2. Properties and Applications
Citric acid (CA) exists in two forms: an anhydrous form and a monohydrate form (food grade).
Table 1 outlines the main characteristics of the two forms.
Table 1.
Physicochemical properties of CA (based on
).
Anhydrous CA is found in citrus fruits, mahogany and cotton leaves, and wild pomegranates
[4][9]. Maintaining a plasma citrate concentration of 100–150 micromoles per liter is vital for various normal physiological processes in humans and animals
[10].
CA monohydrate (a food additive E330) is used to enhance the taste, to adjust the acidity, and to improve the efficacy of antimicrobial agents and preservation properties
[3][11][12]. CA monohydrate is an ingredient in pharmaceuticals, cosmetics, and fragrances. The reaction of CA with bicarbonates, releasing CO
2, is used to improve the solubility of poorly soluble drugs
[13]. The composition containing CA is also used to remove metal oxides from the surface of ferrous and non-ferrous metals. CA helps to prevent the boiler fouling
[11]). CA is used as a feed additive in agriculture, livestock, and aquaculture
[14]. It contributes to the remediation of soil contaminated with heavy metals
[12]. CA is also used in electroplating, photography, textiles, etc.
[3][9].
Tributyl citrate, the known ester of citric acid, is a safe and non-toxic plasticizer, mostly used in the production of polyvinyl chloride, food films, cellulose resins, synthetic rubber, toys, and flexographic inks. Tributyl citrate is also used as a stabilizer for resins in cosmetics
[15].
CA salts are also in great demand in various manufacturing sectors. The most in-demand representative of CA salts is tri-substituted sodium citrate. It is utilized in the chemical, metallurgical, food, medicine, and agriculture industries. It substitutes for hazardous polyphosphates in synthetic detergents, does not corrode metals, and decomposes into carbon dioxide and water in wastewater treatment systems
[16][17].
Sodium salts of CA with varying degrees of substitution can be obtained without the need for isolating CA from the culture broth. Titration of the culture broth with NaOH to pH 4.6 produces an equimolar mixture of monosodium citrate and divalent sodium citrate, whereas titration to pH 8.3 yields tri-substituted sodium citrate
[18].
3. History
The species
Y. lipolytica has approximately twenty synonyms, of which Candida lipolytica is the most prevalent
[19].
The yeast
C. lipolytica was initially characterized by Harrison in 1928
[9]. Until the discovery of sexual reproduction, this yeast species was classified as an imperfect fungus. Afterward, it was reclassified as a perfect fungus and received various species names. Initially, it was known as
Endomycopsis lipolytica (1970), then as
Saccharomycopsis lipolytica (1972), and since 1980 as
Y. lipolytica [20].
The first publications on the production of CA by
C. lipolytica yeast came from Japan and the USSR. Tabuchi et al. (1969) reported that the
C. lipolytica No. 228 wild strain could produce 11–34 g/L of CA from glucose, acetic acid, n-butyric acid, oleic acid, fish oil, linseed oil, and soybean oil
[21]. This strain prospered from petroleum hydrocarbons, yielding 62.5 g/L of total citric acid from 60 g/L, with a CA/ICA ratio of 2:1
[22]. During that time, Finogenova and colleagues conducted research on the production of organic acids by various strains of
C. lipolytica grown on n-alkanes. Their research showed that the growth limitation by mineral components resulted in CA production
[23][24][25].
Illarionova and Suetina (1984)
[26] noted that there was considerable scientific and practical interest in the production of CA from non-food substrates between 1968 and 1981. The authors reported that 115 patents had been granted for producing CA from n-alkanes using
C. lipolytica yeast. The majority of these patents were filed by Japan, the USA, France, Germany, the GDR, and the USSR. Japan’s Kyowa Hakko Kogyo Co. and Taked Chemical Industries LTD were the front-runners in developing CA from n-alkanes.
In the UK, Pfizer played a crucial role in advancing the production of CA. The one-step continuous process with
C. lipolytica growing on a mixture of n-paraffins for 304 h was innovative at the time
[27].
In France, the Institut Français du Pétrole and the Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires were responsible for all scientific publications and patents related to the production of CA from n-alkanes
[28][29][30].
In the GDR, the Institut für Technische Chemie (Leipzig) carried out the work
[31][32].
4. Factors Affecting Citric Acid Production
Y. lipolytica yeast cannot produce citric acid (CA) in a complete nutrient medium. The principal condition of CA overproduction is the limitation of yeast growth by mineral components (nitrogen, phosphorus, sulfur, or magnesium) at a simultaneous excess of a carbon source. As microbial cells require large amounts of nitrogen for growth, the studies are typically conducted using nitrogen limitation.
