Citric acid

Citric acid

Citric acid is a weak organic acid found in citrus fruits.

It is a natural preservative and is also used to add an acidic (sour) taste to foods and soft drinks. In biochemistry, it is important as an intermediate in the citric acid cycle and therefore occurs in the metabolism of almost all living things. It also serves as an environmentally benign cleaning agent and acts as an antioxidant.

Citric acid exists in a variety of 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.

properties

The physical properties of citric acid are summarized in the table at right. An important one is calcium citrate or "sour salt", which is commonly used in the preservation and flavoring of food. Additionally, citrates can chelate metal ions, which gives them use as preservatives and water softeners.

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. The anhydrous form crystallizes from hot water, while the monohydrate forms when citric acid is crystallized from cold water. The monohydrate can be converted to the anhydrous form by heating it above 74 °C.

Chemically, citric acid shares the properties of other carboxylic acids. When heated above 175 °C, it decomposes through the loss of carbon dioxide and water.

Citric Acid can also be written (long hand) as "HOOSeOOOOCCH₂C(OH)(COOH)CH₂COOH (aq)"

History

The discovery of citric acid has been credited to the 8th century Islamic alchemist Jabir Ibn Hayyan (Geber). Medieval scholars in Europe were aware of the acidic nature of lemon and lime juices; such knowledge is recorded in the 13th century encyclopedia Speculum Majus (The Great Mirror), compiled by Vincent of Beauvais. Citric acid was first isolated in 1784 by the Swedish chemist Carl Wilhelm Scheele, who crystallized it from lemon juice. Industrial-scale citric acid production began in 1860, based on the Italian citrus fruit industry.

In 1893, C. Wehmer discovered that Penicillium mold could produce citric acid from sugar. However, microbial production of citric acid did not become industrially important until World War I disrupted Italian citrus exports. In 1917, the American food chemist James Currie discovered that certain strains of the mold Aspergillus niger could be efficient citric acid producers, and Pfizer began industrial-level production using this technique two years later.

Production

In this production technique, which is still the major industrial route to citric acid used today, cultures of Aspergillus niger are fed on sucrose to produce citric acid. After the mold is filtered out of the resulting solution, citric acid is isolated by precipitating it with lime (calcium hydroxide) to yield calcium citrate salt, from which citric acid is regenerated by treatment with sulfuric acid.

Alternatively, citric acid is sometimes isolated from the fermentation broth by extraction with a hydrocarbon solution of the organic base trilaurylamine, followed by re-extraction from the organic solution by water.

Uses

As a food additive, citric acid is used as a flavouring and preservative in food and beverages, especially soft drinks; it is denoted by E number E330. Citrate salts of various metals are used to deliver those minerals in a biologically available form in many dietary supplements. The buffering properties of citrates are used to control pH in household cleaners and pharmaceuticals.

Citric acid's ability to chelate metals makes it useful in soaps and laundry detergents. By chelating the metals in hard water, it lets these cleaners produce foam and work better without need for water softening. Similarly, citric acid is used to regenerate the ion exchange materials used in water softeners by stripping off the accumulated metal ions as citrate complexes.

It is used in the biotechnology and pharmaceutical industry to passivate high purity process piping in lieu of using nitric acid, since nitric acid is a hazardous disposal issue once it is used for this purpose, while citric acid is not.

Citric acid is commonly used as a buffer to increase the solubility of brown heroin. Single-use citric acid sachets have been used as an inducement to get heroin users to exchange their dirty needles for clean needles in an attempt to decrease the spread of AIDS and hepatitis^[1]. Other acidifiers used for brown heroin are ascorbic acid, acetic acid, and lactic acid: in their absence, the drug injector will often substitute lemon juice or vinegar.

Citric acid is one of the chemicals required for the synthesis of HMTD; a highly heat, friction, and shock sensitive explosive similar to Acetone peroxide (also known as "Mother of Satan"). Due to this the purchase of large quantities of citric acid may be seen by some governments as an indicator of potential terrorist activity.

Citric acid can also be added to ice cream to keep fat globules separate.

Citric acid can be added to recipes in place of fresh lemon juice.

Citric acid is used along with sodium bicarbonate in a wide range of effervescent formulae, both for ingestion (e.g. powders and tablets) and for personal care (e.g. bath salts, bath beads, and a cleaning method good on grease).

