Microbial production of propionic acid

Propionic acid is fermented by Propionibacterium freudenreichii ssp. shermanii (26), Selenomonas ruminantium (29), Propionibacterium acidipropionici (30), Propionibacterium jensenii (31), Propionibacterium thoenii (32), Veillonella gazogenes (33), Veillonella criceti (34), Veillonella alcalescens (35), Veillonella parvula (36), Megasphaera elsdenii (37), Clostridium homopropionicum (36), Bacteroides spp. and Fusobacterium necrophorum (38). Propionic acid can also be a by-product of biological fermentation for vitamin B12 (dimethyl benzimidazole as a precursor) (39), trehalose (from levulinic acid) (40) and porphyrin (by δ-aminolevulinic acid and porphobilinogen) production (41).

Larger amounts of volatile fatty acids are produced by gut microbiota through anaerobic fermentation of dietary fibre, non-volatile fatty acids and proteins (42). Dietary fibre, as the primary substrate of colon microbiota, is metabolized to pyruvate, which is converted to PA (43). Undigested carbohydrates in small intestine are fermented to propionic, butyric and acetic acids, and gases including H₂, CO₂ and CH₄ are released, together with heat due to exothermic reaction (44). Formation of volatile fatty acids in the intestine depends on different extrinsic and intrinsic factors regarding environmental conditions, substrate availability (e.g. carbon limitation) and bacterial species (45).

In biosynthesis of propionic acid from glycerol, P. acidipropionici has shown higher efficiency in terms of conversion yield and fermentation time than other strains such as Propionibacterium acnes and Clostridium propionicum (22, 46). Mutation of P. acidipropionici has lead to the increase of H⁺-ATPase expression and resistance to pH changes (14). However, it should be considered that high propionic acid concentration causes carboxylate inhibition during fermentation. Excess propionic acid can be excluded by using extractive fermentation. Low acid concentration ensures higher product yield and lower amounts of by-products (37). For propionic acid extraction, only undissociated acids are drawn out by hexane solution as the solvent (47). To find a solution to the major problem of organic acid production, acid recovery of ten solvents was examined. Alcohols and 1-butanol were considered as the best recovery solution and cost-effective extractor, respectively (48).

Supercritical carbon dioxide (solvent) and tri-n-octylamine (reactant) with high pressure (16 MPa) can be applied for PA extraction from aqueous solutions at low temperature (35 °C). These methods with 94.7% extraction efficiency are superior to the physical extraction of organic acids (49).

Recovery of propionic acid by electrodialysis from cell-free fermentation medium leads to higher product concentration (50). As opposed to aerobic fermentation, anaerobic fermentation is difficult to monitor, which could be overcome by measuring the oxidoreduction potential as an easy and cost-effective method (51). Interconversion of NADH/NAD⁺ redox pair can be used for regulation of propionic acid production through oxidoreduction potential control (52).

Besides environmental pollution from fossil resources, irreversible fuels should be substituted as their prices get higher due to depletion of petroleum (53) and necessity of specific catalysts (54). However, industrial production of propionic acid by fermentation cannot be feasible unless process cost is eqiuvalent to the production of a PA by petrochemical routes such as ethylene carbonylation, hydrocarbons and propanol oxidation (55, 56). The production of PA from industrial wastes such as glycerol or molasses makes biomass-based PA economically competitive to fossil-based PA (22, 57).

Choice of microorganism

Propionibacteria are pleomorphic catalase- and Gram-positive, anaerobic, aerotolerant bacteria that produce propionic acid as the main product via fermentation by Wood-Werkman cycle (68). There are two main pathways for the fermentation of PA from pyruvate: via decarboxylation of succinate or conversion of acrylate with lactate (precursor) (69). Three biotin-dependent carboxylases have shown to control carbon flux through the dicarboxylic acid pathway in the cycle. Their combination with glucose and glycerol as carbon sources results in increased acid concentration and higher productivity (70). Productivity can also be improved by the application of metabolic engineering, for which Escherichia coli is the most widely used host (71). Phosphoenolpyruvate carboxylase enzyme from Escherichia coli has been cloned into Propionibacterium freudenreichii. Higher propionic acid yield was produced at a faster rate by a mutant strain of Propionibacterium freudenreichii than by the wild-type (72). High propionate concentration has been achieved through fermentation of glycerol by E. coli, which is comparable to anaerobic fermentation by Propionibacterium (73).

