Biotechnology of Citric Acid Production PDF

Biotechnology of Citric Acid Dr. Production Joham Sarfraz Ali. Food Biotechnology “ BTE-682”  Introduction Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid) is a tricarboxylic acid. It is naturally found in fruits such as lemon, orange, pi...

Biotechnology of Citric Acid Dr. Production Joham Sarfraz Ali. Food Biotechnology “ BTE-682”  Introduction Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid) is a tricarboxylic acid. It is naturally found in fruits such as lemon, orange, pineapple, plum, and pear; in the seeds of different vegetables; and in animal bone, muscle, and blood. Citric acid produced from fruits is known as “natural citric acid,”. In 1893 Wehmer investigated citric acid production from sucrose with strains of Mucor and Penicillium and this experiment was not successful. Difficulties included the selection of proper microorganisms, microbial degeneration, contamination, long fermentation time, high plant construction cost, and an insufficient spread between raw material cost and the price of finished product In 1917 Currie described the first production of citric acid with Aspergillus niger. He reported that different strains of A. niger, grown in a low pH medium supplemented with 15% (w/v) sucrose plus nutrients, converted 55% of the sugar to citric acid The anhydrous form is produced at a temperature exceeding 36.6°C while the monohydrate is produced at temperatures of up to 36.0°C. The current annual world production of citric acid among 35 countries is  Uses of citric acid Uses of citric acids Food Industry It is also used for metal cleaning, as an acidulant in soft drinks and confectionary, and can serve as a buffer and pH stabilizer, as well as a fat antioxidant in various food products. In the food industry it is used as a preservative in that it prevents color and flavor deterioration, inhibits oxidation, protects ascorbic acid from oxidation, inactivates oxidative enzymes, acts as an antioxidant, and is an emulsifier in dairy products. Citric acid is used in the preparation of soft drinks, desserts, jams, jellies, candies, wines, and frozen fruits. It also provides a tart taste in soft drinks. Pharmaceutical Industry In the pharmaceutical industry, it is used in syrups, astringents, and effervescent tablets and powders. It is also used in blood transfusions. Chemical Industry In the chemical industry, its uses include water conditioning, metal Food and pickling, and used as a foam inhibitor for vinyl sheeting and polyester resin. Also, it is used as a nontoxic plasticizer to make plastic film. beverages Citric acid can be used to adjust pH levels that are neutral or high, thus Pharmaceuticals permitting its wide application in industry in areas such as electroplating, leather tanning, and reactivation of oil wells where pores Other industries of the sand face have become clogged with iron. and applications Detergent industry Finally, citric acid is used in the detergent industry to replace phosphates and in the removal of sulfur in stack gases and other Microorganisms used for the production of citric Fungi acid Citric acids production stability is difficult to maintain because of spontaneous mutation and autolysis of the strains during fermentation. Soluti Long term on These problems can be avoided by periodic reisolation from Produce stabilit y at lowspores, single temperature storage(4.0–7.0°C), and avoidance of high High concent of “foggy” patches sterile rati on sporulat ion mycelium (fungal mat lacking spores) when mass transfers of citric acid are made Strain propert Other species of Aspergillus which have been found to accumulate Fungal species citric acid include strains of A. awamori, A. fenicis, A. ies Resistan fonsecaeus, A. luchensis, A. fumaricus, A. wentii, ce to Good growth A. saitoi, A. usami, A. phoenicus, A. lanosus, A. foetidus, and A. other microorg and flavus as well as ani sm substrat e some strains of Penicillium such as P. janthinellum, P. Short ferment simplicissimum, Today, , and acid almost all citric P. restrictum. produced by fermentation is atio n time manufactured by strains of A. niger in submerged culture.  