) is of great value in polyester industry. Herein, we report a series of Pt/WOx/Al2O3 catalysts for selective hydrogenolysis of glycerol in a fixed‐bed reactor and obtain the highest space‐time yield of 1,3‐PDO (191.7*10−3 g1,3‐PDO h−1 g−1cat.) to date. Both Pt and W have substantial effects on the 1,3‐PDO yield with the optimum Pt/W atomic ratio of 1/2∼1/4. Spectroscopy characterizations as well as chemisorption experiments reveal that at the medium domain size of WOx, hydrogen spillover can take place to the greatest extent due to the improved dispersion of Pt and the suitable reducibility of WOx. Dehydration/dehydrogenation tests of 2‐butanol suggest that strong Brønsted acid sites are created via hydrogen dissociation at the Pt−WOx interface and spillover to the neighboring oxygen atom. Such in situ formed protons are critical to the selective cleavage of secondary C−O bonds of polyols.

Reaction of Glycerol with Trimethyl Orthoformate: Towards the Synthesis of New Glycerol Derivatives

The reactivity of glycerol with trimethyl orthoformate is here described with an emphasis on developing a reliable synthetic approach for glycerol valorization. The glycerol based orthoester 4-(dimethoxymethoxy)methyl)-2-methoxy-1,3-dioxolane (3) was synthesized, under catalytic as well as catalyst-free conditions, by taking advantage of the thermodynamically controlled equilibrium between intermediates. Both Brønsted and Lewis acid catalysts accelerated the attainment of such an equilibrium, particularly Brønsted acidic ionic liquids BSMImHSO4 and BSMImBr were the most effective compounds for this reaction. The kinetic profiles allowed the proposal of a mechanism that accounts for the selectivity of the reaction.

Integration of sorption-enhanced steam glycerol reforming with methanation of in-situ removed carbon dioxide - An alternative for glycerol valorization

Coupling CO2 desorption and methanation in the presence of hybrid materials offers a promising alternative to convert the CO2in-situ removed during sorption-enhanced steam glycerol reforming (SESGR) avoiding the high energy-intensive CO2 sorbent regeneration. The all-inclusive integrated process exemplifies an option for glycerol valorization via consecutive SESGR and CO2 conversion by catalytic hydrogenation. The dual-function catalyst performing successively, in the same reactor, SESGR and CO2 desorption/conversion encompasses reforming/methanation catalyst (10%Ni5%Co) and dispersed nano-sized CaO on γ-Al2O3. Simultaneous CO2 desorption/conversion integrated process is explored in a fixed-bed reactor via an unsteady-state, two-scale, non-isothermal model, highlighting the impact of key parameters on the process performance. At large adsorbent/catalyst mass ratio (2.0) and high CaO conversion (0.5) in preceding SESGR, CO2 is released and hydrogenated for an extended period with extra hydrogen consumption, without a balance between CO2 desorption and CO2 hydrogenation rates. Increasing pressure (3.0 MPa) and gas velocity offers a match between these competitive reaction rates, resulting low CO2 concentration in the exit stream. Desorption/methanation thermal behavior controls the magnitude of CO2 low concentration period and the methanation efficiency.

Sulfonated graphenes: Efficient solid acid catalyst for the glycerol valorization

Heterogeneous acid catalysts were obtained based on the functionalization of reduced graphene oxide. The synthesis involves i) the obtaining of graphene oxide by the modified Hummers method ii) the reduction of graphene oxide by three different routes, through the use of hydrazine dihydrochloride, Zn/HCl and ascorbic acid, and iii) the grafting of sulfonic groups in the surface of graphene oxides with 4-diazonium benzenesulfonate. These solids were characterized and evaluated in the etherification of glycerol with tert-butyl alcohol, finding in general glycerol conversions higher than those obtained with a sulfonic resin (Amberlyst® 15). In addition, the selectivity depends on the reduction route used to obtain the catalyst; A larger amount of oxygen groups remaining after the reduction, helps the formation of poly-substituted ethers. These solids also showed stability in their use, converting them into highly efficient catalysts in the valorization of glycerol.

Investigating the Influence of Reaction Conditions and the Properties of Ceria for the Valorisation of Glycerol

The reaction of aqueous glycerol over a series of ceria catalysts is investigated, to produce bio-renewable methanol. Product distributions were greatly influenced by the reaction temperature and catalyst contact time. Glycerol conversion of 21% was achieved for a 50 wt.% glycerol solution, over CeO2 (8 m2 g−1) at 320 °C. The carbon mass balance was &gt;99 % and the main product was hydroxyacetone. In contrast, at 440 °C the conversion and carbon mass balance were &gt;99.9 % and 76 % respectively. Acetaldehyde and methanol were the major products at this higher temperature, as both can be formed from hydroxyacetone. The space-time yield (STY) of methanol at 320 °C and 440 °C was 15.2 and 145 gMeOH kgcat−1 h−1 respectively. Fresh CeO2 was prepared and calcined at different temperatures, the textural properties were determined and their influence on the product distribution at iso-conversion and constant bed surface area was investigated. No obvious differences to the glycerol conversion or product selectivity were noted. Hence, we conclude that the surface area of the CeO2 does not appear to influence the reaction selectivity to methanol and other products formed from the conversion of glycerol.

