Malic Acid Production Research Articles

Changes in metabolites in maize seedlings under chlorsulfuron and cadmium stress

SUMMARYThe herbicide chlorsulfuron is commonly applied to cereals and may persist in alkaline soil long enough to damage subsequent sensitive crops. Sewage water, a potential source of the heavy metal cadmium (Cd), is used to irrigate agricultural soils in many developing countries. The current work evaluated the effects of the residual herbicide chlorsulfuron and Cd on maize seedlings, with particular attention to the mechanism of their action on plant growth. Maize seeds were planted in soil that had been sprayed with chlorsulfuron and Cd, after which residues in both seedlings and the soil were measured. The chlorsulfuron dose was correlated with the amount of residue found in seedlings but not in the soil. In all, 39 metabolites were identified in seedlings using the Automated Mass spectral Deconvolution and Identification System software program and the retention index method. The combination of chlorsulfuron and Cd significantly reduced multiple metabolites in the shikimate pathway, malic acid and citric acid production in the tricarboxylic acid cycle, and lactic acid, glucose, aspartic acid, asparagine and 3-glycerophosphoric acid production. In addition, chlorsulfuron caused an increase in multiple amino acids, including tyrosine, methionine and asparagine, and a marked decrease in caffeic and cinnamic acids (the secondary metabolites derived from the shikimate pathway and galactose). Finally, chlorsulfuron and Cd stress markedly increased shikimate acid, decreased 3-glycerophosphoric acid and caused negative correlations between the amounts of phenylalanine and tyrosine and those of quinic- and cinnamic-acid. In conclusion, chlorsulfuron and Cd did not have a synergistic effect on maize seedlings; rather, the combination of these pollutants had an antagonistic effect on some amino acids.

A comparative evaluation of different types of microbial electrolysis desalination cells for malic acid production

The aim of this study was to investigate different microbial electrolysis desalination cells for malic acid production. The systems included microbial electrolysis desalination and chemical-production cell (MEDCC), microbial electrolysis desalination cell (MEDC) with bipolar membrane and anion exchange membrane (BP-A MEDC), MEDC with bipolar membrane and cation exchange membrane (BP-C MEDC), and modified microbial desalination cell (M-MDC). The microbial electrolysis desalination cells performed differently in terms of malic acid production and energy consumption. The MEDCC performed best with the highest malic acid production rate (18.4±0.6mmol/Lh) and the lowest energy consumption (0.35±0.14kWh/kg). The best performance of MEDCC was attributable to the neutral pH condition in the anode chamber, the lowest internal resistance, and the highest Geobacter percentage of the anode biofilm population among all the reactors.

Fungal biotransformation of crude glycerol into malic acid.

Malic acid production from the biodiesel coproduct crude glycerol by Aspergillus niger ATCC 9142, ATCC 10577 and ATCC 12846 was observed to occur with the highest malic acid level acid being produced by A. niger ATCC 12846. Fungal biomass production from crude glycerol was similar, but ATCC 10577 produced the highest biomass. Fungal biotransformation of crude glycerol into the commercially valuable organic acid malic acid appeared feasible.

Adaptation and transcriptome analysis of Aureobasidium pullulans in corncob hydrolysate for increased inhibitor tolerance to malic acid production.

Malic acid is a dicarboxylic acid widely used in the food industry, and is also a potential C4 platform chemical. Corncob is a low-cost renewable feedstock from agricultural industry. However, side-reaction products (furfural, 5-hydroxymethylfurfural (HMF), formic acid, and acetic acid) that severely hinder fermentation are formed during corncob pretreatment. The process for producing malic acid from a hydrolysate of corncob was investigated with a polymalic acid (PMA)-producing Aureobasidium pullulans strain. Under the optimal hydrolysate sugar concentration 110 g/L, A. pullulans was further adapted in an aerobic fibrous bed bioreactor (AFBB) by gradually increasing the sugar concentration of hydrolysate. After nine batches of fermentation, the production and productivity of malic acid reached 38.6 g/L and 0.4 g/L h, respectively, which was higher than that in the first batch (27.6 g/L and 0.29 g/L h, respectively). The adapted strain could grow under the stress of 0.5 g/L furfural, 3 g/L HMF, 2g/L acetic acid, and 0.5 g/L formic acid, whereas the wild type did not. Transcriptome analysis revealed that the differentially expressed genes were related to carbohydrate transport and metabolism, lipid transport and metabolism, signal transduction mechanism, redox metabolism, and energy production and conversion under 0.5 g/L furfural and 3 g/L HMF stress conditions. In total, 42 genes in the adapted strain were upregulated by 15-fold or more, and qRT-PCR also confirmed that the expression levels of key genes (i.e. SIR, GSS, CYS, and GSR) involved in sulfur assimilation pathway were upregulated by over 10-fold in adapted strain for cellular protection against oxidative stress.

