Citric acid is an intermediate product that is colorless, odorless, readily soluble in water and alcohol, solid at room temperature and melts at 153ºC. It occurs when carbohydrates are oxidized to carbon dioxide in the Krebs cycle (1). Its molecular weight is 192.12 g/mol, which has three different values of p
Citric acid exists widely in nature and is present in fruits such as lemon, orange, pineapple, plum, peas, peach and also in bones, muscles and blood of animals (5). It was first isolated from lemon juice by the Swedish chemist Carl Scheele in 1784 (6). However, the first commercial citric acid was produced from calcium citrate in England in 1860 (7). In addition, it was reported that Wehmer was the first researcher who demonstrated that
Citric acid is accepted as GRAS by the JECFA (9) and is widely used in the food and pharmaceutical industries (10). 70% of the citric acid produced is used in the food industry, 12% in the pharmaceutical and the remaining 18% in other industries (11,12). This organic acid has a wide area of application in the food industry due to its properties such as pleasant flavor, non-toxic agent, antioxidant (13), acidulant, flavouring agent (13, 14, 15), preservative and also emulsifier (14). It has also been used as a crosslinker in the production of detergent co-builder and biodegradable polymers in dishwasher cleaners over the past decade (16). The areas of usage of citric acid in the food industry and its functions are presented in Table 1. Citric acid production is considered a complex process that is affected by many metabolic and morphological changes (17). All the yeasts, molds and bacteria contain the citric acid cycle, but only some of them can increase citric acid production (18). It is reported that the annual production of citric acid is approximately 1.600.000 metric tons and that the biggest producer in the world is China with a share of about 40% (12). It was reported that the global volume of citric acid is over two million tons and its production has increased by 5% annually (19). The global market for citric acid is estimated to reach 3.6 billion dollars by 2020. Moreover, an annual growth rate of 5.5% from 2015 to 2020 is also expected (20). Global demand continues to increase due to its applications in nanotechnology and tissue engineering (21).
Areas of usage of citric acid in the food industry and its functions
Industry | Functions | References |
---|---|---|
Wines and ciders | - Prevention of browning in some white wines |
(10,22,23) |
Soft drinks and syrups | - Provides tartness |
(10) |
Jellies, jams and preservatives | - pH adjustment |
|
Animal feed | - Feed complementation | (10,23) |
Gelatin desserts | - pH adjustment | (22) |
Animal fats and oils | - Producing synergetic effect with other antioxidants | |
Frozen fruits | - Inactivation of oxidative enzymes |
|
Meat products | - Antioxidant | (24) |
- Prevention of coagulation or clotting of fresh blood in slaughterhouses | (25) | |
Seafoods | - Prevention of discoloration and the development of off odors and flavors by chelating trace metals | |
- Maintaining the stability and flavor by inactivating endogenous enzymes |
(26) | |
Candies | - Provides tartness |
(27) |
Dairy products | - Emulsifier in ice creams and processed cheeses |
(22,27) |
Citric acid is described as the most important organic acid produced in tonnage by microbial processes (27, 28, 29). In the world, more than 90% of citric acid production is obtained through fermentation (6,10,23).
At the end of the 1960s, it was determined that
Maximum citric acid production by several yeast strains at different fermentation conditions
Strain | Substrate | ISC (g/L) | N and P sources | Agitation rate (rpm) | Cult. | Time | pH | T (ºC) | CAmax (g/L) | ICA (g/L) | References |
---|---|---|---|---|---|---|---|---|---|---|---|
1KY 6166 | %10 | (NH4)2SO4, KH2PO4 | 600 | 100 | 5.5 | 30 | 87 | (57) | |||
2NR- | n-paraffin | 166◆ | KH2PO4, Fe(NO)·9HO 332 | 1000 | F | 168 | - | 26 | 12 | - | (71) |