Efficient valorization of waste glycerol to 2,3-butanediol using Enterobacter cloacae TERI BD 18 as a biocatalyst

This study demonstrates the effective use of a bacterial isolate as a biocatalyst converting wastes to 2,3-butanediol. The developed microbial catalyst Enterobacter cloacae TERI BD 18 was robust as it can grow in wastes with high concentration of impurities showing the potential for developing an integrated biorefinery process for producing 2,3-butanediol. Valorization of rice straw, waste glycerol, molasses, distillery effluent, dairy waste and waste gas was observed. Maximum 2,3-BD production was observed at initial pH 7, temperature 37 °C, agitation speed of 350 rpm and 3% inoculum size. The production of 41.44 g L−1 of 2,3-BD with a yield 0.48 g g−1 was achieved in 13L bioreactor using waste glycerol as a sole source of carbon. An ecofriendly and green method for 2,3-BD production has been proposed by implementing wild type bacterial strain E. cloacae TERI BD 18 as biocatalyst utilizing wastes as feedstock (with no added nutrients) in 13L bioreactor.

Influence of Boron, Tungsten and Molybdenum Modifiers on Zirconia Based Pt Catalyst for Glycerol Valorization.

The influence of boron, tungsten and molybdenum modifiers on zirconia-based Pt catalyst was studied for glycerol valorization. Zirconia modified supports were prepared by impregnation of ZrO2 with either boric, silicontungstic or phosphomolybdic acids to obtain supports with enhanced Brönsted acidic properties. The modified supports were subsequently impregnated with chloroplatinic acid to obtain Pt-based catalysts. Pt incorporation resulted in the increase in Lewis acidity of the solids, being more significant for the Pt//W/ZrO2 catalyst. Reduced Pt catalysts were tested for the liquid-phase glycerol hydrogenolysis, observing a synergistic effect between catalyst acid sites and metal function that proved to be crucial in glycerol hydrogenolysis. The Pt//W/ZrO2 catalyst was the most active catalyst in this reaction, being the only leading to 1,3-PDO (45% sel., 160 °C) while Pt//Mo/ZrO2 is the best option for 1,2-PDO (49% sel., 180 °C). Reusability studies carried out for Pt//W/ZrO2 showed that catalytic activity dropped after the first use, remaining constant for the second and subsequent ones. Selectivity to reaction products also changes during reuses. Therefore, the selectivity to 1,2 PDO increases in the first reuse in detriment to the selectivity to n-propanol whereas the selectivity to 1,3-PDO remains constant along the uses. This behavior could be associated to the lixiviation of W species and/or catalyst fouling during reaction runs.

Aluminum(III) triflate-catalyzed selective oxidation of glycerol to formic acid with hydrogen peroxide

Glycerol is a by-product of biodiesel production and is an important readily available platform chemical. Valorization of glycerol into value-added chemicals has gained immense attention. Herein, we carried out the conversion of glycerol to formic acid and glycolic acid using H2O2 as an oxidant and metal (III) triflate-based catalytic systems. Aluminum(III) triflate was found to be the most efficient catalyst for the selective oxidation of glycerol to formic acid. A correlation between the catalytic activity of the metal cations and their hydrolysis constants (Kh) and water exchange rate constants was observed. At 70 °C, a formic acid yield of up to 72% could be attained within 12 h. The catalyst could be recycled at least five times with a high conversion rate, and hence can also be used for the selective oxidation of other biomass platform molecules. Reaction kinetics and 1H NMR studies showed that the oxidation of glycerol (to formic acid) involved glycerol hydrolysis pathways with glyceric acid and glycolic acid as the main intermediate products. Both the [Al(OH)x]n+ Lewis acid species and CF3SO3H Brønsted acid, which were generated by the in-situ hydrolysis of Al(OTf)3, were responsible for glycerol conversion. The easy availability, high efficiency, and good recyclability of Al(OTf)3 render it suitable for the selective oxidation of glycerol to high value-added products.

Glycerol to Glyceraldehyde Oxidation Reaction Over Pt-Based Catalysts Under Base-Free Conditions.

Glycerol valorization through partial oxidation is a good way of obtaining many different molecules with high added value such as glyceric acid, tartronic acid, dihydroxyacetone, etc. Among the potential products, glyceraldehyde is an interesting chemical compound for its various applications in different domains such as organic chemistry, medical, and cosmetic industries. In the present paper, we studied the effect of different supports on the glycerol oxidation reaction in a batch reactor applying base-free conditions. The tested catalysts were Pt-based materials deposited on various supports (i.e., SiO2, TiO2, ZSM-5, γ-Al2O3), which were synthesized using a deposition method followed by a chemical reduction. The catalysts were extensively characterized (BET, ICP, XRD, TEM, XPS), highlighting differences in terms of specific surface areas, textural properties, and Pt nanoparticles sizes. We evidenced a direct relation between glycerol conversion and glyceraldehyde selectivity (i.e., an increase in glycerol conversion leads to a decrease in glyceraldehyde selectivity). The Pt/γ-Al2O3 catalysts exhibited the highest activity, but their selectivity to glyceraldehyde significantly decreased with time on stream. Pt/SiO2 presented the highest selectivity to glyceraldehyde owing to a slower reaction rate, which allows envisioning technical opportunities to continuously extract the formed glyceraldehyde from the mixture.

