F:\JPRfeb 2010\june 2010\paid j

9 downloads 0 Views 317KB Size Report
Jun 6, 2010 - Citric acid, C3H4OH (COOH)3, was first isolated from lemon juice by a Swedish ... fermentation of cane sugar or molasses in the presence of a fungus, Aspergillus ... that contains one water molecule for every molecule of citric acid. .... final concentration of citric acid and dry weight is not related to inocu-.
Research Article ISSN: 0974-6943

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6),

Available online through www.jpronline.info Citric acid production from cane ,ollases using submerged fermentation by Aspergillus niger ATCC9142 Chaturvedi Madhusudan1*, Chugh M Rishi 2, Manoj singh2 Research scholar, Institute of Pathology, ICMR, Safdarjang hospital campus, New Delhi-110029 JRF, Institute of Pathology, ICMR, Safdarjang Hospital campus, New Delhi-110029 ,Research scolar, Dept. of Marine and Costal studies, school of energy sciences, Madurai Kamraj University, Madurai Received on: 17-03-2010; Revised on: 15-04-2010; Accepted on:13-05-2010 ABSTRACT Citric acid, C3 H4 OH (COOH)3, was first isolated from lemon juice by a Swedish chemist, Carl Wilhelm Scheele, in 1784. Citric acid is manufactured by fermentation of cane sugar or molasses in the presence of a fungus, Aspergillus niger ATCC 9142. Fermentation of sugar by the mold Aspergillus niger ATCC 9142 is the chief commercial source of the acid. Fermentation results in the breakdown of complex organic substances into simpler ones through the action of catalysis. This project involves the production of citric acid from fungal strain of Aspergillus niger ATCC 9142, using various sources like cane molasses, beet molasses, sweet potato and grape sugar by employing various methods such as submerged and surface fermentation. The recovery of citric acid from fermented broth is generally performed through three procedures – precipitation, extraction and adsorption (mainly using ion-exchange resins). The main aim of the project is to achieve a cost reduction in citric acid production by using less expensive substrates.

Keywords: Aspergillus niger, citric acid, potato dextrose agar INTRODUCTION History of citric acid: Citric acid fermentation was first observed as a fungal product by Wehmer in 1893 by a culture of Penicillium glaucum on sugar medium. It was the work of Currie which opened up the way for successful industrial production of citric acid. In 1916, he found that numerous strains of Aspergillus niger produced significant amounts of citric acid. The most important finding was that A. niger grew well at pH values around 2.5–3.5 and high concentrations of sugars favour citric acid production. The first citric acid fermentations were carried out in surface cultures. In general, citric acid is commercially produced by submerged microbial fermentation of molasses; the fermentation process using Aspergillus niger is still the main source of citric acid worldwide. Although methods were well developed to synthesise citric acid using chemical means, better successes were achieved using microbial fermentations, and over the period of time, this technique has become the method of ultimate choice for its commercial production over chemical synthesis (1). It was necessary to consider raw material much more carefully. Several works were dedicated to the optimization of the conditions for the utilization of cheap material like sugar cane molasses, beet molasses, starch and hydrolysate starch (2).

At room temperature, citric acid is a white crystalline powder. It can exist either in an anhydrous (water-free) form, or as a monohydrate that contains one water molecule for every molecule of citric acid. Chemically, citric acid shares the properties of other carboxylic acids Citric acid is a weak organic acid found in citrus fruits and vegetables, but it is most concentrated in lemons and limes, where it can comprise as much as 8% of the dry weight of the fruit. Citric acid i.e. 2-hydroxy 1,2,3 propane tricarboxylic acid (CH2COOH.COH.COOH.CH2COOH) is ubiquitous in nature and exists as an intermediate in the citric acid cycle (Krebs cycle) when carbohydrates are oxidized to carbon dioxide. Molasses is a desirable raw material for citric acid fermentation because of its availability and relatively low price. The present investigation deals with the study of citric acid fermentation by Aspergillus niger ATCC 9142. Cane-molasses and few other substrates were employed as the basal fermentation media under the surface and submerged fermentation conditions. The study revealed the nutritional status of the organism and basic fermentation parameters.