Y. lipolytica yeast produces CA and isocitric acid (ICA) at the same time. The CA/ICA ratio varies depending on the carbon source used. CA is predominantly produced in the medium with glucose and glycerol, while approximately equal amounts of CA and ICA are formed with vegetable oils and n-alkanes. ICA is produced when grown on ethanol. The excretion of CA and ICA into the medium commences only after the exhaustion of nitrogen from the medium, and following the transition of the culture from the logarithmic growth phase to the growth retardation phase. The stationary phase cells remain viable, continuing to synthesize the acids until complete depletion of the carbon source
[4][20][33][34][35][36][37][38][39][40].
Y. lipolytica yeast can produce CA from different carbon sources with varying degrees of efficiency.
Figure 1 presents the maximal yields (Y
CA) obtained from different substrates: rapeseed oil
[41], n-paraffines
[42], sunflower oil
[43], raw glycerol
[44], extract of Jerusalem artichoke tubers
[45], ethanol
[46], glucose
[47], the mixture of glucose and acetate
[48], inulin
[49], glucose hydrol
[50], the mixture of glucose and oleic acid
[51], the mixture of glycerol and olive mills
[52], sucrose
[53], pure glycerol
[54], the mixtue of glucose and olive mills
[55], xylose
[56], galactose
[57], expired “waste” glucose
[58], aspen waste
[46], grape must
[59], carrot juice
[60], waste cooking oil
[61], fructose
[62], the mixture of fructose and whey
[59], waste bread hydrolysate
[63].These values were found among the wild, mutant, or genetically modified strains of
Y. lipolytica.
Figure 1. Maximal citric acid production yields (in g/g) on various carbon sources. The yields are displayed at the top of the bars.
Y. lipolytica yeast gives maximum yields from n-paraffins (1.44 g/g)
[42] and vegetable oils (1.42–1.5 g/g)
[41][43]. High yields can be obtained using glucose (0.86 g/g)
[47] and ethanol (0.87 g/g)
[46] as carbon sources.
Y. lipolytica yeast can utilize industrial waste. For instance, the mutant strain
Y. lipolytica NG40/UV7 can produce CA with a yield of 0.95 g/g from glycerol-containing waste from the biodiesel industry
[44].
Y. lipolytica yeast can utilize plant sources and waste from the food industry. For instance, the mutant strain of
Y. lipolytica A-101-1.14 can produce CA with a yield of 0.95 g/g from starch-derived glucose hydrol
[50]. The wild strain
Y. lipolytica ACA-YC 5033 can utilize the combination of olive mill wastewater with glucose or glycerol and produce CA with a yield of 0.63–0.69 g/g
[52][55]. Ra et al.
[45] achieved a yield of 0.91 g/g using
Jerusalem artichoke tuber extract. Recombinant strains of
Y. lipolytica can utilize sucrose
[53][64] and inulin
[49].
Y. lipolytica can use aspen waste
[46], xylose
[56], galactose
[57], fructose
[62], whey
[59], grape must
[59], carrot juice
[60], waste cooking oil
[61], waste bread hydrolysate
[63], sunflower waste cooking oil
[65], and straw hydrolysate
[66]. Diamantopoulou et al.
[58] converted the expired “waste” glucose to CA with a yield of 0.5 g/g. Promising carbon sources for large-scale CA production include low-cost corn syrups and corn steep liquor derived from the saccharification of corn starch
[67][68]. Gao et al.
[69] used corn stover treated with glycerol-assisted instantaneous catapult steam explosion to produce CA and mannitol. Although industrial and agricultural wastes are affordable carbon sources, making them economically preferable raw materials, their processing is challenging and arduous. These wastes can contain trace metals that can inhibit yeast growth and CA synthesis. Researchers often employ chemical pretreatment of substrates and use distilled water instead of tap water when working with waste materials
[36].
High yields can be obtained using a dual-substrate medium. Venter et al.
[70] found that acetate enhanced CA production; its addition to sunflower oil resulted in a 37-fold increase in CA production and a 2-fold increase in the CA/ICA ratio in
Y. lipolytica UOFS Y-1701. Robak et al.
[48] found that the addition of acetate to the glucose medium improved the specific production rate, yield, and productivity of CA biosynthesis by
Y. lipolytica.
It should be noted that studies comparing the effect of different carbon sources on citrate production are limited. Rywinska et al.
[71] found that pure glycerol was a superior substrate compared with crude glycerol and glucose. Celik et al.
