Malic Acid Production Research Articles

Enhanced malic acid production using Aspergillus niger coupled with in situ product recovery

In the present investigation, recovery of malic acid from the fermentation broth was performed by using in situ reactive extraction method employing different combination of amine and solvent systems. Totally six solvent mixtures were tested for toxicity on Aspergillus niger PJR1. Further, effect of solvent mixture concentration on separation of malic acid was investigated. Solvent system consisting of 2 M of trioctylamine in 1-octanol was found to be non-toxic to A. niger PJR1 and resulted in the maximum partition coefficient of 0.75 when the solvent mixture to liquid ratio of 1:2 used. A. niger PJR1 from crude glycerol using in situ reactive batch fermentation resulted in the maximum malic acid titer of 115.67 ± 3.5 g/L with the productivity of 0.53 g/L.h after 216 h. Further, fed batch extractive fermentation with crude glycerol resulted in malic acid titer of 131.48 ± 3.4 g/L with the productivity of 0.45 g/L.h after 288 h. Thus reactive extraction combined with in situ fermentation could become effective method for enhanced malic acid production.

Poly(malic acid) production from liquefied corn starch by simultaneous saccharification and fermentation with a novel isolated Aureobasidium pullulans GXL-1 strain and its techno-economic analysis

In this study, a novel Aureobasidium pullulans GXL-1 strain without melanin secretion was isolated for efficient polymalic acid (PMA) production. The PMA produced by GXL-1 was characterized, and its molecular mass was determined to be 1.621 kDa by gel permeation chromatography. Liquefied corn starch was shown to replace glucose for PMA production by GXL-1 through simultaneous saccharification and fermentation. The PMA titer obtained from batch fermentation was up to 49.0 ± 1.6 g/L in a 10 L fermentor, and the PMA yield and productivity obtained from repeated-batch fermentation were up to 0.50 g/g and 0.34 g/L·h, respectively. Furthermore, process design and techno-economic analysis were performed at an annual output level of 5000 metric tons by SuperPro Designer. Results showed that the production cost of $2.046/kg and payback period of 6.9 years were achieved by repeated-batch fermentation; this provides an economically feasible strategy for industrial-scale production of PMA.

Kinetic Modelling of the Coproduction Process of Fumaric and Malic Acids by Rhizopus arrhizus NRRL 1526

The production of organic acids by biotechnological processes has experienced a notable impulse with the advent of first and second generation biorefineries and the need of searching for renewable and sustainable feedstock, such as biomass. Fumaric acid is a promising biomonomer for polyamide production and a well-known acidulant and preservative in food and feed industries. Malic acid is a well-known food acidulant with a high market share. The biotechnological Fumaric and Malic acid production via fungi of the Rhizopus genus is being explored nowadays as a process for the valorization of food and food-related waste to obtain food ingredients and key platform chemicals of the so-called biochemical biorefinery. In this work, a preliminary study is performed to find reproducible conditions for the production of the acids by Rhizopus arrhizus NRRL 1526 by controlling fungi morphology and inoculum conditions. Afterwards, several production runs are performed to obtain biomass, glucose, and acid concentration data at different processing time values. Finally, an unstructured, unsegregated model including a logistic-type equation for biomass and potential-type equations for the substrate and the products is fitted to experimental data. We find that the production of the organic acids is mainly non-associated with fungal growth.

Enhanced Production of Malic Acid by Co-localization of Phosphoenolpyruvate Carboxylase and Malate Dehydrogenase Using Synthetic Protein Scaffold in Escherichia coli

To produce malic acid from non-oxidative pathway route in Escherichia coli using two key enzymes and synthetic scaffold complex. E. coli was engineered to produce malic acid from glucose by co-localization of two key enzymes phosphoenolpyruvate carboxylase (Ppc) and malate dehydrogenase (MdhA) with synthetic scaffold complex. Scaffold plasmid has produced the maximum concentration of 3.51 g/L malic acid from 10 g/L glucose in 48 h of culture. pH 5.5 and temperature 30°C were optimum for malic acid production without any engineering of competing metabolic pathways. E. coli mutant strains and different concentrations of glucose also tested. When 50 g/L glucose was used as substrate, 20.4 g/L of malic acid was produced.