RL-Y-1095 | 157◆ | Urea, KHPO, 24Fe(NO)·9HO 332 | 120 | 9.8 | (18) | ||||||
1ATCC 20367 | Molasses | %18 | Molasses + KH2PO4 | 200 | S | 144 | 6.5 | 91.4 | (59) | ||
3ATCC 20177 | 120 | 139.5 | - | 50.1 | 1.8 | (17) | |||||
240 | NH4Cl, KH2PO4 | 1000 | 58 | 5.0 | 30 | 74.2 | 3.9 | (79) | |||
2H222 | 100 | F | 92 | 6.0 | 41 | 3.05 | (50) | ||||
2D 1805 | NH4NO3, KH2PO4 | 950 | 80 | 4.7 | 95 | 10 | (80) | ||||
2Y 1095 2 | 150 | YE, NHCl, KHPO424 | 800 120 | S | 128 168 | 5.5 7.0 | 27 30 | 78.5 22.8 | 7 | (55) (1) | |
2NCIM 3472 | 100 | NaNO3, KH2PO4 | 160 | - | 5.5 | 8.4 | - | (75) | |||
2PR32 | Glucose | 60 | YE, CSL, KH2PO4, KHPO24 | 200 | 240 | 6.5 | 28 | 111.1 | (56) | ||
2VKM Y-2373 | 30 | (NH4)2SO4, KH2PO4, KHPO, Ca(NO)2432 | - | F | 144 | 6.0 | 30 | 85 | 4.7 | (81) | |
2K57 | 110 | 800 | - | 6.0 | 72.12 | (64) | |||||
2ACA-DC 50109 | 149.5 | YE, (NH4)2SO4, KHPO, NaHPO2424 | - | S | 555 | 6.5 | 28 | 42.9 | - | (65) | |
2M1 | 100 | 200 | 144 | 6.0 | 29 | 27 | (82) | ||||
2H222 | 150 | (NH4)2SO4, KH2PO4 | 171 | 132.6 | 16.4 | (83) | |||||
2H222-S4 (p67ICL1) T5 | Sucrose | 100 | Proteose peptone, NHCl, KHPO424 | - | F | 191 | 6.8 | 140 | 4 | (67) | |
2A-101-1.14 | Glucose hydrol | 400◆ | 700 | 80 | 5.5 | 100 | - | (34) | |||
21.31 | 200 | - | 132 | 124.5 | 3.9 | (84) | |||||
257 | 160 | S | 418 | 5.2 | 30 | 32.8 | - | (54) | |||
2Wratislavia AWG7 | 200 | YE, NHCl, KHPO424 | 600 | 550 | 97.8 | 5.1 | (85) | ||||
2Wratislavia 1.31 | Glycerol | 5.5 | 92.8 | - | (72) | ||||||
2Wratislavia AWG7 | 150 | 800 | F | - | 85.7 | 3.1 | (86) | ||||
2A-101-B56-5 | 100 | 72 | 6.8 | 57.15 | 0.4 | (87) | |||||
2NG40/UV7 | 20 | (NH4)2SO4, KH2PO4, KHPO, Ca(NO)2432 | 192 | 5.0 | 28 | 115 | 4.6 | (51) | |||
2LGAM S(7)1 | 120 | 185 | 236 | 5-6 | 35.1 | (88) | |||||
2ACA-50109 DC | 164 | 180 | S | 600 | 6.1 | 62.5 | - | (89) | |||
2NRRL YB- 423 | 40 | YE, (NH4)2SO4, KH2PO4, NaHPO24 | 200 | 240 | 2288 | 21.6 | 1.9 | (90) | |||
2ACA-50109 DC | 104.9 | 200250 - | F | 6.0 | 33.55 | (91) | |||||
2ACA-YC | GGllyycceerrooll | 120 | 180 | 375 | 50.1 | - | (92) | ||||
5033 | S | ||||||||||
2NCIM 3589 | 54.4 | YE, KH2PO4, Na2HPO4 | 150 | 72 | 5-6 | 77.4 | (93) | ||||
2Wratislavia K1 | 150 | YE, NH4Cl, KH2PO4 | - | 168 | 5.5 | 30 | 110 | 3.1 | (46) | ||
2A-101-1.22 | 125 | 1129 | 124.2 | 7.2 | (94) | ||||||
2N15 | 170 | YE, (NH4)2SO4, KH2PO4, KHPO, Ca(NO)2432 | 800 | 144 | 98 | 3.3 | (95) | ||||
2VKM Y-2373 | Ethanol | <1.2 | (NH4)2SO4, KH2PO4, K2HPO4, Ca(NO3)2, yeast autolysate | - | 145 | 4.5 | 28 | 116.8 | - | (76) | |
2N1 | <1 | (NH4)2SO4, KH2PO4, yeast autolysate | F | - | 21.6 | 6.4 | (74) | ||||
2VKM Y-2373 | <10 | (NH4)2SO4, KH2PO4, K2HPO4, Ca(NO3)2.4H2O | 144 | 5.0 | 29 | 22.6 | 90.5 | (68) | |||
2NTG9 | Canola oil | 15 | NH4CI, KH2PO4, KHPO24 | 800 | 216 | 30 | 137.5 | 49.2 | (78) | ||
2NG40/UV7 | Rapese- | YE, (NH4)2SO4, KH2PO4, KHPO, Ca(NO) | 168 | 4.5 | 175 | 5.6 | (35) | ||||
2187/1 | ed oil | 20 | 2432 (NH4)2SO4, KH2PO4, | - | 144 | 5.0 | 28 | 135 | (45) | ||
2N 15 | K2HPO4, Ca(NO3)2 | 4.5 | 150 | - | (96) | ||||||
2Y-UOFS 1701 | Sunf- | 30 | YE, NH4Cl, K2HPO4 | 160 | 240 | 5.8 | 26 | 18.7 | (97) | ||
2TEMYL3 | lower oil | 100 | (NH4)2SO4, KH2PO4, K2HPO4, Ca(- NO3)2.4H2O | 150 | S | 408 | 5.5 | 27 | 66.2 | 46.8 | (98) |
2SWJ-1b | Waste cooking oil | 80 | - | 250 | F | 336 | - | 28 | 31.7 | 6.5 | (66) |
2B9 | Whey+ a | 20a | 150 | 120 | 5.5 | 20 | 33.3 | 4.9 | (99) | ||
257 | Whey+ b | 150b | YE, NHCl, KHPO | - | 290 | 5.2 | 30 | 49.23 | 2.57 | (100) | |
Fructose | 200 | 424 | 191 | 65.1 | 5.58 | (101) | |||||
2ACA-DC | 65c | - | S | - | 5-6 | 28.9 | (102) | ||||
50109 2ACA-YC | OMW + c | 35c | YE, (NH4)2SO4, KH2PO4, | 180 | 144 | 6.0 | 28 | 18.9 | - | (103) | |
5033 | 80c | Na2HPO4 | 384 | 5-6 | 52 | (104) |
1: Candida zeylanoides, 2: Yarrowia lipolytica, 3: Candida oleophila, ISC: Initial substrate concentration, N: Nitrogen, P: Phosphorus, Cult: Cultivation, S: Shake flask, F: Fermenter, Time: Fermentation time, h: hours, T: Fermentation temperature, CAmax: Maximum citric acid production, ICA: Isocitric acid, -: unspecified, ◆: ml/L YE: Yeast extract, CSL: Corn steep liquor, OMW: Olive mill wastewater