Catalytic hydrothermal conversion of macroalgae-derived alginate: effect of pH on production of furfural and valuable organic acids under subcritical water conditions

The hydrothermal decomposition of sodium alginate was analyzed in subcritical water as a function of pH in order to investigate the effects of catalysts on the production of value-added chemicals. A base-catalysed reaction at pH 13 promoted the decomposition of alginate, resulting in the production of lactic acid, fumaric acid and malic acid as major species. At pH 1, monomers (mannuronic acid and guluronic acid), furfural and glycolic acid were predominantly produced by the acid-catalysed hydrothermal decomposition of alginate. Increasing the reaction temperature enhanced both the acid- and base-catalysed reactions, albeit by varying degrees dependent upon the catalyst type. Our results demonstrate that optimizing the reaction temperature and pH is critical for the efficient conversion of seaweed-derived biomass into valuable products.

Malic acid production using a biological electrodialysis with bipolar membrane

Malic acid is a valuable organic acid and important acidulant in food and pharmaceutical industries and can be produced using electrodialysis with bipolar membrane (EDBM). The aim of this study was to investigate the possibility of malic acid production using a system integrating EDBM and biochemical process, i.e., the microbial electrodialysis and chemical-production cell (MEDCC). With an applied voltage of 1.0V, the MEDCC successfully conversed 0.3M malate into 0.23M malic acid, which was about four times as that produced in the EDBM (0.06M). The maximum current density in the MEDCC was five times higher than that in the EDBM (10 vs. 1.9A/m2). The specific electric consumption for the malic acid production in the MEDCC was 0.34kWh/kg, which was only 10–30% of that in the reported EDBMs. In the MEDCC, the energy from the anode biodegradation provided about 50% of the total energy demand (0.68kWh/kg) during the malic acid production. Our results show that the MEDCC should be a promising method for organic acid production with advantages of lower electrical consumption and diverse substrates utilized by exoelectrogens.

Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates of Saccharomyces cerevisiae sake strains.

Research on the relationship between mitochondrial membrane potential and fermentation profile is being intensely pursued because of the potential for developing advanced fermentation technologies. In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential correlate with malic acid production. To define the underlying biochemical mechanism, we determined the activities of enzymes required for malic acid synthesis and found that pyruvate carboxylase and malate dehydrogenase activities in strains that produce high levels of malic acid were elevated compared with the standard sake strain K901. These results inspired us to hypothesize that decreased mitochondrial membrane potential was responsible for increased malic acid synthesis, and we present data supporting this hypothesis. Thus, the mitochondrial membrane potential of high malic acid producers was lower compared with standard strains. We conclude that mitochondrial membrane potential correlates with malic acid production.

High levels of malic acid production by the bioconversion of corn straw hydrolyte using an isolated Rhizopus delemar strain

The microbial fermentation of malic acid, which is one of the most important organic acid platforms used widely in food and chemical engineering, has attracted considerable interest. A malate production strain was isolated, a mutation was induced, and regulation of the metabolic network was then conducted. The identification results showed that the malic acid production strain, HF- 119, belonged to Rhizopus delemar. An analysis of the metabolic pathway showed that the malic acid flux of this strain occurred through three main pathways, and many byproducts, such as succinic acid, fumaric acid and ethanol, were produced. Although corn straw hydrolyte was used, the metabolism of xylose was not as rapid as that of glucose. Subsequently, breeding of the strains and regulation of the metabolic network resulted in an increase in malate yield, and the strain HF-121 produced more than 120 g/L malic acid within 60 h. The ability to produce malic acid from biomass hydrolyte highlights the industrial development potential of this strain.