Biochemical conversion of biodiesel by-product into malic acid: A way towards sustainability

Crude glycerol, one of the ever-growing by-product of biodiesel industry and is receiving the closest review in recent times because direct disposal of crude glycerol may emerge ecological issues. The renewability, bioavailability and typical structure of glycerol, therefore, discover conceivable application in serving the role of carbon and energy source for microbial biosynthesis of high value products. This conceivable arrangement could find exploitation of crude glycerol as a renewable building block for bio-refineries as it is economically as well as environmentally profitable. In this review, we summarize the uptake and catabolism of crude glycerol by different wild and recombinant microorganism. The chemical and biochemical transformation of crude glycerol into high esteem malic acid by various microbial pathways is also additionally discussed. An extensive investigation in the synthesis of high-value malic acid production from various feed stock which finds applications in cosmeceutical and chemical industries, food and beverages, and to some extent in the field of medical science is also likewise studied. Finally, the open doors for unrefined crude glycerol in serving as a promising abundant energy source for malic acid production in near future have been highlighted.

Malic acid production from renewables: a review

AbstractMalic acid derived from fossil resources is currently applied in the food and beverage industries with a medium global production capacity. However, in the transition from a fossil‐based to a bio‐based economy, biotechnologically produced l‐malic acid may become an important platform chemical with many new applications, especially in the field of biopolymers. In this review, currently used petrochemical production routes to dl‐malic acid are outlined and insights into possible bio‐based alternatives for microbial l‐malic acid production are provided. Besides ecological reasons, the possibility to produce enantiopure l‐malic acid by microbial fermentation is the biggest advantage over chemical synthesis. State‐of‐the‐art and open challenges concerning production host engineering, substrate choice and downstream processing are addressed. With regard to production hosts, a literature overview is given covering the leading natural production strains of Aspergillus, Ustilago and Aureobasidium, as well as Escherichia coli as the most important engineered recombinant host. The utilization of renewable substrates as an alternative to glucose is emphasized in particular as a key aspect for a competitive bio‐based production. Out of the alternative substrates discussed in this review, the industrial side‐streams crude glycerol and molasses seem to be most promising for large‐scale l‐malic acid production. © 2019 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

BackgroundLignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered.ResultsUsing the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207.ConclusionsThis is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

BackgroundIn filamentous fungi, transport of organic acids across the mitochondrial membrane is facilitated by active transport via shuttle proteins. These transporters may transfer different organic acids across the membrane while taking others the opposite direction. In Aspergillus niger, accumulation of malate in the cytosol can trigger production of citric acid via the exchange of malate and citrate across the mitochondrial membrane. Several mitochondrial organic acid transporters were recently studied in A. niger showing their effects on organic acid production.ResultsIn this work, we studied another citric acid producing fungus, Aspergillus carbonarius, and identified by genome-mining a putative mitochondrial transporter MtpA, which was not previously studied, that might be involved in production of citric acid. This gene named mtpA encoding a putative oxaloacetate transport protein was expressed constitutively in A. carbonarius based on transcription analysis. To study its role in organic acid production, we disrupted the gene and analyzed its effects on production of citric acid and other organic acids, such as malic acid. In total, 6 transformants with gene mtpA disrupted were obtained and they showed secretion of malic acid at the expense of citric acid production.ConclusionA putative oxaloacetate transporter gene which is potentially involved in organic acid production by A. carbonarius was identified and further investigated on its effects on production of citric acid and malic acid. The mtpA knockout strains obtained produced less citric acid and more malic acid than the wild type, in agreement with our original hypothesis. More extensive studies should be conducted in order to further reveal the mechanism of organic acid transport as mediated by the MtpA transporter.

Khelatisasi Ion Aluminium oleh Asam Organik Eksudat Akar Brachiaria

Aluminum toxicity is one of the major factors inhibiting plant growth in acid soils. Brachiaria grass adapt to high Al concentration. This experiment was conducted to study exudation of low molecular weight organic acids (LMWOA) activated by Al, from Brachiaria roots and its potential in chelating Al. Three Brachiaria species, i.e. B. decumbens, B. ruziziensis and B. brizantha, planted in sterile sand culture and were treated with 5 Al concentrations (0, 100, 200, 300 and 400 μM). After two-month experiment, three kinds of LMWOA, i.e, malic, citric, and oxalic acids, produced by the three Brachiaria-root exudates were measured in the sand culture. The production of malic acid was higher than that of citric and oxalic acid. Those organic acids were influenced by Al concentration; the higher Al concentration the higher organic acid content would be. The organic acids were also proved to form Al-organic compounds effectively of which B. decumbens and B. brizantha were more effective in chelating Al at relatively low Al (100 μM) and at relatively high Al concentration (300 μM and 400 μM), respectively.

