Citric Acid Production of Yeasts: An Overview

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 C. oleophila ATCC 20177 under submerged continuous system using glucose. As a result, maximum citric acid concentration and citric/isocitric acid ratio were determined as 57.8 g/L and 15.6 respectively at pH 5.0. In addition, Kamzolova et al. (45) investigated citric acid production capacity of Y. lipolytica 187/1 with submerged batch fermentation. The strain was cultivated in a fermentor. They determined that 135 g/ L citric acid was obtained when rapeseed-oil was used (under nitrogen limitation conditions). In another study conducted on an acetate-negative mutant of Y. lipolytica Wratislavia K1, highest citric acid production (110 g/ L) was determined after 168 h of fed-batch cultivation with the total glycerol concentration of 250 g/L (46).

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 A. niger is the most commonly used microorganism in this method, it has been stated that some yeasts species such as Saccharomyces cerevisiae and Zygosaccharomyces bailii could also be used in this type of production (6). Imandi et al. (49) used the statistical experimental design for the optimization of citric acid production by Y. lipolytica NCIM 3589 in SSF using pineapple waste. Finally, the optimum conditions were found to be yeast extract 0.34 (w/w%), moisture content of the substrate 70.71 (%), KH₂PO₄ 0.64 (w/w%) and Na₂HPO₄ 0.69 (w/w%). Finally, they determined that citric acid concentration at these optimum conditions was 202.35 g/kg ds.

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 Y. lipolytica NG40/UV7 and citric acid concentration decreased gradually with temperature alteration (51). Anastassiadis and Rehm (52) studied the influence of temperature on citric acid production by C. oleophila and they determined that the maximum citric acid concentration (63.5 g/L) and citric/isocitric acid ratio (28.8) were achieved at 30ºC.

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 Y. lipolytica 57 at the end of the 418 h. Rane and Sims (5) detected that citric acid production by C. lipolytica Y 1095 depend on the initial glucose concentration and fermentation time. They obtained 13.6 g/L citric acid with 50 g/ L initial glucose concentration at 27 h. Additionally, citric acid concentration was increased to 78.5 g/L with 150 g/ L initial glucose concentration at the end of the 128 h. In a study conducted on the citric acid production by Y. lipolytica PR32, it was reported that when glucose concentration in the medium was 50 g/ L, citric acid concentration formed was 24.76 g/L. However, when glucose concentration in the medium was 120 g/ L, citric acid concentration was increased to 64.58 g/ L with a yield of 0.72 g/g and a productivity of 0.45 g/L/h (56).

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 Y. lipolytica, C. guilliermondii and C. oleophila have the possibility to use a wider carbon source than fungi (especially, n-alkanes, glucose, raw glycerol, ethanol and galactose). Furthermore, yeasts can use less refined substrates (45). Hattori and Suzuki (57) used 10% (w/v) n-alkane as substrate in a fermentor, and the citric acid concentration was determined as 87 mg/ml at pH 5.5 after 100 h for C. zeylanoides KY6166. It has been stated that molasses and invert sugar mixtures may also be used for citric acid production by yeast, although fructose assimilation levels of some yeasts are low (58). Glucose syrups from starch hydrolysis, sugar beet molasses and low-quality sugarcane by-products are the most preferred substrates in industrial productions. The yield of citric acid production increases depending on the initial sugar concentration in batch processes or glucose feeding rate in the chemostat process. It is reported that the highest productivities are usually obtained when sugar is used at the level of 14-2%, since it was stated that this level leads to the suppression of ketoglutarate dehydrogenase (8). In a study, C. tropicalis ATCC 20115, C. oleophila ATCC 20373 ve ATCC 20177, C. zeylanoides ATCC 20367, Y. lipolytica ATCC 20237, ATCC 20346, ATCC 46330 and Saccharomycopsis lipolytica IFO1658 were examined for citric acid production. Fermentation was carried out in shake flasks. As a result, when sugarcane molasses was used as a substrate 91.4 g/L of citric acid was achieved with C. zeylanoides ATCC 20367 (59).

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 Candida oleophila ATCC 20177. They remarked that ammonium nitrogen was the limiting factor for citrate formation. It was also stated that nitrogen content of C. oleophila biomass decreased during the production phase. According to obtained results, low intracellular nitrogen content and NH4+ concentration were found effective for the production.