Yea st Although A. niger is the traditional producer of citric acid, in the past 30 years researchers have been attracted to the use of yeasts as citric acid producers Higher The variety of yeasts that produce citric acid belongs to the producti genus Candida, Saccharomyces, Hansenula, Pichia, vi ty Trichosporon, Torula, Rhodotorula, Sporobolomyces, Debaryomyces, Nematospora. Torulopsis, Kloeckera,  Saccharomyces, and Candida are widely used for the Short production of citric acid. Metal ferment Advantages Yeasts have some advantages compared to A. ions ati on toleran time nigerTheir 1. strains. capabilities can lead to significant reductions in Advantages of ce than substrate and waste treatment costs and product recovery yeast fungi costs. 2. The advantages of using n-alkanes over carbohydrates are potentially lower costs for substrates and higher citric Disadvantages acid concentrations. Insensit The major disadvantage of using yeasts is the production of Contam ive to isocitric acid during fermentation. The amount of isocitric ina tion molass acid produced depends on the yeast strains used, the chemical tolerant es composition of the substrates, the fermentation system, and variatio generally the conditions under which fermentation takes place. To overcome this problem, some methods have been developed to ns reduce the amount of isocitric acid produced.  Yea st Citric acid production on the laboratory scale: Citric acid production by yeasts in the laboratory employs two phases:  A preliminary growth phase on a complete medium.  A production phase without nitrogen sources. In some cases, the medium is supplemented with limited amounts of nitrogen (1.0 g/L) to keep cellular activity at an acceptable level. Citric acid production on an industrial scale:  The exponential growth phase,  The citrate production lag phase.  The linear production phase. The production phase is connected to a preliminary reduction in intracellular nitrogen content. Fermentation runs from 3 to 6 days, with pH controlled at 4.5– 6.5. Citric acid concentrations varied from 20 to 60 g/L, depending on the strain used, the substrate, the fermentation system, and the Bacteri Bacterial fermentation is a: aerobic at 30–37°C for 2–5 days, depending on the strain and the composition of medium used. Generally, citric acid production by bacteria was 50–100% lower than that by fungi or yeasts Little information is available on the production of citrate by bacteria, as most of the references in the literature are to patents. Bacteria generally include Bacillus, Brevibacterium, Arthrobacter, Corynebacterium, Klebsiella, Aerobacter, Pseudomonas, and Micrococcus. Among these, B. subtilis, B. licheniformis, B. flavum, and A. paraffinens are the most promising. B. licheniformis grown in medium containing glucose, urea, calcium carbonate, and ammonium sulfate or glutamate (pH 7.0) produced 42.0 g/L of citric acid. A. paraffinens, Corynebacterium sp., and Bacillus  BIOSYNTHESIS OF CITRIC Citric acid ACID biosynthesis Embden–Meyerhof– involves both the Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle.Citric acid is formed by condensation of acetyl-coenzyme A with oxaloacetic acid. Because A. niger produces both invertase and hexokinase (HXK), which convert sucrose to fructose-6-phosphate, sucrose can be used as a source for the production of citric acid. Verhoff and Spradlin reported that only 10% of the glucose is available for the production of mycelia and CO2, both of which occur primarily during the growth phase early in fermentation. Thus, practically all the glucose entering into the cell during the citric acid production phase must be converted to citric acid. The key enzymes that were responsible for the biosynthesis of citric acid from sucrose by A. Biosynthe sis of citric acid: Citric Acid Production Pathway in Yeasts (N- Alkanes) FACTORS AFFECTING CITRIC ACID PRODUCTION Nutrients The composition of the media used for the production of citric acid by A. niger depends on the strain of the microorganism used and the type of process. Generally, strains that can use one carbon source efficiently fail to show good acid production when cultured in a medium containing another. Aspergillus niger grows well in media containing Carbohydrates (sucrose, glucose, fructose, maltose, mannose, and starch) Nitrogen (as ammonium or nitrate ions), Phosphate, Low amounts of potassium, Magnesium, Sulfate  Carbohydra It has been tes found that citric acid yields are much higher when A. niger strains are grown in simple synthetic media rather than in complex media. The initial sugar concentration plays an important role. The highest citric acid concentrations were observed in cultures grown at high initial sugar concentrations (15−20% w/v). Further increase of sugar concentration (i.e., 250 g/L)) resulted in a decrease of acid concentration by 15%. The decreased concentration of acid encountered with the highest concentration treatment was probably due to osmotic effects. The source of carbohydrate has been shown to have a marked effect on citric acid production by A. niger. Xu et al. found that sucrose and maltose were better carbon sources for citric acid production by A. niger than glucose and fructose. Enzyme activities, such as pyruvate carboxylase, 2- oxoglutarate dehydrogenase, and ACH (aconitase), play a crucial role in citric acid production. The activity of these enzymes varies with the sugar source.  