a: Lactose, b: Fructose, c: Glucose
Citric acid is produced in the stationary phase of growth. The accumulation and release of citric acid in the cell are realized by different mechanisms (43). However, in a study carried out with
SmF is one of the methods used for commercial citric acid production (32). While SmF can be applied in batch, fed-batch or continuous systems, the batch system is the most preferred method (6,27). Stirred tanks with 40-200 m3 capacity or larger airlift fermenters with 200-900 m3 capacity can be used for SmF (15). The bioreactors to be used for production should be acid-resistant because of the corrosive effect of citric acid and the decreasing pH value during fermentation (32). Tower fermenters are mainly preferred in terms of price, size and operation. Fermenters are also equipped with an aeration system to maintain the high dissolved oxygen level. Cooling can be performed by an external water film (27). This fermentation takes 6-8 days depending on growth of microorganism (31).
Anastassiadis and Rehm (44) researched citric acid production by
SmF requires dilution of carbon source, pre-treatment of the appropriate nutrients and sterilization in line or in the bioreactor. This method offers advantages such as less space (32), sophisticated control mechanism (25), high productivity and yield, lower labour costs (6,25,47) and lower contamination risk (6). On the other hand, the factors such as high-cost medium, sensitivity to metal inhibition, contamination risk and high amount of post-recovery wastewater generation are considered as disadvantages for this method (25).
SSF fermentation is considered as an alternative method in which agro-industrial wastes can be evaluated for citric acid production (27,47). This type of fermentation has an advantage about bioconversion of organic solid wastes through the production of biologically active metabolites both at the laboratory and industrial scale (48). Nevertheless, this fermentation type is a small-scale operation (15). Moreover, it is considered to be the simplest method for citric acid production (6,10,27). This method was primarily applied through raw materials such as fruit wastes and rice bran (6,10). The Erlenmeyer conical flasks, glass incubators, trays, rotating and horizontal drum bioreactors, packed-bed column bioreactor, single-layer packed-bed and multi-layer packed-bed systems are used for production with SSF (23). In this fermentation, the solid substrate is adjusted to 70% moisture level, the initial pH of the medium and the incubation temperature are adjusted to about 4.5-6.0 and 28-30°C respectively (depending on the microorganism used). Fermentation lasts for 96 h under optimum conditions (10). Although
The use of yeast with high nitrogen and phosphorus requirements is not appropriate due to the lower diffusion rate of nutrients and metabolites that occur at lower water activities in the SSF (10). This method involves difficulties in control of process parameters such as pH, moisture, temperature, nutrients. Other disadvantage is higher recovery product costs due to the use of products with a higher impurity. On the other hand, this method has many advantages such as the use of simple technology, higher yield, wider range of low cost medium resembling natural habitat for various microorganisms, better oxygen circulation, less susceptibility to the inhibition of trace elements. Use of lower energy and cost requirements, lower risk of bacterial contamination and less post-recovery waste are other advantages of this method (25). It is also among the advantages that trace elements do not affect citric acid production as much as in SmF (10). As well as the advantages mentioned above, SSF requires less water usage in the upstream process and produces less waste water formation during the downstream process. On the other hand, it does not require the addition of some nutrients because of the use of agro-industrial residues (23).