When Applied to hair, Citric Acid opens up the cuticle. It is generally not recommened if you have any artificial hair coloring done, unless you are trying to strip the color out. It can be used in Shampoo(While the cuticle is open, it allows for deeper penetration of the cleaning agents.); And, it is used in the product Sun-In to bleach hair (While the cuticle is blasted open, it allows the bleaching effects of the sun to penetrate directly to the hair, taking out natural color; and, also causing a tremedous amount of damage).

Safety

Citric acid is recognized as safe for use in food by all major national and international food regulatory agencies. It is naturally present in almost all forms of life, and excess citric acid is readily metabolized and eliminated from the body.

Interestingly, despite its ubiquity in the body, intolerance to citric acid in the diet is known to exist. Little information is available as the condition appears to be rare, but like other types of food intolerance it is often described as a "pseudo-allergic" reaction.

Contact with dry citric acid or with concentrated solutions can result in skin and eye irritation, so protective clothing should be worn when handling these materials.

Alleged cancer claims

There have been erroneous reports that E330 is a major cause of cancer. It is thought that this has been brought about by misunderstanding and confusion over the word Krebs.

Citric acid is one of a series of compounds involved in the physiological oxidation of fats, proteins, and carbohydrates to carbon dioxide and water.

This series of chemical reactions, which is central to nearly all metabolic reactions and the source of two-thirds of the food-derived energy in higher organisms was discovered by the German-born British biochemist Sir Hans Adolf Krebs. Krebs received the 1953 Nobel Prize in Physiology or Medicine for the discovery, and as well as being known as the tricarboxylic acid cycle (its correct name), it is also known as the citric acid cycle or the Krebs cycle. Hence, citric acid is fundamental to the Krebs cycle and Krebs is the German word for cancer.

Production of citric acid by Aspergillus niger using cane molasses in a stirred fermentor

s: Aspergillus niger, blackstrap molasses, citric acid, fermentation, filamentous fungi, kinetic study. The present investigation deals with the kinetics of submerged citric acid fermentation by Aspergillus niger using blackstrap molasses as the basal fermentation media. A laboratory scale stirred fermentor of 15-L capacity having working volume of 9-L was used for cultivation process and nutritional analysis. Among the 10 stock cultures of Aspergillus niger, the strain GCBT7 was found to enhance citric acid production. This strain was subjected to parametric studies. Major effects were caused due to oxygen tension (1.0 l/l/min), pH value (6.0) and incubation temperature (30ºC). All fermentations were carried out following the growth on 150 g/l raw molasses sugars for 144 hours. Ferrocyanide (200 ppm) was used to control the trace metals present in the molasses medium. Ammonium nitrate (0.2%) was added as nitrogen source. Maximum citric acid production (99.56 ± 3.5a g/l) was achieved by Aspergillus niger GCBT7. The dry cell mass and sugar consumption were 18.5 and 96.55 g/l, respectively. The mycelia were intermediate round pellets in their morphology. The specific productivity of GCBT7 (qp = 0.074 ± 0.02a g/g cells/h) was several folds higher than other strains. The specific production rate and growth coefficient revealed the hyperproducibility of citric acid using mutant GCBT7.

 Citric acid fermentation is one of the rare examples of industrial

fermentation technology where academic discoveries have worked in tandem with industrial know-how, in spite of an apparent lack of collaboration, to give rise to an efficient fermentation process. The current world market estimates suggest that upwards of 4.0 x 10⁵ tonnes citric acid per year may be produced (Kristiansen et al. 1999). Citric acid is a major product but the upward trend in its use seen over many years is an annual 2-3% increase. The demand for this particular metabolite is increasing day by day which requires a much more efficient fermentation process for higher yield product (Moreira et al. 1996). When applied to appropriate mass balances, it is possible to predict the utilization of substrates and the yield of individual products. Fermentation media for citric acid biosynthesis should consist of substrates necessary for the growth of microorganism, primarily the carbon, nitrogen and phosphorus sources. Moreover, water and air can be included as fermentation substrates (Singh et al. 1998; Haq et al. 2001). The basic substrates for citric acid fermentation using submerged technique of fermentation are beet or cane-molasses (Pazouki et al. 2000). The present investigation deals with the kinetic study of citric acid fermentation. Cane-molasses was employed as the basal fermentation media in the stirred fermentor under the submerged fermentation conditions. The study revealed the nutritional status of the organism and basic fermentation parameters.

Materials and Methods

Organism and culture maintenance. Twelve stock cultures of Aspergillus niger were obtained from the culture collection of Biotechnology Research Laboratories, Government College, Lahore. These cultures have previously been developed (in our labs) by alternate treatment of ultraviolet irradiations (1.6 x 10² J/m²/S) and nitrosomonas (100 mg/ml) for different time intervals (5-45 min). The cultures of Aspergillus niger were 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. All the culture media, unless other wise stated, were sterilized in autoclave at 15-lbs/inch²pressure (121ºC) for 15 min.

