Production of polymalic acid and malic acid byAureobasidium pullulansfermentation and acid hydrolysis (2025)

ARTICLE 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 PMAproducing 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. Fedbatch 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 858C, 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.

Optimization ofL-malic acid production byAspergillus flavus in a stirred fermentor

Biotechnology and Bioengineering, 1991

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fez+ ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C4 acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO3 in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD+-malate dehydrogenase, fumarase, and citrate synthase.

Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export

Applied and Environmental Microbiology, 2008

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox-and ATP-neutral, CO 2 -fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose) ؊1 . A previously engineered glucose-tolerant, C 2 -independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter ؊1 at a malate yield of 0.42 mol (mol glucose) ؊1 . Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13 C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Characterization and Optimization of L-Malic Acid Production by Some Clinical Isolates of Aureobasidium pullulans

Research in Molecular Medicine, 2020

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Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid

Microbial cell factories, 2018

Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δm...

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

Applied Microbiology and Biotechnology, 2013

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.

Sequential Mixed Cultures: From Syngas to Malic Acid

Frontiers in Microbiology, 2016

Synthesis gas (syngas) fermentation using acetogenic bacteria is an approach for production of bulk chemicals like acetate, ethanol, butanol, or 2,3-butandiol avoiding the fuel vs. food debate by using carbon monoxide, carbon dioxide, and hydrogen from gasification of biomass or industrial waste gases. Suffering from energetic limitations, yields of C 4-molecules produced by syngas fermentation are quite low compared with ABE fermentation using sugars as a substrate. On the other hand, fungal production of malic acid has high yields of product per gram metabolized substrate but is currently limited to sugar containing substrates. In this study, it was possible to show that Aspergilus oryzae is able to produce malic acid using acetate as sole carbon source which is a main product of acetogenic syngas fermentation. Bioreactor cultivations were conducted in 2.5 L stirred tank reactors. During the syngas fermentation part of the sequential mixed culture, Clostridium ljungdahlii was grown in modified Tanner medium and sparged with 20 mL/min of artificial syngas mimicking a composition of clean syngas from entrained bed gasification of straw (32.5 vol-% CO, 32.5 vol-% H 2 , 16 vol-% CO 2 , and 19 vol-% N 2) using a microsparger. Syngas consumption was monitored via automated gas chromatographic measurement of the off-gas. For the fungal fermentation part gas sparging was switched to 0.6 L/min of air and a standard sparger. Ammonia content of medium for syngas fermentation was reduced to 0.33 g/L NH 4 Cl to meet the requirements for fungal production of dicarboxylic acids. Malic acid production performance of A. oryzae in organic acid production medium and syngas medium with acetate as sole carbon source was verified and gave Y P/S values of 0.28 g/g and 0.37 g/g respectively. Growth and acetate formation of C. ljungdahlii during syngas fermentation were not affected by the reduced ammonia content and 66 % of the consumed syngas was converted to acetate. The overall conversion of CO and H 2 into malic acid was calculated to be 3.5 g malic acid per mol of consumed syngas or 0.22 g malic acid per gram of syngas.

Microbial organic acids production, biosynthetic mechanism and applications -Mini review

2017

Organic acid constitute a significant portion of the fermentation market in the world, and microbial production is an important economic alternative to chemical synthesis for many of them. Thus, in order to address the growing market demands of organic acids with the passage of time, it needs to develop new strategies or discoveries for new or novel microbial strains for high level production of commercially important organic acid such as; gluconate, malate, and citrate. In present review, through cumulative analysis of the current microbial strains and their biosynthetic mechanisms for production of these acids, we present guidelines for future developments in this fast-moving field.

Key Process Conditions for Production of C 4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

Applied and Environmental Microbiology, 2009

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter −1 of malate at a yield of 0.42 mol (mol glucose) −1 in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO 3 , was required for efficient C 4 dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO 3 dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate ...

l -Malic acid production within a microreactor with surface immobilised fumarase

Microfluidics and Nanofluidics, 2011

In recent years, microreactors have evolved from a technical novelty to a useful tool in many scientific laboratories. On the other hand, their implementation in industry proceeds with a slower pace. In this work, we present a microreactor system for continuous l-malic acid production that demonstrates potentials of microfluidic systems for production of this widely used organic acid. APTES and glutaraldehyde were used to covalently immobilise fumarase on glass microreactor walls in order to allow product–catalyst separation. The system was tested at different substrate concentrations and flow rates and conversion up to 80% could be obtained in appropriate conditions. The reaction was precisely predicted by the developed mathematical model. Kinetic studies were performed with both free and immobilised enzyme and the later was found to retain approximately 25% of free enzyme activity and had the activity half-life of 9 days.