Veillonella criceti as a Gram-negative bacterium can convert lactate to propionate with high productivity rate of 39 g/(L·h) (74). Bacillus coagulans and Lactobacillus zeae are able to convert glucose or other carbon sources to lactate (74, 75). The mutant strain of Bacillus coagulans has shown high final titre (145 g/L), yield (0.98 g/g) and d-lactate purity (99.9%) (76). To avoid product and substrate inhibition, PA (product) and lactate (substrate) should be removed from fermentor and kept at low concentrations (74).

Control of the pH during fermentation

Fermentation encounters feedback inhibition via propionic acid. This event can be controlled by different methods including choosing acid/propionate-tolerant strains, pH control by the inclusion of buffers or bases and pH adjustment and shift control strategies (37, 77, 78).

At constant pH, lactate exhibits higher product yield than glucose and lactose and limits succinic acid production. Moreover, pH control is easier when using lactate as a carbon source in immobilized cell bioreactors (continuous type) (61).

The production of propionic acid can be improved by controlling pH during fermentation (57, 79). Since the optimum pH for growth of Propionibacterium is higher than for Clostridium, a pH shift from 6 to 8 leads to a higher proportion of propionic than butyric acid from glucose medium (80). In Swiss-type cheese, reducing lactose and higher pH values (5.20-5.35) leads to acceleration of PA fermentation (81).

The acid-tolerant mutant strain of Propionibacterium acidipropionici has been physiologically (82) and molecularly (55) studied by genome shuffling and proteomics, respectively. Understanding the details of acid tolerance mechanisms and factors contributing to changes in acid accumulation may lead to an increase in propionic acid production by regulation of the fermentation process (83).

Results of serial studies demonstrate that genome shuffling can be used to produce the mutant by inactivated protoplast fusion, and acid-tolerant mutant bacteria are affected by proton pump of the membrane, glutamate decarboxylase and arginine deaminase (82). The pH change caused by the production of acid metabolites affects the membrane and cell wall structures (73). Therefore, the effect of pH is an important issue in fermentation process due to the high sensibility of biological materials. Many studies have been performed to find optimum pH for the growth of Propionibacterium. By using the strategy of pH adjustment in two stages (pH maintained at 6.5 for 48 h and then at 6.0), it was possible to increase PA yield significantly (from 14.58 to 19.21 g/L) compared to the production at constant pH=6.0 (23).

The type of substrate is another parameter that influences propionic acid yield since its conversion ratio can be directly affected. Depending on the type of substrate, the pH control might become harder to manage. It was stated that lactate, as carbon basis, presented some advantages compared to glycerol and sugarcane molasses (84). It was noticed that when glycerol and sugarcane molasses were used, faster pH variation was observed; however, it was slow when lactate was used (19).

Temperature

Temperature is an important factor in all fermentation processes that affects overall process yield by directly influencing biochemical performance. Many genera of Propionibacterium have been studied, and each genus requires different optimum temperature. In the oldest available literature, the optimum temperature was determined in the range 14–40 °C (85). In the following studies, the optimum temperature for PA production was recorded mostly between 30 and 40 °C (86, 87).

Carbon source

Many different types of carbon sources as the substrate can be considered as the most expensive conventional raw materials in the fermentation process (Table 1 (11, 14, 23, 24, 30, 58, 74, 88-96)).

To maintain high productivity and reduce the cost of production, many studies have been carried out to evaluate the possible use of cheap renewable agro-industrial sources and wastes (i.e. sugarcane molasses and glycerol) (9, 26). The use of cheap substrates including corn gluten, corn steep liquor, sulphite and wood pulp waste liquors, lignocelluloses, flour hydrolysates and whey as carbon sources (88) can be an alternative for a more viable product.