Cofactor and CO2 donor regulation involved in reductive routes for polymalic acid production by Aureobasidium pullulans CCTCC M2012223

Polymalic acid (PMA) is a water-soluble polyester with many attractive properties for biomedical application. Its monomer L-malic acid is widely used in the food industry and also a potential C4 platform chemical. Cofactor and CO2 donor involved in the reductive routes were investigated for PMA production by Aureobasidium pullulans. Biotin as the key cofactor of pyruvate carboxylase was favor for the PMA biosynthesis. Na2CO3 as CO2 donor can obviously improved PMA titer when compared with no CO2 supplier NaOH, and also exhibit more advantages than the other donor CaCO3 because of its water-soluble characteristic. A combinational process with addition of biotin 70mg/L and Na2CO3 as the CO2 donor was scaled-up in 50L fermentor, achieving the high product 34.3g/L of PMA and productivity of 0.41g/Lh. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application.

Process characterization and influence of alternative carbon sources and carbon-to-nitrogen ratio on organic acid production by Aspergillus oryzae DSM1863

L-Malic acid and fumaric acid are C4 dicarboxylic organic acids and considered as promising chemical building blocks. They can be applied as food preservatives and acidulants in rust removal and as polymerization starter units. Molds of the genus Aspergillus are able to produce malic acid in large quantities from glucose and other carbon sources. In order to enhance the production potential of Aspergillus oryzae DSM 1863, production and consumption rates in an established bioreactor batch-process based on glucose were determined. At 35 °C, up to 42 g/L malic acid was produced in a 168-h batch process with fumaric acid as a by-product. In prolonged shaking flask experiments (353 h), the suitability of the alternative carbon sources xylose and glycerol at a carbon-to-nitrogen (C/N) ratio of 200:1 and the influence of different C/N ratios in glucose cultivations were tested. When using glucose, 58.2 g/L malic acid and 4.2 g/L fumaric acid were produced. When applying xylose or glycerol, both organic acids are produced but the formation of malic acid decreased to 45.4 and 39.4 g/L, respectively. Whereas the fumaric acid concentration was not significantly altered when cultivating with xylose (4.5 g/L), it is clearly enhanced by using glycerol (9.3 g/L). When using glucose as a carbon source, an increase or decrease of the C/N ratio did not influence malic acid production but had an enormous influence on fumaric acid production. The highest fumaric acid concentrations were determined at the highest C/N ratio (300:1, 8.44 g/L) and lowest at the lowest C/N ratio (100:1, 0.7 g/L).

Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68

Malic acid is a C₄ dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C₄ dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))⁻¹ h⁻¹ and with a yield of 1.49 mol mol⁻¹. Intracellular fluxes were obtained using ¹³C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.

Influence of culturation method on enzymes concerned with the production of malic acid of yeasts

Influence of culturation method on enzymes concerned with the production of malic acid of yeasts

Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid

Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.

Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions

Malic acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity malic acid producers like the malic acid producer Aspergillus flavus have so far been disqualified because of special growth requirements or the production of mycotoxins. As A. oryzae is a very close relative or even an ecotype of A. flavus, it is likely that its high malic acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the malic acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on malic acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 ± 1.05 g liter(-1), using peptone than the one of 22.27 ± 0.46 g liter(-1) obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of the Saccharomyces cerevisiae yeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic malic acid production pathway from pyruvate via oxaloacetate to malate, which suggests that malic acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.

Isolation of a high malic and low acetic acid-producing sake yeast Saccharomyces cerevisiae strain screened from respiratory inhibitor 2,4-dinitrophenol (DNP)-resistant strains

We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+).

Efficient production of polymalic acid from raw sweet potato hydrolysate with immobilized cells of Aureobasidium pullulans CCTCC M2012223 in aerobic fibrous bed bioreactor

AbstractBACKGROUNDPolymalic acid (PMA) is a water‐soluble polyester with many attractive properties for biomedical application. Its monomer L‐malic acid is widely used in the food industry and is also a potential C4 platform chemical. Hydrolysate of raw sweet potato as a low‐cost renewable feedstock was applied for the production of PMA by immobilized Aureobasidium pullulans cells in an aerobic fibrous bed bioreactor system (AFBB).RESULTSUnder the initial hydrolysate sugar concentration 120 g L‐1, yeast extract 5 g L‐1, and citrate 7.5 g L‐1, PMA production in a shake flask was improved. Comparison of the kinetics of fed‐batch immobilized‐cell fermentation using the AFBB with those of batch or fed‐batch free‐cell fermentation in a 5 L fermentor showed that immobilized‐cell fermentation gave much higher PMA production and productivity. The maximum PMA production and productivity in AFBB were 57.5 g L‐1 and 0.34 g L‐1 h, which was 30.6% and 21.4% higher than that in free‐cell mode.CONCLUSIONRaw sweet potato is an efficient renewable feedstock for PMA fermentation. This present work demonstrated the great potential of AFBB and renewable feedstocks for the economical production of PMA and malic acid at industrial scale. © 2013 Society of Chemical Industry