Nitrogen and Phosphate concentrations In addition to carbohydrates, nitrogen and the phosphate concentrations have a strong influence on citric acid production. Generally, a nitrogen or phosphate concentration less than 0.2% (w/v) in the medium appears to be adequate. High concentrations of nitrogen (0.8 g/L) resulted in a reduction by 100% in citric acid production Amino acids and vitamins In addition to the above nutrients, A. niger needs amino acids and vitamins for growth and for production of citric acid. Lal and Srivastava studied the effect of amino acids on citric acid production by A. niger. They found that the presence of glutamic acid and aspartic acid stimulated citric acid production to the extent of 79.6% and 76.7%, respectively. Lysine was effective in increasing yield by 62%.However, serine could not influence the yield (50.4%) to a greater extent, while the effect of cysteine was found to be detrimental. Hamissa et al. (34), however, found that the addition of certain amino acids to the medium completely inhibited citric acid biosynthesis by C. lipolytica Y 1095. When the medium was supplied with vitamins such as thiamine, nicotinic acid, and nicotinamide, concentration of citric acid increased. The optimum concentration of thiamine favoring Trace the yield was 6.0 mg/L. elements Play a significant role. High concentrations of trace metals (5.0 mg/L) decrease concentration of citric acid while low concentrations (1.0 mg/L) improve production of the acid. Heavy Metals The effect of heavy metals, such as copper and cadmium, on citric acid production varies with the strain used and the composition of the medium. In some cases, the addition of certain metals may enhance citric acid Micronutrients production. Micronutrients like FeSO4 7H2O and MnSO4 4H2O are found to be more suitable for citric acid production in certain strains.  Inhibitors: Purpose: Inhibitors are added to the medium to accumulate enhance thea productionmetabolic of a normally metabolized. specific product intermediate that or is Effectiveness: Inhibitors are generally Inhibitors increasing effective in the product and reducing concentration of the the yield and related desiredof undesired stimulant products. s Stimulants: Chemical substances that increase the concentration of the product when added to the medium. The most important stimulants used for improving citric acid yield by A. niger are methanol and ethanol. Other Inhibitors and Stimulants: 1.Addition of inhibitors such as calcium fluoride, sodium fluoride, potassium fluoride, hydrogen peroxide, naphthaquinone, methylene blue, sodium malonate, potassium ferricyanide, iodoacetate, sodium azide, and sodium arsenate increased citric acid concentration. 2.Cyanide (CN)-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathways, catalyzed by the alternative oxidase (AOX), contribute to citric acid production. Overall Impact: The addition of inhibitors and stimulants can significantly impact citric acid production by influencing the metabolism of sugars and key enzymes in the TCA  To prepare inoculum,  Growth Medium: A. niger is grown in standard media for molds.  Cultivation Conditions: It is on potato usually dextrose agar (PDA) slants cultivated dishes ,at 28–30°C for 3– or in petri  5Spore days. Suspension: The spores suspended obtained in sterile are Tween Inoculum water  80. containing 0.1% Inoculation Methods: Inoculation is using spores carried out of A. niger or pregrown  mycelia. Submerged Fermentation for mycelia Mycelia: When pellets are used, submerged they are grown fermentation in medium that for 2–3has the same days in production composition as the medium.  