Propionibacterium is capable of consuming various sources of carbon such as glucose (89), fructose (16), sucrose (9), lactose (90), glycerol (91) and molasses (86). Different productivity and conversion yield can be achieved dependent on the type of applied carbon source. Propionic acid productivity based on glycerol (46), hemicellulose (97) and glycerol/lactate (19) as carbon sources is 0.18, 0.28 and 0.113 g/(L·h), respectively. Contrary to lactic acid and glucose, higher glycerol concentration results in increased productivity and lower conversion yield (46). Glycerol feed with the constant rate of 0.01 L/h (72-120 h) has led to the maximum PA production by P. acidipropionici that can be scaled up to industrial level (30). The productivity of PA is higher in fermentation of glycerol than in fermentation of glucose. In contrast, the increase in glucose/glycerol mass ratio increases the vitamin B12 productivity (98).

By application of vegetable oil, biodiesel industry provides a great amount of glycerol as a by-product, which can be considered as an economically viable feedstock for industrial production of PA (23). Glycerol can be used in PA fermentation as a carbon source (26, 89). Although it is an excellent reducing agent, which favours the production of PA (99), it might lead to redox imbalance in the metabolism that might affect the cell evolution and lower the yield when used as the sole carbon source in fermentation. Besides low price and availability of glycerol, its advantage is that higher yields could be obtained due to the higher average degree of reduction of carbon atoms (κ=4.67) than with glucose (κ=4) (100). Consequently, by using glycerol, the yield of PA and recovery rate from glycerol will increase while acetic acid formation will decrease (100:1) (14, 30). Therefore, reusing agro-industrial waste obtained from biodiesel can reduce the total cost of PA production up to 70% (19).

Coral et al. (19) used various carbon sources to test the effect of substrate on PA fermentation by 9 strains of Propionibacterium. Lactate showed the highest PA productivity and yield. Additionally, lactate enhanced the rate of PA production compared to molasses; it is not degraded via glycolytic route; therefore, acid biosynthesis is easier. Another advantage of lactate is that due to low pH variation during its degradation, there is no demand for constant control of pH, which is needed for glycerol and sugarcane molasses.

Mixed carbon source

Although common practice for propionic acid fermentation is usually the use of a single carbon source, this is generally not enough for the growth of Propionibacterium and production of PA. Therefore, the application of mixed carbon sources could be proposed as an effective strategy for increasing PA production through kinetics alterations.

Jerusalem artichoke-based media, which contain different carbohydrates as mixed carbon sources, were used with the addition of 10 g/L yeast extract for the production of propionic acid by P. acidipropionici, with propionic acid concentration and productivity of 40 g/L and 0.26 g/(L·h), respectively (88). The mixture of glucose and glycerol yielded 29.2 g/L of propionic acid (89). The yield was quite low, and the used medium was relatively expensive. Co-fermentation of glucose and glycerol at a suitable mass ratio gave higher yield and concentration of propionic acid.

Fermentation time

Each microorganism has a particular growth phase, which depends on the fermentation variables including pH, temperature, culture medium or desired properties of the final product. Fermentation time strongly depends on the growth rate of the microorganism. Selection of appropriate strains of Propionibacterium is also one of the most considerable factors. However, in propionic acid fermentation, its concentration could reach its maximum at a certain time of production; however, any prolongation of the bioprocess might cause a decrease in the final PA concentration. With prolongation of fermentation time, productivity is reduced due to the accumulation of inhibitory factors in the fermentor. Therefore, the optimization of fermentation duration is essential in order to obtain maximum productivity (23).