Recent advances in microbial production of malic acid from renewable byproducts

In the last few years, ecofriendly malic acid production has received a potential platform for the bio-based chemicals to replace the dependency of fossil based resources. The main goal of this paper is to explore the feasibility of efficient production of malic acid from cost effective alternative renewable byproducts as feedstock. To replace the traditional method of malic acid production from petroleum-based compounds such as maleic acid, the efficiency of fermentation technology for malic acid production using various microorganisms has been improved. To date, glucose is designated as the best substrate for malic acid production. However, few reviews concerning about malic acid production by employing various microbial strains were reported. The current knowledge on the biosynthesis of malic acid has assisted to improve malic acid production using various microbial strains. But, there is still need for the continuous production and replacement of low-cost substrates to increase the yield of malic acid. This review provides an overview about progress, achievements, merits, challenges and future perspectives in malic acid production from cost effective alternative substrates. Thus, malic acid production can be economical using renewable byproducts like crude glycerol by employing appropriate microorganism.

Kinetic Study on the Preparation of Fumaric Acid from Maleic Acid by Batch Noncatalytic Isomerization.

In recent years, the increase in demand for fumaric acid from industry has resulted in an increased need for a high-selectivity process for the conversion of maleic acid to fumaric acid. A highly selective conversion of fumaric acid was achieved without a catalyst by a simple one-step hydrothermal reaction. In addition, the competitive conversion of maleic acid, fumaric acid, and malic acid was first systematically investigated in detail without using a catalyst. The products were characterized by X-ray diffraction and Fourier transform infrared, which demonstrated that the product was fumaric acid. The highly selective conversion of fumaric acid was achieved, and the yield of fumaric acid could reach 92%. Furthermore, a reaction kinetic model was put forward to study the competitive transformation process. The kinetic model predictions were found to agree well with the experimental data. The kinetic parameters were used to explain the changes in the content of every substance at different reaction temperatures and reaction times. In addition, the initial maleic acid concentration in the reaction was also considered as an influencing factor. These results can facilitate the conditional control and product control of industrial processes for the production of fumaric acid or malic acid using latter without a catalyst.

Metabolic response to drought in six winter wheat genotypes.

Wheat is one of the most important cereals, whose growth and development is strongly limited by drought. This study investigated the physiological and metabolic response of six winter wheat cultivars to drought with the emphasis on the induction of dominant metabolites affected by the treatment and genotypes or both. The plants were exposed to a moderate (non-lethal) drought stress, which was induced by withholding watering for six days under controlled greenhouse conditions. A decline in CO2 assimilation (Pn) and transpiration rate, stomata closure, a decrease in relative water content (RWC) and increase of malondialdehyde content were observed in drought-treated plants of all cultivars. These changes were most pronounced in Ellvis, while Soissons was able to retain the higher RWC and Pn. Among the studied metabolites, sugars (sucrose, glucose, fructose, several disaccharides), organic acids (malic acid, oxalic acids), amino acids (proline, threonine, gamma-aminobutyric acid (GABA), glutamine) and sugar alcohols such as myo-inositol accumulated to higher levels in the plants exposed to drought stress in comparison with the control. The accumulation of several metabolites in response to drought differed between the genotypes. Drought induced the production of sucrose, malic acid and oxalic acid, unknown organic acid 1, unknown disaccharide 1, 2 and 3, GABA, L-threonine, glutamic acid in four (Soissons, Žitarka, Antonija or Toborzó) out of six genotypes. In addition, Soissons, which was the most drought tolerant genotype, accumulated the highest amount of unknown disaccharide 5, galactonic and phosphoric acids. The two most drought sensitive cultivars, Srpanjka and Ellvis, demonstrated different metabolic adjustment in response to the stress treatment. Srpanjka responded to drought by increasing the amount of glucose and fructose originated from hydrolyses of sucrose and accumulating unidentified sugar alcohols 1 and 2. In Ellvis, drought caused inhibition of photosynthetic carbon metabolism, as evidence by the decreased Pn, gs, RWC and accumulation levels of sugar metabolites (sucrose, glucose and fructose). The results revealed the differences in metabolic response to drought among the genotypes, which drew attention on metabolites related with general response and on those metabolites which are part of specific response that may play an important role in drought tolerance.