Fermentation Time The optimum time for the maximum production of citric acid depends on  the strain used,  the chemical composition of the medium,  the fermentation system  the conditions under which fermentation takes place. In the surface culture, fermentation time is usually completed in 10–20 days, while in the submerged culture incubation time is much shorter (5–10 days). In solid-state fermentation the fermentation time depends strongly on the amount of inoculum used,  The moisture content of the substrate,  The initial ph,  The temperature,  And the particle size of the medium. Temperatur e: niger and other A. used in citric acid production have an optimum temperature between 25 and 30°Cfungi in submerged fermentation using synthetic media or molasses. At temperatures above 34°C, the maintenance coefficient decreases, reflecting a deactivation effect of temperature on endogenous metabolism. PH: The initial pH of the fermentation medium plays a crucial role in the biosynthesis of citric acid. For A. niger in synthetic media, the pH is typically adjusted to 2.5–3.5, while for molasses-based mediums, a neutral to slightly acidic initial pH is essential for microorganism germination and growth. Adjustment of pH is carried out using HCl, H2SO4, or NaOH. Aeration and Agitation: Aspergillus niger is an aerobic microorganism and therefore requires oxygen. Aerating and agitating the fermentation broth normally satisfies the oxygen demand of a fermentation process. The effect of agitation and aeration on citric acid production in submerged fermentation is extremely important for the  Surface Surface fermentation was the initial industrial-scale fermentation system used for citric acid Fermentation production. Submerged fermentation in deep tanks is also employed, but only 20% of global citric acid production still uses surface fermentation with molasses. A. niger forms a mycelium layer on the liquid surface in trays made of Process Description: aluminum or stainless steel. Trays are stacked in fermentation rooms supplied temperature with filtered air for oxygen and control. Air supply is sterilized, humidified, and maintained at 0.25 vvm (volume of air per volume of medium). Trays, sterilized and filled with medium, are inoculated with A. niger spores and incubated at 28–30°C for 9–12 days. After reaching maximum citric acid concentration, mycelium is separated from the broth by filtration. The biomass is washed to remove citric acid, and the washings are added to the main liquor. Citric acid in the solution is precipitated as calcium citrate. Chambers are sterilized by washing with NaOH, water, and formaldehyde, followed by sulfur dioxide treatment.   Submerged Fermentation: Microorganisms are grown in fermentation broth.  Fermentor Types: Various fermentors include shake flasks, stainless steel tanks, agitated reactors, sparged towers, loop bioreactors, bubble columns, tower fermentors, disk Submerge  fermentors, Oxygen and rotating Rising Transfer: disk contactors. air bubbles ensure thorough mixing and oxygen transfer in d the fermentor; oxygen can be used instead of air for improved transfer in unagitated Fermentati vessels.  Foam Control: Foaming is countered by on mechanical or chemical foam breakers.  Cell Recycle Reactors: Mechanical devices separate cells from liquid, returning cells to the fermentor, conserving substrate, enabling continuous operation, and increasing productivity. Continuous Culture In continuous culture the substrate is fed to the fermentor at a constant rate, and the culture is harvested at the same rate, so that the culture volume remains constant. Continuous culture is superior to batch culture in productivity, uniformity of operation, and ease of automation but is more susceptible to contamination. Very little published information is available on the production of citric acid by free cells of A. niger in continuous culture. Fed Batch Culture Fed batch culture is a batch culture fed continuously or sequentially with substrate without the removal of fermentation broth. Compared to conventional batch culture, fed batch culture has several advantages including a very low concentration of residual sugars, higher dissolved oxygen in the medium, decreased fermentation time, higher productivity, and reduced toxic effects of the medium components that occur at high concentrations. Solid-State Fermentation Solid-state fermentation (SSF) is generally defined by the growth of the microorganism in a low water activity environment on an insoluble material that acts both as a physical support and a source of nutrients. However, it is not necessary to combine the role of support and substrate, but rather to reproduce the conditions of low water activity and high oxygen transference by using a nutritionally