[72] reported that sunflower oil was superior to glucose and glycerol. When comparing rapeseed oil, glucose, glycerol, ethanol, glycerol-containing biodiesel industry waste, and glucose-containing aspen waste, it was shown that rapeseed oil, ethanol, and crude glycerol allowed high CA production (100–140 g/L), whereas aspen waste resulted in a low CA concentration (31.2 g/L)
[46].
The choice of the optimum initial carbon source concentration depends on the strain, the composition of the nutrient medium, and the cultivation conditions. Usually, the optimal yields are obtained at initial carbon concentrations of 50–250 g/L
[73], with the exception of ethanol (0.1–1.0 g/L)
[46].
Y. lipolytica yeast requires nitrogen, phosphorus, sulfur, potassium, magnesium, calcium, and trace elements in its nutrient medium. The ideal dosage of these elements is dependent on the physiological characteristics of the strain and the carbon source
[36][73].
Y. lipolytica yeast can use both inorganic salts, such as NH
4Cl and (NH
4)
2SO
4, and organic compounds, such as yeast extract and peptone, as nitrogen sources for CA production. It is crucial to select an appropriate nitrogen concentration that guarantees enough cell density in the culture medium. In general,
Y. lipolytica yeast grown on pure substrates such as glucose and glycerol requires between 630 and 1200 mg/L of nitrogen
[73]. Kamzolova et al.
[74] found that the volumetric productivity (Q
CA) was very high (1.11 g/L·h) at a nitrogen concentration of 1200 mg/L. However, waste with a high nitrogen content can be utilized without adding nitrogen to the medium. Ra et al.
[45] reported that CA production from
Jerusalem artichoke tuber extract requires no nitrogen source. Moreover, Carsanba et al.
[63] observed similar results with waste bread hydrolysate. Da Silva et al.
[75] showed that the addition of ammonium sulfate to the culture medium shifts the metabolic pathway to isocitrate acid production in
Y. lipolytica grown on crude glycerol. However, it was necessary to add nitrogen and magnesium to accumulate CA from waste cooking oil
[61].
The carbon-to-nitrogen (C/N) ratio plays a critical role in the regulation of CA biosynthesis. Carsanba et al.
[76] studied the effect of initial C/N ratios (167, 367, and 567) on CA production by
Y. lipolytica K57 grown on glucose. The best C/N ratio was found to be 367, which resulted in the highest CA concentration, yield (Y
CA), and CA production rate (q
CA). Levinson et al.
[77] found that the best CA/ICA ratios in
Y. lipolytica grown on glycerol were achieved between C/N ratios of 343 and 686. Another study found that
Y. lipolytica W29 could produce CA from high fructose syrup at a C/N mass ratio of 75, but, after 15 days at a C/N ratio of 150, CA production stopped while lipid accumulation increased
[68].
Y. lipolytica yeast uses KH
2PO
4, K
2HPO
4, and Na
2HPO
4 as sources of potassium and phosphorus. The concentrations vary from 100 to 2400 mg/L
[33][48][74][75].
For intensive production of CA,
Y. lipolytica yeast require specific microelements. These microelements can either activate or inhibit enzymes involved in the metabolism of
Y. lipolytica. To activate alcohol dehydrogenase when
Y. lipolytica is grown on ethanol, it is necessary to maintain a Zn
2+ concentration of at least 1 mg/g of cells
[78].
Y. lipolytica also requires iron. Under iron deficiency, most of the acetyl-CoA does not participate in the TCA cycle but undergoes condensation to form ethyl acetate
[79]. In contrast, the increased iron concentration leads to activation of the TCA cycle, respiratory chain, and oxidative phosphorylation in
Y. lipolytica [80]. However, the substantial increase in Fe
2+ ion concentration has minimal effect on yeast growth and total amounts of acids. But, it alters the balance between these acids, favoring the formation of ICA
[81][82]. The positive effect of copper ions on the metabolism of
Y. lipolytica is well studied. The addition of 2.5–5 mg/L Cu
2+ to
Y. lipolytica yeast grown on glycerol leads to increased production of erythritol, CA, and α-ketoglutaric acid
[83]. In addition, CA production is stimulated by Mn
2+ [84]. Researchers often use tap water rather than a microelement solution
[48][53][56][85].
Y. lipolytica is unable to synthesize the thiamine molecule, i.e., the pyrimidine moiety, and requires thiamine to be added to the medium. In the absence of thiamine, the yeast produces pyruvic acid and α-ketoglutaric acid rather than CA
[86][87][88][89][90][91]. A sufficient quantity of thiamine (100–200 µg/L) should be added to the growth medium. Yeast extract can replace thiamine
[86][87][88][89][90][91]. However, an excessive amount is not recommended because nitrogen is also present, not just vitamins.
Information regarding the impact of growth temperature on CA production is limited. Morgunov et al.