Although the optimum temperature for citric acid production varies depending on the organism and equipment used, it generally varies between 26 and 35°C (18). Previous studies about the production of citric acid have indicated that 28 and 30°C were usually selected as a fermentation temperature (Table 2). Moeller et al. (50) reported that the maximum citric acid concentration (41 g/L) was obtained at 30°C. They found that the citric acid concentration decreased rapidly at 34°C compared to at 32°C. In another study, maximum citric acid was achieved at 28°C by glycerol-grown
The type and concentration of the carbon source are important for the process (10,23) and yeasts can metabolize various carbon sources such as hydrocarbons, molasses, ethanol, vegetable oils, glycerol, galactose and glucose etc. (13). The presence of carbohydrates that are fermented more quickly by the microorganism is essential for production of citric acid (10,23). The yields from glucose (g/g) decrease significantly at the concentration below 100 g/L, and that only small amounts of citric acid are produced at sugar concentration below 50 g/L (53). Karasu-Yalçın et al. (54) reported that maximum citric acid concentration and productivity were achieved at 160 g/ L initial glycerol concentration, the highest product yield was obtained at 120 g/ L initial glycerol concentration for the
The fact that many natural substrates containing carbohydrate can be used more efficiently and afford in citric acid production is important (42). It has been indicated that the yeasts such as
As can be seen in Table 2, in previous studies, ethanol, canola oil, sunflower oil, rapeseed oil, glycerol, molasses, n-paraffin, fructose, sucrose, glucose and glucose hydrol were used as carbon sources for citric acid production by yeasts.
Citric acid production is influenced by the concentration and nature of the nitrogen source (10,23). Substances such as ammonium sulphate, ammonium chloride, malt extract, peptone, urea (47), yeast extract, beef extract, dry corn-steep, soybean (60), ammonium, sodium and potassium nitrate (2) are used as nitrogen source. However, Darvishi et al. (29) reported that urea was the best nitrogen source for citric acid production. On the other hand, some media such as molasses may not need additional nitrogen sources because they are rich in nitrogen (8). Ammonium nitrate at a concentration higher than 0.25% has been found to cause oxalic acid accumulation. But, it is stated that ammonium sulphate increases citric acid production without causing oxalic acid accumulation (2).
It was determined that citric acid production by yeasts starts after depletion of the nitrogen source (58). Nitrogen restriction coupled with a high concentration of glucose is very important for the citric acid production by yeasts. Citric acid is released by a specific energy-dependent transport system induced by intracellular nitrogen restriction (61). Because citrate synthase is negatively affected by the presence of ammonium ions (41). Souza et al. (62) determined that the citric acid production began after the stationary growth phase, when extracellular nitrogen was a limiting factor for yeast growth. Anastassiadis et al. (17) calculated citric acid concentration as 80 g/ L in fed-batch fermentation with
Good adjustment of the high C/N in production with yeasts plays an important role in citric acid biosynthesis. Nitrogen deficiency in the culture substrate leads to a rapid decrease in intracellular AMP. The excessive decrease of AMP concentration in the cell reduces the isocitrate dehydrogenase activity which converts isocitric acid to α-ketoglutaric acid. Such conditions lead to intracellular accumulation of large quantities of isocitrate (63). Carsanba et al. (64) studied for the citric acid production by
It was determined that the presence of phosphate in the medium has a significant effect on citric acid yield. Low phosphate levels have an enhancing effect on citric acid production and this effect is effective at the level of enzyme activity. Moreover, it has been stated that excess of phosphate leads to a decrease in carbon dioxide fixation and causes the formation of some sugar acids and the stimulation of growth (10). Additionally, production of citric and isocitric acid has usually been actualized under the restriction of yeast growth by nitrogen or other mineral components, such as sulphur, phosphorus and magnesium (45). Liu et al. (66) obtained citric acid from waste cooking oil using
The initial pH value should be adjusted and optimized according to the microorganism, substrate and production technique (23). There are many studies showing that the initial pH value of the medium for yeasts generally varies between 5.0 and 6.5 (Table 2). It is indicated that pH is an effective factor for the productions by