inert material soaked with a nutrient solution. In recent years, SSF technology has gained more attention. Solid-state fermentation offers numerous advantages for the production of microbial products. It requires less economic investment, an important aspect when methods for waste treatment are being encouraged. It has lower energy requirements and produces less wastewater than submerged fermentation. In addition, increasing environmental concerns regarding the disposal of solid wastes and  Immobilized Cells In the past few years, the immobilization of microbial cells has been studied with greater interest, and immobilized cells have been used for the production of organic acids, amino acids, antibiotics, enzymes, and other compounds. The production of citric acid by immobilized A. niger or yeasts cells compared with free cell systems has several advantages. The cells can be reused for long periods and transferred simply by draining the supernatant and replacing with fresh medium. The fermentation process can be controlled more easily. A less expensive fermentor design is required. Continuous fermentation takes place at a high dilution rate without washout and with higher product yield. Working at dilution rates greater than the growth rate of contaminating microorganisms also helps to overcome infection problems. Immobilized microbial cells are more stable than free cells. In the case of A. niger, the immobilization process generally leads to a consistent decrease in medium viscosity, thus enhancing nutrient and oxygen transfers which makes repeated batch and continuous processes possible. However, immobilized cell systems have some problems such as possible metabolic changes, a need to ensure diffusion of substrates and products, contamination of the medium with free cells, the cost of the immobilization matrix, and, in the case of A. niger, a layer of the mycelium forms outside of beads, which prevents the diffusion of substrate and air into the beads  Substra tes Defined Chemically Media Molasis Cerial constituents Fruit extracts Hydrocarbons Agricultural waste The main techniques are old and involve the treatment of microorganisms with UV, gamma rays, nitrogen mustard, UV nitrous acid, UV ethyleneamine, UV nitrosoguanidine, and UV N-nitroso-N-methyl urea. Mutations were carried out in some strains of A. niger and Y. lipolytica. Advantages: The mutant strains of A. niger have some advantages over the parent strains:  They produce higher concentrations of citric acid.  Fermentation time is shorter.  They increase metabolic flux through the pathway leading to citric acid formation. There is a direct increase of the flux through the main pathway (i.E., By overproduction of the enzymes involved in the production of the acid).  They tolerate high concentrations of heavy metals and produce large amounts of citric acid when they are grown in molasses solution Disadvantages : Mutant strains of A. niger have some disadvantages as well. During the screening of mutants for high citric acid concentration producers, there is a lack of a precise and quick method by which citrate producing strains can be easily selected. The variability of the strains is decreased after repeated mutagen 1. ​Fermentation Process: Fermentation can be conducted Recovery through surface or submerged techniques. Aspergillus niger is commonly Process: 5. Filtration and Purification: Solution is filtered to remove calcium sulfate. used in industrial-scale citric acid production. Decolorization with charcoal and ion exchangers improves purity. 2. Broth Filtration: Broth 6. Concentration and Crystallization: obtained from fermentation is filtered to remove Purified solution is concentrated through evaporation. mycelia and other impurities. Low-temperature crystallizers are used to obtain citric Filtrate is then processed for acid crystals. citric acid recovery. Citric 7. acid is Market marketed Forms: in various forms: anhydrous crystalline, 3. ​Precipitation of Citric Acid: monohydrate, or as a crystalline Citric acid is precipitated from sodium salt. the filtrate as calcium citrate. 8. Solvent Extraction Precipitation is achieved by (Optional): Solvent extraction methods can be employed for citric adding lime slurry at elevated acid extraction. Solvents like 2-butanol, tributyl temperatures. phosphate, or amines may be used. 4. ​Wash and Regeneration: Extracted citric acid is concentrated and crystallized. Precipitate is washed to remove soluble impurities. Sulfuric acid is added to