Citric acid

Microorganisms

*Corresponding author. Madhusudan chaturvedi Research scholar, Cell Biology Laboratory Institute of Pathology, ICMR, Safdarjang Hospital Campus, New Delhi. Tel.: + 91-09555017287 Telefax: +91E-mail:[email protected]

Microbial Production of Citric Acid

A large number of microorganisms including fungi and bacteria such as Arthrobacter paraffinens, Bacillus licheniformis and Corynebacterium ssp., Aspergillus niger, A. aculeatus, A. carbonarius, A. awamori, A. foetidus, A. fonsecaeus, A. phoenicis and Penicillium janthinellum; and yeasts such as Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, Hansenula anamola and Yarrowia lipolytica have been employed for citric acid production (3-8). Among the mentioned strains, the fungus A. niger has remained the organism of choice for commercial production because it produces more citric acid Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), per time unit. The problem in the production of citric acid for yeasts is the simultaneous formation of isocitric acid. The main advantages of using A. niger are its ease of handling, its ability to ferment a variety of cheap raw materials, and high yields.

citrus fruits, and was crystallized from lemon juice by Scheele in 1784. Approximately 70% of citric acid produced is used in the food and beverage industry for various purposes, 12% in pharmaceuticals and about 18% for other industrial uses (13).

The improvement of citric acid producing strains has been carried out by mutagenesis and selection. The most employed technique has been by inducing mutations in parental strains using mutagens (6,7,9). Mutants of Aspergillus niger are used for commercial production (10). To obtain hyper-producer strains, UV treatment can frequently be combined with some chemical mutagens. The single- spore technique and the passage method are the principal methods of selecting strains. The first one has the disadvantage that mineral acid and organic acids (gluconic and oxalic acids) simulate the presence of citric acid (5-7, 11).

The estimated world production of citric acid was reported as 350.000 tons/year in 1986 (14). It however was recently reported that the world market requirement of citric acid is around 500.000 tons/year (15). Although mainly A. niger has been used in the citric acid production process, other strains of fungi apart from A. niger, various kinds of yeast and some bacteria are known to accumulate citric acid in the medium (16). Steel et al. (17) recommended that between 120x103 and 280x103 pellets per liter (obtained from spore inoculated shake flasks) is a suitable inoculums level, although Kristiansen (18) indicated that the final concentration of citric acid and dry weight is not related to inoculum size, as long as it was kept below 106 spores/ml culture. Yigitoglu (19) therefore employed a less complex method for inoculum.

Different methods of fermentation can lead to different yields of citric acid production by the same strain. Thus, a strain which produces good yields in the solid fermentation or liquid surface is not necessarily good producer in the submerged fermentation. In that way, each method and raw material of industrial interest should be tested with known producer strains (8). In any technique used in citric acid production the inoculation of microorganism is done by means of spores which are added into the fermentation medium (8). Spores can be inoculated either mixing them with the air, which is introduced in substrate, or in form of a spore suspension. Spores are produced in glass bottles on solid substrates at optimum temperature (6). The type of sporulation medium and time of incubation affect spore viability and citric acid production by mycelia grown from A. niger. It was mentioned that potato dextrose agar gives high citric acid yields. Viability increases with time of incubation, but higher production of citric acid was achieved in less than 7 days of spore incubation (12). Aspergillus niger

There is a general agreement in the literature that the pelleted form is desirable for acid production. An ideal pellet configuration, pellets of 1.2 to 2.5 mm diameter after five days, was described early (20). Gomez et al. (21) showed that the pelleted form is favorable due to pellet cultures have low culture viscosity causing improving bulk mixing and aeration conditions and lower oxygen consumption than in the cultures composed mainly of filamentous (dispersed) forms.Strain improvement by mutation in order to achieve higher yields and higher trace metal tolerance is a continual aim of industrial producers. Its importance can be illustrated by the 500 fold increase in penicillin production from Penicillium chrysogenum due to mutation (22). McKay et al. (23) increased the production of citric acid yields from glucose by Yarrowia lipolytica IFO 1658 two fold and by Candida guillermondii NRRL Y-448 from galactose, six fold via ultra-violet mutagenesis and subsequent selection. James et al. (24) produced a mutant via multiple X-ray and UV irradiation of spores, and mutant strains showing a six fold increase in citric acid yield compared to the parent strain. The biochemical pathways related to citric acid accumulation and the role of the tricarboxylic acid cycle (TCA) in fungi has been well established (13). Citric acid accumulation can be divided into three processes (14):

Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. Aspergillus niger is cultured for the industrial production of many substances. Various strains of A. niger are used in the industrial preparation of citric acid and gluconic acid and have been assessed as acceptable for daily intake by the World Health Organisation. Aspergillus is utilized industrially in a number of ways. Most sodas and soft drinks contain citric acid as a main ingredient. It is too expensive to isolate the citric acid from citrus fruits so it is produced in large-scale fermentation vats utilizing Aspergillus niger. REVIEW OF LITERATURE:Citric acid is a 6-carbon containing tricarboxylic acid which was first isolated from lemon juice. It is a natural component of many