Coupling of glycerol-APR and in situ hydrodeoxygenation of fatty acid to produce hydrocarbons

Glycerol valorization by coupling APR to yield hydrogen with hydrogenation of a model compound as tandem reactions is approached using Pt-Ni/Al2O3 as efficient catalyst. Oleic acid is used as a C18 fatty acid model compound for in situ hydrogenation reaction. The combination of the two catalytic transformations to a single tandem process appears particularly interesting since this protocol reduces the number of synthetic steps towards the target compound and avoids external hydrogen supply.Glycerol reforming is carried out in a catalytic fixed bed batch reactor under subcritical water condition between 175 and 275 °C (2–6.21 MPa as autogenous pressure). The coupled reactions were carried out using 1 wt% of catalyst, adding oleic acid to the mixture of water/glycerol and without any external hydrogen supply. In APR, maximum glycerol conversion of 80.3% was achieved using Pt-Ni/Al2O3 catalyst at 200 °C with H2-selectivity close to 80% and CO formation below to 3.7%. In the coupled reactions, no hydrogen is observed in the gas-off, which is associated to the hydrogen consumption by oleic acid at 275 °C; reaching a hydrogenation conversion value close to 91.3% and deoxygenation conversion of 32% giving a C17 paraffinic like-hydrocarbon.

Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species.

SummaryThe use of renewable waste feedstocks is an environment‐friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by‐products. Glycerol (1,2,3‐propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by‐product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole‐cell biocatalysts with both wild‐type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value‐added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost‐effective substrate. The unique genetic and metabolic features of glycerol‐grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate – and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.

Valorization of crude glycerol based on biological processes for accumulation of lipophilic compounds.

Bacteria that are capable of accumulating lipids in their cells as storage compounds can also produce polyhydroxyalkanoates of high technological value, depending on the specific culture conditions. The objective of this study was to utilize crude glycerol from biodiesel (CGB) as a substrate, which is a major byproduct from biodiesel production, to produce lipophilic compounds. Bacillus megaterium INCQS 425 was cultivated and evaluated for the production of lipophilic compounds and the properties of these compounds were investigated. Cultivation of the bacteria in a medium with a C:N ratio of 0.60:1 favored the accumulation of lipids by (17.5%) comprising mainly palmitic acid (13.08%), palmitoleic (39.48%), and especially oleic acid (37.02%), which imparts good characteristics to biodiesel. Meanwhile, cultivation of the bacteria in a medium with a C:N ratio of 4:1 favored the accumulation of polyhydroxyalkanoates (PHA) (3.31gL-1) mainly comprising medium and long chain PHA. Low crystallinity (<30%) and excellent thermal properties make them suitable for processes that demand high temperatures, such as extrusion. The lipids produced in the present study had satisfactory oxidative stability for the production of quality biodiesel. The polyhydroxyalkanoates produced in the study are of low cost and have promising thermal properties that justify its technological potential, thereby configuring highly competitive bioproducts.

13C metabolic flux analysis-guided metabolic engineering of Escherichia coli for improved acetol production from glycerol

BackgroundBioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. 13C metabolic flux analysis (13C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied 13C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by Escherichia coli. Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.ResultsWe performed 13C-MFA and measured intracellular pyridine nucleotide pools in a first-generation acetol producer strain (HJ06) and a non-producer strain (HJ06C), and identified that engineering the NADPH regeneration is a promising target. Based on this finding, we overexpressed nadK encoding NAD kinase or pntAB encoding membrane-bound transhydrogenase either individually or in combination with HJ06, obtaining HJ06N, HJ06P and HJ06PN. The step-wise approach resulted in increasing the acetol titer from 0.91 g/L (HJ06) to 2.81 g/L (HJ06PN). To systematically characterize and the effect of mutation(s) on the metabolism, we also examined the metabolomics and transcriptional levels of key genes in four strains. The pool sizes of NADPH, NADP+ and the NADPH/NADP+ ratio were progressively increased from HJ06 to HJ06PN, demonstrating that the sufficient NADPH supply is critical for acetol production. Flux distribution was optimized towards acetol formation from HJ06 to HJ06PN: (1) The carbon partitioning at the DHAP node directed gradually more carbon from the lower glycolytic pathway through the acetol biosynthetic pathway; (2) The transhydrogenation flux was constantly increased. In addition, 13C-MFA showed the rigidity of upper glycolytic pathway, PP pathway and the TCA cycle to support growth. The flux patterns were supported by most metabolomics data and gene expression profiles.ConclusionsThis research demonstrated how 13C-MFA can be applied to drive the cycles of design, build, test and learn implementation for strain development. This succeeding engineering strategy can also be applicable for rational design of other microbial cell factories.