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 Y. lipolytica K57 in batch bioreactor. Finally, maximum citric acid yield (0.77 g/g glucose), titre (72.3 g/L citric acid) and productivity (0.04 g/g h) were obtained with C/N ratio of 367. Papanikolaou et al. (65) examined the citric acid production of Y. lipolytica ACA-DC 50109 using different C/N ratios (110, 138, 172 and 500 mol/mol). It was determined that when C/N ratio and initial glucose concentration were increased, biomass and citric acid concentrations were increased also. Maximum citric acid concentration (42.9 g/L) and biomass (7.2 g/ L) were calculated with 149.5 initial glucose concentration and 500 C/N ratio. Additionally, Anastassiadis et al. (44) pointed out that biomass-specific nitrogen feed rate was the most effective factor influencing continuous citric acid production at yeast-based productions.

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 Y. lipolytica SWJ-1b and they indicated that the optimal concentration of ammonium sulphate added for the production was 0.2 g/L. In this condition, it was reported that 21.4 g/L citric and 5.8 g/ L isocitric acid were obtained respectively. It was also found that extra ammonium sulphate was needed when waste cooking oil was used for citric acid production. Previous studies about the production of citric acid have shown that ammonium sulphate, ammonium chloride, yeast extract, potassium dihydrogen phosphate and dipotassium hydrogen phosphate were commonly used as nitrogen and phosphorus source (Table 2).

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 Y. lipolytica and neutral pH values are favored for citric acid productions (41). Förster et al. (67) obtained citric acid by Y. lipolytica H222-S4 (p67ICL1) T5 strain using sucrose in the fermentation medium. Fermentation was carried out in a bioreactor. According to the data obtained, when the production medium was adjusted at values of pH 5.0, 6.0 and 6.8, citric acid concentrations were calculated as 87, 127 and 140 g/ L respectively. Moeller et al. (50) investigated the effect of pH value on the citric acid production on Y. lipolytica H222. As a result of the study, maximum citric acid (24.91 g/ L), selectivity of the production (89.87%), yield (0.22 gCA/gglucose) and volumetric citric acid productivity (0.27 gCA/L h) were calculated at pH 6.0. Kamzolova et al. (68) examined the role of different pH values on the production of citric acid by Y. lipolytica VKM Y-2373 strain. According to obtained results, they stated that lowest (12.1 g/L) and the highest (40.15 g/L) citric acid concentration were obtained at pH 7.0 and pH 4.5, respectively.

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 Y. lipolytica Wratislavia AWG7 and Y. lipolytica Wratislavia 1.31. As an important result it was found that an increase in agitation and aeration rate had not any effect on dissolved oxygen concentration and citric acid production. Considering the research results, maximum citric acid concentration (92.8 g/L) and yield (0.63 g/g) were calculated by Y. lipolytica Wratislavia 1.31 at 0.24 vvm. Crolla and Kennedy (71) investigated the effect of fermentor agitation on citric acid production by C. lipolytica using n-paraffin. Eventually, it was determined that maximum citric acid concentration (12 g/ L) was achieved using 1000 rpm at 7 days with fed-batch fermentation. Sabra et al. (73) proved that control of dissolved oxygen concentration enhanced the citric acid production by Y. lipolytica in cultures grown on glucose in mono or dual substrate fermentations. Moreover, it was determined that dissolved oxygen concentration had no significant effect on the production which performed using glycerol.

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 C. lipolytica NCIM 3472 was decreased with methanol addition to glucose based medium. On the other side, it was determined that using molasses (35 kg/ m3) with methanol (3% v/v) increased the citric acid concentration. Arzumanov et al. (76) studied citric acid production by mutant strain Y. lipolytica VKM Y-2373 with using ethanol as the sole carbon source. Maximum citric acid concentration was calculated as 116.8 g/L in the batch system.

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 Yarrowia lipolytica N1 using ethanol. It was proved that the production of citric and isocitric acid from ethanol required high concentrations of zinc and iron ions. At the same time, it was stated that intracellular iron concentration determined whether citric acid or isocitric acid was predominantly formed. In another study, it was proved that iron and copper salts had inhibition effects on citric acid production. It was reported that addition of manganese salt caused a decrease for the citric acid concentration. Maximum citric acid concentration (41.63 g/ L) was achieved by Y. lipolytica 57 by addition of 0.008 g/L of zinc sulphate (77).

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 S. lipolytica NTG9 using canola oil. As a result, it was determined that iron and zinc were found to have inhibitory effects on the production. Additionally, it was found that manganese stimulated citric acid synthesis. Liu et al. (66) investigated the effects of different magnesium amounts on the citric acid production obtained by Y. lipolytica SWJ1b using waste cooking oil. They reported that best concentration for the production was determined as 1 g/ L under non-optimized conditions.