1. The breakdown of hexoses to pyruvate and acetyl-CoA by glycolysis, 2. Formation of oxaloacetate, 3. Condensation of acetyl-CoA and oxaloacetate to citric acid. As citric acid synthesis involves the condensation of an acetyl unit with oxaloacetate, it is quite important to generate sufficient oxaloacetate in order for production to continue. Regeneration of oxaloacetate involves fourmechanisms (16): 1. The direct carboxylation of pyruvate catalyzed by malic enzyme provides malate which is readily oxidized into oxaloacetate through malic dehydrogenase; 2. The carboxylation of pyruvate catalyzed by pyruvate carboxylase, Mg+ Pyruvate + CO2+H2O+ATP

Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Oxaloacetate +ADP+Pi

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), 3. The carboxylation of phospho-enol pyruvate (PEP) catalyzed by PEP carboxykinase, PEP + ADP+CO2

Oxaloacetate +ATP

4. via the glyoxylate cycle involving the key enzymes isocitrate lyase and malate synthase, Isocitrate

Succinate + Glyoxalate

Co-enzyme A + malate

Glyoxalate + Acetyl-Co-A

Oxaloacetate Pyruvate carboxylase is an important enzyme for citric acid production. It is poorly regulated, only weakly inhibited by 2-Oxoglutarate and not influenced by acetyl-CoA (25). Phosphofructokinase was the regulatory enzyme of citric acid production in A. niger (26). The enzyme was inhibited by high concentrations of citrate and ATP but activated by ADP, AMP, inorganic phosphate and ammonium ions. During citric acid production ammonium ions overcome the inhibition of PFK by citrate and ATP. Aconitase and isocitrate dehydrogenase are very important key enzymes in citric acid fermentation. The activity of these enzymes decrease to very low levels during the production stage which cause faulty operation of the cycle whilst the activity of citrate synthase increases (13) MATERIALS AND METHODS:Production of citric acid using fermentation technology (and not the chemical synthesis) by Aspergillus niger ATCC 9142. The work basically includes two types of fermentation viz. surface and submerged fermentation. The entire work has been performed on a small lab scale level (and not on an industrial level). METHODOLOGY Raw material used: – Cane molasses, Beet molasses, Brewery waste, Sugar. Culture selection and maintenance The mother culture of Aspergillus niger ATCC 9142 is obtained from the National Collection Of Industrial Microorganisms (National Chemical Laboratory) Pune, Maharashtra. The sub cultured cultures of Aspergillus niger are maintained on sterilized potato dextrose agar medium (Diced potato 200 g/l, Dextrose 20 g/l and Agar 15 g/l), pH 4.5 and stored at 5ºC in the refrigerator (the sub culturing method has been described below). All the culture media, unless other wise stated, are sterilized in autoclave at 15-lbs/inch2 pressure (121ºC) for 15 min. Although mainly A. niger is used in the citric acid production process, the reason for choosing A. niger over other potential citrate producing organism are: cheap raw materials (molasses) used as substrate; easily available; cost effective; high consistent yields etc. Sub culturing of Aspergillus niger

Boil finely diced potatoes in water until thoroughly cooked and filter it through cheesecloth. After filtration add water to filtrate to make the volume to 0.5 L. Heat the filtrate to dissolve the added agar and add the glucose before sterilization. Composition of Potato Dextrose Agar:Diced potatoes.............150.0 g Glucose..................... 10.0 g Agar........................ 7.5 g Distilled water..............0.5 L 1.The prepared media is distributed into 6 test tubes (up to half of the capacity of each test tube). Autoclave at 1210C for 15 minutes. Keep in slanting position and allow them to solidify. Incubate two of the slants (as blank) to check if any type of contamination is there or not. Incubate the slants at 20-250C for 4 days. 2.After cross checking of contamination is over, the remaining slants are sub-cultured using the mother culture. A loop full quantity from the mother culture is taken on an inoculation loop and streaked (rubbed in zigzag fashion) onto the prepared slants in an aseptic environment. Now, incubate these slants at 20-250C for 4-5 days. It is to be noted that subcultures (from the mother culture) can not be used more than five times. Treatment of raw material Cane molasses is the main raw material used in the present study. Cane molasses contains 20% water, 62% sugar contents, 10% non-sugar contents, and 8% inorganic salts (ash contents), making a blackish homogenous liquid with high viscosity. Ash contents include ions such as Mg, Mn, Al, Fe and Zn in variable ratio. Sugar content is diluted to about 25% sugar level. a.The cane molasses is heated to boiling temperature by keeping in autoclave for 1-2 hours to prevent fermentation that might be caused due to contamination. b.Then the solution is cooled and filtered through normal filter paper. c.1N H2SO4 is added to the molasses, the same boiled for half an hour, cooled, neutralized with lime-water (CaO) and was left to stand over night for clarification. H2SO4 was added to breakdown complex sugars in juice to simpler sugars so that microorganisms can utilize it. d.Filter the solution again using normal filter paper to remove the impurities of the first phase. The following elements are added in the flask containing the molasses. KH2PO4 2.5 gm NH4NO3 1.05 gm MgSO4.7H20 0.5 gm FeCl3 0.01 gm ZnSO4 0.0025 gm e.Then the pH of the solution is adjusted using H2SO4 since no further breakdown is required. The pH was finally adjusted to 5.