Oxygen is a very important parameter for microbial growth and metabolite production (69). It is reported that the formation of all organic acids increases under high aeration and that the high oxygen requirement results from the metabolic balance of citrate formation and the high sugar concentration used (70). The dissolved oxygen concentration also affects the concentration and type of organic compounds produced by yeasts (69). Since citric acid production is also an aerobic process, the oxygen plays an important role in production (10,22,23). Citric acid yield increases depending on the increase in aeration rate, while the fermentation time is shortened (2). The interruption in aeration during batch fermentation was found to be quite harmful (10). It is very important to determine an optimal agitation rate for good production (100-180 g/L). On the other hand, high levels of agitation (1000 and 1200 rpm) can result in a shear stress on the cell-insoluble substrate interface on cell walls. Furthermore, it may lead to less cell-substrate interaction or decrease the cell viability (71). Another disadvantage of very high aeration rate (>1.0 vvm) is the low partial pressure of dissolved CO2 in the broth. CO2 is important as a substrate for pyruvate carboxylase, but excessive aeration may cause some losses. Furthermore, it was emphasized that high CO2 levels were not found useful for the final concentrations of citrate and biomass (8). The aeration of the medium is applied at the same concentration throughout the fermentation process. It is found economically favorable to choose low oxygen levels at the beginning of fermentation. However, the combination of oxygen with air in the SmF increases citric acid production, but this is economically unviable. During the growth phase, high aeration levels may cause a large amount of foam formation. For this reason, it is emphasized that antifoaming agents should be added and mechanical defoamers should be used to solve this problem (23).
Rywińska et al. (72) used glycerol as substrate for citric acid production by
As can be seen in Table 2, for citric acid production, agitation rates range from 120 to 200 rpm for shake flask cultivation and 200 to 1000 rpm for fermenter type.
It has been reported that ethyl alcohol, as well as carbohydrates, can be used in citric acid production and thus, high yields can be obtained (74). Lower alcohols such as methanol, ethanol, n-propanol, isopropanol and methyl acetate inhibit citric acid production when they are used in pure form, but if added as crude carbohydrates increase the production. The alcohols have been reported to neutralize the negative effects of metals in citric acid production (generally in amounts about 1 to 5%) (10,23). The use of methanol and ethanol in the optimal amount varies depending on the strain and composition of the medium. It was stated that alcohols are effective on membrane permeability in microorganisms by affecting the phospholipid composition (10,47). Pazouki et al. (75) reported that citric acid production by
Metal ions such as zinc, iron, copper, manganese and magnesium affect citric acid production (10). Some important integral components of metalloenzymes such as zinc and iron are involved in the yeast metabolism. Zinc is present as a component of dehydrogenases, aldolases, polymerases and proteases. It was determined that the need of zinc ions for yeasts increased in processes in which ethanol was used as a substrate. Moreover, it was indicated that NAD-dependent ADH activity under zinc restriction decreases significantly in different yeast species. Zinc is important because it acts as a stabilizer of the cell structure and some organelles (74).
Iron ions have been found to be effective on the activities of enzymes such as AIDH, AH, catalase, peroxidases and the components of the mitochondrial electron transfer chain. On the other hand, it was stated that iron ions that are present at intracellular concentrations determine yeast requirements for oxygen (74). It has also been reported that iron restriction may cause the inactivation of aconitase in the tricarboxylic acid cycle (15). Additionally, the high efficiency of citric acid may be provided with a small excess of copper ions (10).
Finogenova et al. (74) examined the citric acid production by mutant
Manganese significantly affects the idiophase metabolism. While cell growth increases in the presence of manganese, sugar consumption and acidogenesis decrease drastically. Manganese deficiency results in the suppression of anaerobic and Krebs cycle enzymes, except for citrate synthase and this event leads to an overflow of citric acid. On the other hand, magnesium has been described as a metal involved both in citric acid production and cell growth (2).
Good et al. (78) studied the effect of micronutrients on citric acid production by
The fact that the demand for citric acid with the developing industry is increasing from year to year makes citric acid production more important. The mechanism of microbial citric acid production needs to be well understood to meet the demand and to make production more affordable and efficient. The most species widely used in citric acid production is