Process optimization and kinetic analysis of malic acid production from crude glycerol using Aspergillus niger

In the present work, optimization of crude glycerol fermentation to produce malic acid by using Aspergillus niger was investigated using response surface methodology and artificial neural network. Kinetic investigation of bioconversion of crude glycerol into malic acid using Aspergillus niger was studied using Monod, Mosser, and Haldane-Andrew models. Crude glycerol concentration, initial pH and yeast extract concentration were found to be significant compounds affecting malic acid production by Aspergillus niger. Both dry cell weight and malic acid titre were found decreased with increase in crude glycerol concentration. Haldane-Andrew model gave the best fit for the production of malic acid from crude glycerol with µmax of 0.1542 h−1. The maximum malic acid production obtained under optimum conditions was 92.64 + 1.54 g/L after 192 h from crude glycerol using Aspergillus niger.

Development and benefit evaluation of fermentation strategies for poly(malic acid) production from malt syrup by Aureobasidium melanogenum GXZ-6

Malt syrup, as a low-cost substrate without any pretreatment, was proved to be able to replace maltose for ploymalic acid (PMA) production by Aureobasidium melanogenum GXZ-6. The PMA titer of 55.53 ± 1.72 g/L was obtained by batch fermentation in a 10-L fermentor with addition of malate, citrate and sodium malonate. Then, a higher PMA titer of 124.07 ± 2.28 g/L was obtained in fed-batch fermentation, which increased by 123.43% than that from batch fermentation. Moreover, repeated-batch fermentation with three batches gave a PMA titer of 64.06 g/L on average with a higher yield of 0.81 g/g and productivity of 0.56 g/L·h. Fermentation process and economics analysis were performed by SuperPro Designer for a 2000 metric tons plant. Results showed that PMA production cost was as low as $ 1.716/kg by fed-batch fermentation, which provides an economical strategy for large-scale PMA production.

Microbial production of dicarboxylic acids from edible plants and milk using GC-MS

ObjectiveThe present experiment was designed to assess the contents of organic acids such as citric acid, succinic acid, fumaric acid, and malic acid in edible food plants and milk under the influence of Eschericia coli.MethodsGas chromatography-mass spectrometry (GC-MS) was used to estimate the contents of organic acids in edible plants and milk. Two microliters of samples was injected into the GC-MS, and the contents of organic acids were computed using standard curves.ResultsMaximum citric content (204 mg/g DW, 24 h E. coli treatment) was observed in tomato followed by papaya (175 mg/g DW). Papaya and grapes (715 and 504 mg/g DW, 24 h E. coli treatment respectively) can be good sources of succinic acid. Malic acid content was highest in E. coli-treated milk (168 mg/g DW). In general, there was a decrease in average citric acid and increase in succinic acid contents in the food sources tested on treatment with E. coli.ConclusionIt was found that among the tested raw food items and milk, with or without E. coli inoculation, tomato and papaya hold a good potential for citric acid production, grapes and papaya for succinic acid, and milk for malic acid production. The study can be a basis for utilization of vegetables, fruits, and milk for the production of dicarboxylic acids to boost the agrarian economy.

A tandem bio-chemo catalytic approach to the sustainable production of malic acid from glucose

A tandem bio-chemo catalytic approach to the sustainable production of malic acid from glucose

Malic acid production from biodiesel derived crude glycerol using morphologically controlled Aspergillus niger in batch fermentation

In the present investigation, the effects of crude glycerol concentration, spore inoculum concentration, yeast extract concentration and shaking frequency on seed morphology of Aspergillus niger PJR1 on malic acid production were investigated and dispersed fungal mycelium with higher biomass (20.25 ± 0.91 g/L) was obtained when A. niger PJR1 grow on crude glycerol. Dry cell weight under dispersed fermentation was 21.28% higher than usual pellet fermentation. The optimal crude glycerol, nitrogen source and nitrogen source concentration were found to be 160 g/L, yeast extract and 1.5 g/L, respectively. Batch fermentation in a shake flask culture containing 160 g/L crude glycerol resulted in the yield of malic acid 83.23 ± 1.86 g/L, after 192 h at 25 °C. Results revealed that morphological control of A. niger is an efficient method for increased malic acid production when crude glycerol derived from biodiesel production is used as feedstock.