f.Distribute the sample into two flasks with each containing 500 ml of the First of all, 500 ml of potato dextrose agar medium is made. For cane molasses. making the PDA, the following method is followed. Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), g.Again autoclave the cotton plugged flasks at 1100C for 10 minutes. This sterilization step was carried out in horizontal autoclave.

3.Filter the same using muslin cloth and then again filter using filter paper.

Inoculum’s preparation

4.Add Ca(OH)2 or CaCO3 to the filtrate until the solution gets neutralized.

1.500 ml of molasses medium (Sugar 15%, pH 6.0) in 1.0 L cotton wool plugged Erlenmeyer flask, is now sterilized. 2. 5 ml of 0.9 % normal saline is transferred to well sporulated Aspergillus niger slants. The inoculation needle is rubbed to ensure proper mixing of the culture with saline. 3.Small amount of mixed solution from the well sporulated slant is aseptically transferred to the flask containing raw material (molasses). Normal saline is used so as to prevent the death of the spores. Fermentation

5.Filter again and collect the precipitate this time. Wash the same with distilled water and again filter through Whattman 42 filter paper. Keep the flasks in shaker again for continued washing for 24 – 36 hours. Filter again using filter paper. 6The precipitate is filtered and washed with water several times. It is then treated with H 2SO4. The solution is again filtered to remove CaSO4. The mother liquor containing citric acid is decolourized by charcoal and passed through ion exchange resin columns. The liquor is concentrated in vacuum and finally run into low temperature crystallizers where citric acid crystallizes as citric acid monohydrate, the details of which have been described below.

One of the flasks is incubated at 300C in a rotary incubator shaker at 300 rpm for 5 days. This is for submerged fermentation.

The other flask was incubated in cooling incubator at 280C for 5 days. This is done for surface fermentation. It is to be noted here that the final concentration of citric acid and dry weight is not related to inoculum size, as long as it was kept below 106 spores/ml culture. Growth form The pelleted form is desirable for acid production. An ideal pellet configuration, pellets of 1.2 to 2.5 mm diameter after five days, was described early. The pelleted form is favorable due to pellet cultures have low culture viscosity causing improving bulk mixing and aeration conditions and lower oxygen consumption than in the cultures composed mainly of filamentous (dispersed) forms. Furthermore, problems of wall growth and pipe blockage are reduced and separation of biomass from culture liquid by filtration is considerably enhanced by the pelleted growth form.

Charcoal Treatment The filtrate is collected after treating with Sulphuric acid. In a beaker, add 20 gms of charcoal powder to it, mix properly and again filter with filter paper. Charcoal absorbs the free acidic part and other impurities. The filtrate is then passed through the filter paper again and then finally through the chromatography column.

Steps followed after fermentation 1.Take out both the flasks from the shaker incubator and cooling incubator. 2.Heat both of them up to 90 – 950C to kill the mycelium.

Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), Preparation of chromatography column A 50 ml burette, ion exchange resin 244 and glass wool are taken. Now, a little amount of glass wool is put into the burette. Distilled water is added to it. Distilled water is passed through the burette to settle down the glass wool. Resin powder is mixed in a separate beaker with distilled water & allowed to settle down. The top layer (supernatant) is discarded and solid part (precipitate) is put into the burette till 43 mark and again distilled water is added from top. Distilled water is added till the column is properly settled and colorless drops start passing out from the burette. Ion exchange Resin treatment Now, the filtrate after the charcoal treatment is passed through this column. The clear solution obtained is now taken to Rotor evaporator for vacuum distillation. We finally obtain the citric acid powder which is then dried by using vacuum oven.