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end-product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA-producing Aureobasidium pullulans strain ZX-10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high-titer equivalent to 87.6 g/L of malic acid and high-productivity of 0.61 g/L h in free-cell fermentation in a stirred-tank bioreactor. Fed-batch fermentations with cells immobilized in a fibrous-bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA-900 anion-exchange resins, achieving a ∼100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first-order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application.

Alteration of Media Composition and Light Conditions Change Morphology, Metabolic Profile, and Beauvericin Biosynthesis in Cordyceps bassiana Mycelium

Metabolic alterations of Cordyceps bassiana mycelium were investigated under the following culture medium and light conditions: dextrose agar supplemented with 0.5% yeast extract (SDAY) medium with light (SL), SDAY medium without light (SD), nut medium without light (ND), and iron-supplemented SDAY medium without light (FD). The levels of asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, ornithine, and proline were significantly higher under SD and SL conditions. The levels of most of the alcohols, saturated fatty acids, unsaturated fatty acids, fatty acid esters, sterols, and terpenes were higher under the ND condition than in the other conditions, but beauvericin was not detectable under the ND condition. The FD condition was favorable for the enhanced production of aminomalonic acid, malic acid, mannonic acid, and erythritol. Thus, the metabolic characteristics of C. bassiana can be manipulated by varying the cultivation conditions, rendering this fungus potentially favorable as a nutraceutical and medicinal resource.

Control strategy of pH, dissolved oxygen concentration and stirring speed for enhancing β‐poly (malic acid) production by Aureobasidium pullulans ipe‐1

AbstractBACKGROUND: β‐poly(malic acid) (PMLA) can be used as a pro‐drug or for a drug‐delivery system. Effects of pH, dissolved oxygen concentration (DO) and stirring speed were investigated to improve PMLA production by A. pullulans ipe‐1.RESULTS: The strain produced a high PMLA concentration when pH and DO remained at about 6.0 and above 70%, respectively, and the yeast‐like cells were the main PMLA producers. To further promote PMLA production, the cultivation could be divided into three phases. In phase I, cell growth was accelerated by maintaining high DO (>70%) with a constant stirring speed of 800 rpm. In phase II, PMLA production was increased by controlling DO at 70% using the automatically controlled stirring speed. In phase III, PMLA production on per gram of glucose (Yp/s) was enhanced by keeping DO at 70%, and using a low stirring speed to decrease cell growth. Compared with batch cultures, a higher PMLA yield was obtained with this strategy, i.e. PMLA production and Yp/s increased by 15% and 18%, respectively.CONCLUSION: Control strategies for pH, DO and stirring speed provide a good reference for process development and optimization of PMLA production. © 2012 Society of Chemical Industry

Intensification of β-poly(l-malic acid) production by Aureobasidium pullulans ipe-1 in the late exponential growth phase

β-Poly(malic acid) (PMLA) has attracted industrial interest because this polyester can be used as a prodrug or for drug delivery systems. In PMLA production by Aureobasidium pullulans ipe-1, it was found that PLMA production was associated with cell growth in the early exponential growth phase and dissociated from cell growth in the late exponential growth phase. To enhance PMLA production in the late phase, different fermentation modes and strategies for controlling culture redox potential (CRP) were studied. The results showed that high concentrations of produced PMLA (above 40 g/l) not only inhibited PMLA production, but also was detrimental to cell growth. Moreover, when CRP increased from 57 to 100 mV in the late exponential growth phase, the lack of reducing power in the broth also decreased PMLA productivity. PMLA productivity could be enhanced by repeated-batch culture to maintain cell growth in the exponential growth phase, or by cell-recycle culture with membrane to remove the produced PMLA, or by maintaining CRP below 70 mV no matter which kind of fermentation mode was adopted. Repeated-batch culture afforded a high PMLA concentration (up to 63.2 g/l) with a productivity of 1.15 g l(-1) h(-1). Cell-recycle culture also confirmed that PMLA production by the strain ipe-1 was associated with cell growth.