Dual Use of a Biopolymer From Durian (Durio zibethinus) Seed as a Nutrient Source and Stabilizer for Spray Dried Lactobacillus Plantarum

The search for natural and sustainable biopolymers is increasing. Biopolymers are used in applications, such as to encapsulate and stabilize probiotic bacteria. Currently, this process requires many steps, including the separation of bacteria from the culture media after fermentation, which is energy intensive. In this study, we developed a strategy to use a natural biopolymer from durian seed, durian seed gum (DSG), to address these issues. DSG was used to serve dual roles, firstly as a nutrient source, and secondly as an encapsulating agent. DSG was used in synergy with reconstituted skim milk (RSM), to be a complete nutrient source, for probiotic bacteria Lactobacillus plantarum (L. plantarum). Usually RSM requires supplementation, with costly yeast extract. DSG was characterized to be a suitable nutrient source as it contains polysaccharides, organic acids, amino acids and fatty acids. In the presence of DSG and RSM, the growth of L. plantarum increased from 8.73 log CFU/mL to 13.86 log CFU/mL, at the end of 72 h. In comparison, when grown with commercial gum arabic (GA) and RSM, growth of L. plantarum reached a lower 11.49 log CFU/mL at 72 h. Metabolomics revealed that several metabolites, including lactose, depleted after fermentation, in the DSG and RSM treatment group, as compared to GA and RSM treatment group. This suggested that glycolysis were up regulated. This correlated with the increased growth, lactic acid, malic acid and fatty acids production by L. plantarum when it was grown on DSG and RSM. Next, the entire culture of L. plantarum with DSG and RSM was taken for spray drying, without the need to separate the bacteria from the culture media. DSG and RSM was able to stabilize L. plantarum, to remain viable at 10 log CFU/g after storage for 10 weeks. On the other hand, the viability was 8.8 log CFU/g when L. plantarum was spray dried with GA and RSM. This study demonstrated an innovative process and low-cost strategy to produce spray dried probiotic powder. DSG could be a potential low cost and sustainable replacement for GA, to develop other functional foods.

Pseudomonas sp. AF-54 containing multiple plant beneficial traits acts as growth enhancer of Helianthus annuus L. under reduced fertilizer input

Plant growth promoting rhizobacteria (PGPR) are capable to increase the growth and yield of crops in eco-friendly and sustainable manner. To evaluate the response of sunflower towards inoculation with PGPR, a sunflower root associated bacterium AF-54 isolated from Diyar Gali Himalayan Mountain region, Azad Jammu and Kashmir (AJK), identified as Pseudomonas sp. by 16S rRNA sequence analysis and was characterized using polyphasic approach. The bacterium produced 23.9 μgmL−1 indole-3-acetic acid in tryptophan-supplemented medium, showed 44.28 nmoles mg-1 protein h−1 nitrogenase activity through acetylene reduction assay and released 48.80 μg mL−1 insoluble phosphorus in Pikovskaya’s broth. During P-solubilization, the pH of the Pikovskaya’s medium decreased from 7 to 3.04 due to the production of acetic acid, malic acid and gluconic acid. Pseudomonas sp. AF-54 showed metabolic versatility by utilizing 79 carbon sources from BIOLOG GN2 plates and resistance to many antibiotics. Furthermore, it inhibited the growth of Fusarium oxysporum in dual culture assay. To evaluate the plant-inoculation response, series of experiments conducted in hydroponic, sterilized soil and fields at AJK, and Faisalabad where inoculated plants with reduced fertilizer showed a significant increase in growth, yield, oil contents and achene NP uptake as compared to non-inoculated control. AF-54 showed extensive root colonization in sterilized and non-sterile conditions documented through yfp-labeling and fluorescent in situ hybridization coupled with confocal laser scanning microscopy. This study concludes that the Pseudomonas sp. strain AF-54 containing multiple plant growth promoting traits can be a potential candidate for biofertilizer production to enhance sunflower crop yield with reduced application of chemical (NP) fertilizers.

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

BackgroundMalate 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.ResultsWe 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 13C-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 Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export.ConclusionsSince more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.