Identification & Assay:Identification of citric acid is initially done on the basis of solubility. This can be done in the following two ways:1.Water solubility test: - Little amount of citric acid is taken in a test tube and distilled water is added to it. If it is very soluble in it, then it passes the solubility test. Otherwise, it is some other acid. 2.Ethanol solubility test: - Little amount of citric acid is taken in a test tube and ethanol is added to it. If it is freely soluble in it, then it passes the solubility test. Otherwise, it is some other acid. Note: Citric acid is very soluble in water, freely soluble in ethanol and sparingly soluble in ether. 3.Calcium chloride test: - To a little amount of citric acid precipitate, water is added. Then pH is adjusted to 7 by using 1N NaOH and 0.1 % CaCl2 is added. If precipitate dissolves, then it confirms the presence of citric acid and if it does not dissolve then citric acid is absent. 4.Potassium permanganate test: - To a little amount of citric acid precipitate, add 25 ml of water. Then 0.5ml H2SO4 and 3ml of KMnO4 are added. Warm the solution on a burner. If the color of KMnO4 disappears, then it confirms the presence of citric acid. Purity and Yield Calculation Citric acid (CA) is determined titrimetrically by using 0.1 N NaOH and phenolphthalein as indicator and calculated in % according to the following formula:-

RESULTS:A successful process depends both on an appropriate strain and optimization of fermentation parameters. In the present work, cultural conditions such as sugar concentration, time profile of citric acid synthesis, incubation temperature, initial pH, agitation intensity and air supply were optimized by Aspergillus niger ATCC 9142 in a laboratory scale. In the present study, the strain of Aspergillus niger ATCC 9142 supported maximum production of citric acid (106.65 g/l) without supplements which is substantial. The addition of nitrogen sources and minerals like iron and phosphate may further increase the production of citric acid, as required for an industrial process. ALKEM LABORATORIES LIMITED, R&D C ENTRE, TALOJA, NAVI MUMBAI (Analysis Report) Citric Acid Monohydrate Formula Mol. Wt Description Solubility

:C6H8O7.H2O :210.13 (192.13 + 18) :A white, crystalline powder or colorless crystals. :Very soluble in water, freely soluble in etha nol, spar ingly soluble in ether Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), Functional use Packing & storage

:Acidulant, dispersing agent, sequestrant : 25 kg in composite paper bag with inner PE bag.Kept in cool and dry place. Analysis : Clarity and color of solution - Test Pass Identification - Test Pass Solubility - Test Pass Organic volatile impurities - Test pass Calcium Chloride test - Test Pass KMnO4 test - Test Pass Mesh : Granular : 12-30 mesh Fine granular : 24-120 mesh % Citric Acid (Yield): 39.44 %

Copper: Copper ions play an important role in reducing the deleterious effect of iron on citric acid production. Copper ions can successfully counteract addition of manganese to citric acid fermentation media and are inhibitors of cellular manganese uptake. Copper is an essential requirement for citric acid production and optimum concentration of Cu 2+ is 40 ppm for high yield. Zinc: Low concentrations of zinc in the fermentation medium are generally favored in most citric acid production media. Zinc plays a role in the regulation of growth and citrate accumulation. At high zinc levels (about 2 µM) the cultures are maintained in growth phase, but when the medium becomes zinc deficient (below 0.2 µM) growth is terminated and citric acid accumulation begins. Addition of zinc to citrate accumulating cultures results in their reversion to growth phase. Sugars: Due to their rapid assimilation by fungus the usual carbon sources are glucose, fructose, or sucrose for high final yield of citric acid. Some sugars such as galactose and arabinose inhibit citric acid production. In most cases, sucrose or its cheaper commercial source molasses is used.

DISCUSSION Nitrogen is a limiting factor in the citric acid production. Nitrogen is usually supplied in the form of ammonium nitrate, which was completely metabolized during fermentation periods. Citric acid started to appear when the nitrogen concentration fell below a low limiting value. It appears that the citric acid was produced by carbon-storing cells. Low dry weight might have been caused by the drastic reduction and denaturation of some enzymes active in the accumulation of carbon in the used pH range. The sugar concentration decreased throughout the fermentation period. This indicated that the cells were still viable. Therefore, there seems to be a link between the storage of carbon and the production of citric acid. Similarly, other factors that affect the fermentation process have been detailed below. Factors affecting the fermentation Process:Medium Constituents:Trace elements: Trace element nutrition is one of the most important factors affecting the yields (grams citric acid per gram sugar) of citric acid fermentation. In particular, the levels of manganese, iron, copper and zinc are quite critical. If the levels of these trace elements are correct other factors have less pronounced effects. Conversely, medium will not allow high production unless the trace element content is controlled carefully. Manganese: Manganese (Mn2+ ions) in the nutrient medium plays a key role in the accumulation of large amount of citrate byA. niger. When the Mn 2+ concentration is maintained below 0.02 mM (which does not affect growth rate or biomass yield) large amounts of citric acid are produced. Manganese deficiency leads to significantly lower lipid content in A. niger whereas there is elevated lipid level in manganese sufficient cultures. Iron: - Up to 1 mg iron per liter medium is essential for high yields of citric acid by A. niger, but that amounts in excess of this interferes with citric acid accumulation. Partial deficiency of iron is necessary for citric acid production. The presence of excess iron favors the production of oxalic acid.