Pre-treatment of molasses. Cane molasses obtained from different Pakistani Sugar Mills was used in the present study. Cane molasses contains water 20%, sugar contents 62%, non-sugar contents 10%, and inorganic salts (ash contents) 8%, making a blackish homogenous liquid with high viscosity. Ash contents include ions such as Mg, Mn, Al, Fe and Zn in variable ratio (Prescott and Dunn's, 1987). Sugar content was diluted to about 25% sugar level. The molasses solution, after adding 35 ml of 1N H₂SO₄ per litre, was boiled for half an hour, cooled, neutralized with lime-water (CaO) and was left to stand over night for clarification (Panda et al. 1984). The clear supernatant liquid was diluted to 15% sugar level.

Vegetative procedure. Hundred ml of molasses medium (Sugar 15%, pH 6.0) containing glass beads, in 1-L cotton wool plugged conical flask was sterilized. One ml of conidial suspension (6.5 × 10⁶ conidia) from the slant culture was aseptically transferred. The conidial count was made by Haemocytometer Slide Bridge. The flask was then incubated at 30ºC in an incubator shaker at 200 rpm for 24 hours.

Fermentation technique. Stainless steel fermentor of 15 L capacity with working volume of 9-L (60%) was employed for citric acid fermentation. Vegetative inoculum was transferred to the production medium at a level of 5% (v/v). The incubation temperature was kept at 30 ± 1ºC throughout the fermentation period of 144 hours. Agitation speed of the stirrer was 200 rpm while aeration rate was maintained at 1.0-4.0 l/l/min. Sterilized silicone oil was used to control foaming during fermentation.

Estimation methods. 'Mycelial dry weight' was determined according to Haq and Daud, 1995. 'Sugar' was estimated colorimetrically by Duboise method (1956). A double beam UV/Vis scanning spectrophotometer (Model: CE Cecil-7200 series, UK) was used for measuring colour intensity. 'Anhydrous citric acid' was estimated using pyridine-acetic anhydride method as reported by Marrier and Boulet, 1958. Kinetics of the research work was studied after Pirt, 1975.

Results and Discussion

Screening of stock-cultures of Aspergillus niger and molasses media

Twelve cultures of Aspergillus niger were screened for citric acid production, following growth on 150 g/l molasses sugar and incubated at 30ºC for 144 hours (found optimum). Of these cultures, Aspergillus niger GCBT7 produced higher citric acid (84.95 ± 4.0 g/l). Dry cell mass and sugar consumption were 20.05 and 91.45 g/l, respectively. Mycelial morphology was in the form of intermediate size round pellets. Three cultures gave 58.14 ± 2.7 - 78.18 ± 3.5 g/l, while four cultures produced 18.86 ± 1.8 - 42.56 ± 2.0 g/l citric acid. The citric acid productivity was greater than the 34 cultures studied by Grewal and Kalra, 1995. Cane-molasses obtained from different Pakistani Sugar Mills was screened for citric acid fermentation using the best culture of Aspergillus niger GCBT7 (Table 2

Nitrogen limitation

Nitrogen constituent has a profound effect on citric acid production because nitrogen is not only important for metabolic rates in the cells but it is also the basic part of cell proteins. Effect of different concentrations of ammonium nitrate (as nitrogen source for mycelial growth) on citric acid productivity by Aspergillus niger GCBT7 is shown in Figure 1. The maximum amount of citric acid (89.64 ± 1.5a g/l) was obtained when the concentration of NH₄NO₃ was kept at 0.2%. Any increase or decrease other than this concentration, resulted in the disturbance of fungal growth and subsequently citric acid production. The growth rate constant (µ = 0.548 ± 0.02a g^-1) indicated that enzyme to substrate ratio was optimum at 0.2% NH₄NO₃. Kristiansen and Sinclair, 1979 used continuous culture and concluded that nitrogen limitation is necessary for citric acid production. Pellet formation in filamentous fungi has been discussed in many cases and among the factors considered to induce it, is the limitation of particular nutrients, including nitrogen. In the present study, the highest values of kinetic parameters i.e., Yp/s = 0.908 ± 0.05a g/g, Qp = 0.618 ± 0.02a g/l/h and qs = 0.036 ± 0.01b g/g cells/h were observed at 0.2% NH₄NO₃.