[54] found that
Y. lipolytica grown on glycerol exhibited optimal CA production at temperatures between 28 and 30 °C, with the yield declining even if the cultivation temperature shifted by just 2 °C in either direction. Moeller et al.
[92] determined that
Y. lipolytica growth was ideal within the 30–34 °C temperature range. However, it was found that CA production reached its maximum levels only at 30 °C. Arslan et al.
[93] introduced the
Y. lipolytica B9 strain that is adapted to low temperatures. This strain successfully produced 33.3 g/L of CA, even at 20 °C.
The pH medium level is a critical factor affecting
Y. lipolytica yeast metabolism. Yeast grows at pH values between 2.5 and 11.5, although CA production only occurs within a pH range of 4.5 to 6.0
[73]. Zhang et al.
[94] found that the wild strain
Y. lipolytica W 29 produced CA at a neutral pH while generating lipids at an acidic pH. The authors have proposed that this pH-dependent mechanism is impacted by CA transport, rather than alterations in enzyme expression for acid production and lipid synthesis. Gao et al.
[69] found the shift from CA production at pH 5.5 to mannitol synthesis at pH 3.5 in
Y. lipolytica CGMCC 2.1506 yeast grown on corn stover enzymatic hydrolysate. Another strain,
Y. lipolytica H222, grown on glycerol, simultaneously produced CA (19.1 g/L), mannitol (18.1 g/L), arabidol (2.3 g/L), and erythritol (6.7 g/L) at pH 3.5, but at pH 5.5 it produced 42.5 g/L CA without polyol accumulation
[95]. In spite of this, some genetically modified strains have a higher pH tolerance. Mirończuk et al.
[96] found that strains overexpressing
GUT1 and/or
GUT2 have the ability to produce substantial amounts of CA from glycerol at pH 3. This capability to synthesize metabolites at a low pH has significant industrial value since it reduces production costs, prevents bacterial contamination, and maintains aseptic conditions. Another genetically modified strain exhibited higher invertase at a pH range of 6.0–6.8, thus increasing the production of CA from sucrose to 127–140 g/L. However, at a pH of 5.0, the yield dropped to 87 g/L
[64].
The production of CA by
Y. lipolytica yeast is affected by aeration, namely the oxygen saturation level (pO
2) in the culture medium. The appropriate pO
2 level is dependent upon the carbon source. For the biosynthesis of CA from n-paraffins, it is necessary to maintain optimum pO
2 levels between 70 and 90% saturation
[23]. To promote CA production from vegetable oils and ethanol, it is essential to maintain high levels of aeration, typically at 50–60% saturation
[41][97]. In order to achieve the highest levels of CA production using other carbon sources, it is important to maintain a minimum pO
2 value of 20% saturation
[72]. Liu et al.
[51] recommended increasing the oxygen saturation level by introducing oxygen vectors such as oleic acid. Bellou et al.
[98] noted that the pO
2 value, rather than the type of carbon source or the nitrogen concentration in the medium, had an effect on the morphology of
Y. lipolytica. Their research showed that, at low or zero pO
2, mycelial and/or pseudomycelial forms were more prevalent than yeast-like forms. Another important oxygenation factor is the initial volumetric oxygen mass transfer coefficient (k
La), which is sensitive to operating conditions such as stirring speed, specific air flow rate, and cell density. Ferreira et al.
[99] showed a 7.8-fold increase in CA production by increasing the initial k
La from 7 h
−1 to 55 h
−1.
Different types of bioreactors have been constructed to obtain an adequate volumetric oxygen mass transfer coefficient without high aeration rates. These bioreactors can provide significant cost savings during operations. It was observed that, using airlift and pressurized bioreactors,
Y. lipolytica W29 could produce 14 g/L and 6 g/L of CA, respectively
[100].
In summary, the essential conditions for CA synthesis by the
Y. lipolytica yeast include nitrogen limitation, carbon excess, temperature control between 28 and 30 °C, pH control of the medium to about 4.5 to 5.5, and adequate aeration.
5. Wild-Type CA Producers
Table 2 presents efficient methods for the production of citric acid (CA) by well-known wild strains of
Y. lipolytica. The strains under consideration were sourced from various countries, including Poland (A-101), the United States (ATCC 76598, ATCC 8661, and ATCC 20346), China (SWJ-1b), Germany (H 222, DSM 8218, and DSM 3286), Greece (ACA-DC 50109 [LGAM S(7)1], ACA-DC 5031, ACA-DC 5033, LMBF-46), France (W29), Russia (VKM Y-2373), and Turkey (K 57).
Table 2.
Citric acid production by wild strains of
Y. lipolytica
.