Nitrogen source: Usually ammonium sulfate or ammonium nitrate is used as a nitrogen source. Physiologically, acid ammonium compounds are preferred since their consumption lowers the pH of the medium to below 2 which is an additional prerequisite of citric acid fermentation. The optimal concentration of ammonium sulfate is 5 kgm-3. However the best initial ammonium sulfate level is 3 kgm-3 by a series of fermentation which were carried out at varying initial concentration of ammonium sulfate between 0.5 and 4 kgm-3. When the concentration of intracellular ammonium ion is between 2 and 3 mmol/g cell the production rate of citric acid is the highest. However when the concentration of intracellular ammonium ion is decreased below 1 mmol/g cell, the citric acid production gets stopped. Phosphate: The effect of phosphate is not very pronounced but the balance between manganese, zinc and phosphate is critical. In any cases of trace metal contamination, phosphate limitation can have a beneficial effect on citric acid yield. Requirement of phosphate for fungal growth is 0.1 to 0.2%. However the presence of copper in the medium could reduce the optimum phosphate concentration. Phosphate plays a key role in secondary metabolite production. When 0.005% phosphate is added to beet molasses, 5-ketogluconic and gluconic acid replaces oxalic acid as secondary products. In addition fermentation time gets significantly reduced. Magnesium: Magnesium is essential for growth and citric acid production due to its role as a cofactor in a number of enzyme reactions in the cell. The optimum concentration of magnesium sulphate to produce maximum citric acid varies from 0.02 to 0.025%. Environmental conditions:Aeration: Aeration has a critical effect on the submerged citric acid production process. Aeration should be 0.6 vvm (liter air per liter medium per minute). The citric acid concentration rises from 30.3 to 48.7 kgm-3 by increasing air flow rate from 0.9 to 1.3 vvm. Citric acid production is also related to oxygen pressure. The yield of citric acid increases by increasing the flow rate of air and the oxygen pressure up to 1.7 atmospheres using pure oxygen for pressures of 1 atm and greater, beyond which citric acid production decreases.

Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), Agitation: Agitation in stirred tank fermenters is critical. Increasing agi- M) and riboflavin (4x10-5 M) stimulates the citric acid formation to the tator speed breaks up pellets, leading to dispersion of more than 95% of extent of 59% and 50% respectively. Biotin (3x10-5 M) produces the pellets, resulting in higher yields of citric acid. Maximum yield is at greatest enhancement, stimulating growth and increasing the producagitator speeds between 400 and 700 rpm. 500 rpm is the optimal agita- tion of citric acid by 66.4%. tion speed for citric acid production. However the higher yield of citric acid 28 kgm-3 is generally obtained in culture agitated at lower impeller Amino acids: The presence of glutamic acid (4x10- 3 M) and aspartic acid speed (300 rpm). (3x10-3 M) stimulates citric acid production by 79 and 76.7% respectively. Presence of lysine (5x10-3 M) and serine (4x10-3 M) also influence the formation of citric acid by 62.3 and 50.4%. The effect of cysteine (in all concentrations) is found to be detrimental.

pH: citric acid yield increases with increasing pH. The optimal initial pH is 6.5. A pH of 2.5 is a clear optimum for final product concentration in a 10 dm3 STR. The optimal final pH for batch fermentation is 1.7. It is recommended that pH should be kept low (below 2.0). According to this at higher pH’s, A. niger accumulates gluconic acid, especially when the pH is around 4.0.

Toxic chemicals: There is slight increase in citric acid formation in the presence of phenol (20 ppm) and b-naphthol (20 ppm). But hydroquinone (with 30 ppm) and o-cresol (with 15 ppm) exhibit maximum citric acid stimulation i.e. 85 and 80 kgm-3 respectively. Acid formation in the presence of resorcinol (with 50 ppm) is 78 kgm-3. The increase in citric acid production may be due to either the direct effect of these phenols on the growth process i.e., metabolism of A. niger, or due to the inhibition of enzymes involved in further metabolism of citric acid. Applications of Citric Acid:Citric acid is a universally used alimentary additive. It is accepted worldwide as GRAS (generally recognized as safe), approved by the Joint FAO/WHO Expert Committee on Food Additives (27,28,29). The food and pharmaceutical industries utilize citric acid extensively because of its general recognition of safety, pleasant acid taste, high water solubility and chelating and buffering properties. In addition to its carboxyl and hydroxyl groups permit the formation of a variety of complex molecules and reactive products of commercial interest. Table 4 presents the main applications of citric acid (27-29, 30). C.R. SOCCOL et al.: Citric Acid Production, Food Technol. Biotechnol. 44 (2) 141–149 (2006) 147 Applications

Industry

function

Incubation temperature: The incubation temperature should be in the range to 28 to 32°C, while 30°C is the optimum for citric acid production.