Rate of citric acid fermentation

The optimal time of incubation for maximum citric acid production varies both with the organism and fermentation conditions. The rate of citric acid biosynthesis was studied (Figure 2a) and the maximum yield of citric acid (94.93 ± 4.2a g/l) was achieved, 144 hours after inoculation. In batch-wise fermentation of citric acid, the production starts after a lag phase of one day and reached maximum at the onset of stationary phase or late. The sugar consumption and dry mycelial weight were 92.94 and 16.15 g/l, respectively. Further increase in incubation period did not enhance citric acid production. It might be due to the decreased available nitrogen in fermentation medium, the age of fungi and depletion of sugar contents. Similar type of work has also been reported by Wieczorek and Brauer, 1998. The kinetics of citric acid production was studied using cultures of Aspergillus niger GCBT2 and GCBT7 and the results have been shown in Figure 2b. The product formation rate of GCBT7 (Qp = 0.659 ± 0.03a g/l/h) was 1.46 folds higher as compared with GCBT2 (Qp = 0.417 ± 0.05c g/l/h). Rajoka et al. 1998 obtained 0.0506 ± 0.06 g/l/h product formation rate, which is 1.58 times lower than the present results.

Incubation temperature

The temperature of fermentation medium is one of the critical factors that have a profound effect on the production of citric acid. A temperature of 30ºC was found to be the best for citric acid fermentation (Qp = 0.667 ± 0.02a g/l/h) in present studies (Figure 3). When the temperature of medium was low, the enzyme activity was also low, giving no impact on the citric acid production (Yp/s = 0.444 ± 0.08ef g/g at 24ºC). But when the temperature of medium was increased above 30ºC, the biosynthesis of citric acid was decreased (Yp/s = 0.528 ± 0.06d at 36ºC). It might be due to the accumulation of by-products such as oxalic acid. The value of specific product formation i.e., Yp/x = 6.020 ± 0.02a g/g by Aspergillus niger GCBT7 is highly significant. Different workers have also used 30ºC as the cultivation temperatures and obtained higher values of actual product (Vergano et al. 1996; Arzumanov et al. 2000). But when values were divided by the time of fermentation, all values were lower than the one supported by the isolate used in these studies.

Initial pH of fermentation medium

The maintenance of a favourable pH is very essential for the successful production of citric acid. Effect of different pH (4.5 - 7.0) on the citric acid production was studied and maximum yield (96.12 ± 3.5a g/l, anhydrous citric acid) was obtained when initial pH of the fermentation medium was kept at 6.0 (Figure 4). Decrease in pH caused reduction in citric acid production (Qp = 0.319 ± 0.03f g/l/h). It might be due to that at low pH, the ferrocyanide ions were more toxic for the growth of mycelium. This finding is an agreement with Pessoa et al. 1982. A higher initial pH leads to the accumulation of oxalic acid.

Inoculum size

Among the factors that determine morphology and the general course of fungal fermentations, the type and size of inoculum is of prime importance. In the present study, Figure 5 shows the effect of vegetative inoculum size (0.5 - 3.5%) on citric acid production by Aspergillus niger GCBT7 in stirred fermentor. Maximum citric acid acid..

Ferrocyanide ion concentration

In the present investigation, the effect of different concentrations of ferrocyanide on the production of citric acid by Aspergillus niger GCBT7 was carried out and their kinetic relations have been shown in Figure 6. The addition of ferrocyanide was made, 24 hours after the inoculation. Maximum citric acid yield (98.28 ± 4.5a g/l, anhydrous citric acid) was obtained at 200 ppm. Further increase in the concentration of ferrocyanide, both the citric acid and the amount of residual sugars were decreased. The amount of mycelial dry weight was continuously decreased by increasing the concentration of ferrocyanide beyond 200 ppm. The specific product formation rate i.e., Yp/x (4.889 ± 0.03d g/g) is highly significant and is 2.56 folds improved as compared with Pirt, 1975. Similarly, the values of growth yield coefficient (Yx/s = 0.191 ± 0.05c g/g) and product formation rate (Qp = 0.611 ± 0.04b g/l/h) indicated higher yields of the product and lower substrate consumption rates. The work is substantiated with the findings of

Concluding Remarks

The culture of Aspergillus niger GCBT7 was selected as the best mould to support maximum production of citric acid without supplements. The observation indicates that it might be possible to manipulate the morphology parameters in order to improve bioreactor performance and process yields. Substrate requirement as well as biomass and product yields are some of the basic parameters that need to be considered in determining the feasibility of the fermentation process