Beverages

Wines andCiders

Duration of fermentation: The citric acid fermentation is completed in 8 days. Extension of the fermentation period does not increase the yields of citric acid. Generally incubation period of about 6-9 days is preferred.

Food

Prevents browning in some white wines. Prevents turbidity of wines and ciders. Used in pH adjustment. Provides tartness. Stimulates natural fruit flavour. As acidulant in carbonated and sucrose based beverages. Used in pH adjustment. Acts as acidulant. Provides the desired degree of tartness, tang and flavour. Increases the effectiveness of antimicrobial preservatives. As emulsifier in ice creams and processed cheese. Acidifying agent and antioxidant in many cheese products. Acts as acidulant. Provides tartness. Minimizes sucrose inversion. Produces dark colour in hard candies. Prevents crystallization of sucrose Protects ascorbic acid byinactivating trace metals. Lowers pH to inactivate oxidative enzymes. Synergist for other antioxidants, as sequestrant. Stabilizing action. Feed complementation Micronutrient evaluation in fertilizers. Enhances Pavailability in plants As effervescent in powders and tablets in combination with bicarbonates. Anticoagulant. Provides rapid dissolution of active ingredients. Acidulant in mildly astringent formulation. Buffering agent. pH adjustment. Antioxidant as a metallic–ion chelator. Acts as buffer agent. Sequestring metal ions. Neutralizes bases. Used in nontoxic, noncorrosive and biodegradable processes that meet current ecological and safety standards. Removes metal oxides from the surface of ferrous and nonferrous metals, for operational cleaning of iron and copper oxides. In electroplating, copper plating, metal cleaning, leather tanning, printing inks, bottle washing compounds, floor cement, textiles, photographic reagents, concrete, plaster, refractories and moulds, adhesives, paper, polymers, tobacco, waste treatment, chemical conditioner on teeth surface, ion complexation in ceramic manufacture.

Soft drinks and syrups Jellies,jams and preservatives Dairy Products

Other factors:-

Candies

Alcohols: Addition of lower alcohols, methanol, ethanol, n-propanol, to crude carbohydrate raw materials increases the yield of citric acid. Optimal concentration of methanol, which is said to be more effective than ethanol, varies from 1 to 4% by volume. However addition of methanol to highly purified, high yielding substrates is deleterious to acid yields. The exact mechanism of the alcohol effect however is unexplained, though it is postulated that addition of methanol increases the tolerance of fungi to Fe2+, Zn2+ and Mn2+.

Frozen fruit Fats and Oils Animal Feed Agriculture Pharmaceutics

Other

Lipids: Addition of natural oils with a high content of unsaturated fatty acids and oleic acid at 2% (v/v) to fermentation media led to increase in the yield by 20% without affecting dry weight of mycelium. A concentration of fatty acid of 0.05 to 0.3% has to be maintained during the fermentation.

Pharmaceuticals

Cosmetics and toiletries Industrial applications Metal cleaning

Vitamins: Ascorbic acid and p-amino benzoic acid, at all concentrations, inhibited growth of citric acid. The presence of thiamine (3x10-5 Journal of Pharmacy Research Vol.3.Issue 6.June 2010

Madhusudan chaturvedi et al. / Journal of Pharmacy Research 2010, 3(6), CONCLUSION:

7.

Citric acid is the largest produced organic acid measured in tonnage. Till now its annual production has reached upto 1.5 million tones and continues to increase more each year because of its enormous application in food, beverage, pharmaceutical and agricultural industries.in traditional processes, submerged fermentation with the help of fungus aspergillus niger, is mostly used for its mass production. However, different new techniques of its production are continuously being used for showing new methods for production of citric acid.Upon the basis of work done so far, it can be predicted that citric acid production by fermentation of molasses and other liquid citrus cannery wastes is entirely feasible. ACKNOWLEDGEMENT: We would like to thanks Dr. L K Yerneni, Dr. D Dhanshekhran and Mr. M D Tiwari for their help. REFERENCES: 1. 2. 3. 4. 5. 6.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

M. Mattey, The production of organic acids, Crit. Rev. Biotechnol. 12 (1992) 87–132. S. Sarangbin, Y. Watanapokasin, Yam bean starch: A novel substrate for citric acid production by the protease-negative mutant strain of Aspergillus niger, Carbohydr. Polym. 38 (1999) 219–224. H.S. Grewal, K.L. Kalra, Fungal production of citric acid, Biotechnol. Adv.13 (1995) 209–234. Y. Ikeno, Y.M. Masuda, K. Tanno, I. Oomori, N. Takahashi, Citric acid production from various raw materials by yeasts, J. Fermentat. Technol. 53 (1975) 752–756. C.P. Kubicek, M. Röhr, Citric acid fermentation, Crit. Rev. Biotechnol. 3 (1986) 331– 373. Pandey, C.R. Soccol, J.A. Rodriguez-Leon, P. Nigam: Production of Organic Acids by Solid-State Fermentation. In: Solid-State Fermentation in Biotechnology – Fundamentals and Applications, Asiatech Publishers Inc., New Delhi, India (2001) pp. 113–126.

24. 25. 26. 27.

28. 29. 30.

L.P.S. Vandenberghe, C.R. Soccol, A. Pandey, J.M. Lebeault, Review: Microbial production of citric acid, Braz. Arch. Biol. Technol. 42 (1999) 263–276. F. Yokoya: Citric Acid Production. In: Industrial Fermentation Series, Campinas, SP, Brazil (1992) pp. 1–82. I.U. Haq, S. Khurshid, S. Ali, H. Ashraf, M.A. Qadeer, M.I. Rajoka, Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses, World J. Microbiol. Biotechnol. 17 (2001) 35–37. W. Jianlong, W. Xianghua, Z. Ding, Production of citric acid from molasses integrated with in situ product separation by ion-exchange resin adsorption, Bioresour. Technol. 75 (2000) 231–234. M. Röhr, C.P. Kubicek, J. Kominek: Citric Acid. In: Biotechnology, Vol. 3 , G. Reed, H.J. Rehm (Eds.), Verlag Chemie, Weinheim, Germany (1983) pp. 419–454. M.G.F. Vergano, M.A. Soria, N.L. Kerber, Influence of inoculums preparation on citric acid production by Aspergillus niger, World J. Microbiol. Biotechnol. 12 (1996) 655–656. Marison IW : “Biotechnology for Engineers Biological Systems in Processes”, p 323, 1988 Kubicek P, Rohr M : CRC Critical Reviews in Biotech, 3, 4, 331, 1986. Bu’lock JD: Biotech Insight, 84, 5, 1990 Kapoor KK, Chaudhary K, Tauro P : In “Prescott and Dunn’s Industrial Microbiology” 4th edition, p 709, 1982. Steel R, Lentz CP, Martin SM : Can J Microbiol, 1, 299, 1955 Kristiansen B : PhD thesis, UMIST, 1976. Yigitoglu M : PhD thesis, University of Strathclyde, 1992. Clark DS : Can J Microbiol, 8:133, 1962. Gomez R, Scnabel I, Garrido J : Enzyme Microbiol Tech, 10:188, 1988. Kelly W: In “Biotechnology for Engineers Biological Systems in Processes” p 219, 1988. McKay IA, Maddox IS, Brooks JB : In “International Biotech Conference on Fermentation Technologies: Industrial Applications”, p 285, 1990. James LW, Rubbo SD, Gardner JF : J Gen Microbiol, 14:223, 1956. Jernejc K, Cimerman A, Perdih A : Eur J Appl Microbiol Biotech, 14:29, 1982. Habison A, Kubicek CP, Rohr M : FEMS Microbiol Lett, 5:39, 1979. Pandey, C.R. Soccol, J.A. Rodriguez-Leon, P. Nigam: Production of Organic Acids by Solid-State Fermentation. In: Solid-State Fermentation in Biotechnology – Fundamentals and Applications, Asiatech Publishers Inc., New Delhi, India (2001) pp. 113–126. L.P.S. Vandenberghe, C.R. Soccol, A. Pandey, J.M. Lebeault, Review: Microbial production of citric acid, Braz. Arch. Biol. Technol. 42 (1999) 263–276. C.R. Soccol, L.P.S. Vandenberghe, Overview of applied solid- state fermentation in Brazil, Biochem. Eng. J. 13 (2003) 205–218. H.S. Grewal, K.L. Kalra, Fungal production of citric acid, Biotechnol. Adv.13 (1995) 209–234.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.3.Issue 6.June 2010