In conventional one-step batch fermentation, production period lasts up to 200 h; however, this period can be prolonged by using advanced bioreactor systems (91). By applying several repeated batch cycles with continuous recycle of cells, production time could be prolonged. During the cell-recycle fermentation for 11 consecutive batches the production time of PA by P. acidipropionici DSM 4900 lasted over 500 h (101). High concentration of PA could be produced by using the fed-batch system. The considerably high concentration of PA (71.8 g/L) was obtained after 12 days of fermentation of hemicellulose hydrolysate and corncob molasses using P. acidipropionici ATCC 4875 (9). Immobilization systems also alter PA production time radically. Immobilization of P. acidipropionici DSM 4900 in polyethylenimine-treated Luffa (PEI-Luffa) allowed a batch fermentation with a total production time of 225.5 h, which is considered longer than with free cells (126.75 h) (91). Recent studies have shown that the production of biofilm and exopolysaccharides (EPS) facilitates immobilization of Propionibacterium freudenreichii and Propionibacterium acidipropionici. The formation of biofilm and EPS can be induced by triggering factors such as NaCl and citric acid (102).

Nitrogen source

Propionibacterium spp. can digest nitrogen sources including peptone, corn steep liquor and yeast extract, which can enhance the PA production (16). Production of PA on corn steep liquor as an agro-industrial effluent exhibits relatively high yield (0.79 g/g) and productivity (5.20 mg/(L·h) (103). Although the addition of different concentrations of nitrogen source in the range 5-40 g/L was reported in various studies, the most frequent were 5 and 10 g/L (104). However, more investigations to find low-cost nitrogen sources is recommended.

Antimicrobial agent

Numerous microorganisms can produce propionic acid via fermentation, while many of them can metabolize it. PA shows inhibitory effect against the microorganisms that metabolize it by accumulation in the cells, blocking metabolic pathways and consequently resulting in the inhibition of enzymes. Depending on the concentration, PA lowers the intracellular pH and inhibits microbial growth due to anion accumulation.

Propionic acid, as a relatively strong organic acid, has been employed as an antimicrobial agent in foodstuffs such as dairy and baking products, and in animal feed preservation. Instead of using antibiotics, which could lead to antibiotic resistance, feed can be treated with PA for its protection from bacterial and fungal degradation (111, 112). PA is added to many poultry feed products to reduce the contamination by Salmonella spp. and undesired mould formation (113, 114). In addition to antimicrobial activity, the PA in feed has shown to improve ruminal productivity by enhancing substrate degradation (8%) and reducing methane production (20%) (115, 116). Unlike acetate, PA reduces the hydrogen transformation into methane (117). Application of lactic acid bacteria (LAB) can improve the production of PA by increasing the concentration of lactic acid and water-soluble carbohydrates in the rumen (118, 119).

Anti-inflammatory agent

Since the last century, there has been increasing need to discover novel anti-inflammatory agents with high efficiency for the treatment of many diseases. Several types of organic acids have been used for this purpose; however, as a prerequisite, in general, only nitrogen-free and non-steroidal compounds have been recognized as useful agents. Propionic acid, in its common chemical structure (C₃H₆O₂), is free from nitrogen so it is widely used to produce anti-inflammatory agents (120, 121).

Many different chemical groups could be added to PA to increase the anti-inflammatory effect. Studies confirmed that PA with an aryl group (profens i.e. 2-arylpropionic acid derivatives) is an important part of non-steroidal anti-inflammatory agents, which are widely prescribed against diseases such as arthritis and rheumatism (122).

Recently, new compounds have been introduced as possible additional agents with PA. Some propionic acid-based drugs used sas anti-inflammatory agents may have gastric ulcerogenic activity, which is an undesired effect for patients. 2-2-Fluoro-4-(2-oxocyclopentyl)methyl]phenyl}propionic acid can be incorporated in the formulation to eliminate this gastric effect (123).

Herbicide

Wide ranges of herbicides have been utilized in modern agricultural methods in order to eliminate the target organisms. However, these herbicides may also affect the beneficial activities of non-target organisms that grow on the crops. Thus, it is essential to use biodegradable, target-specified agents such as derivatives of PA as promising herbicides to avoid agricultural expenses (124).

Far from other available artificial herbicides, propionic acid biodegrades firstly into acetic and formic acids, then to carbon dioxide and water; thus, it does not pose any threat to the environment. It is less caustic and corrosive than formic acid, another common herbicide. If proper formulation and respiratory protection are used, PA does not cause any health hazards during apllication. Propionic acid can control both monocotyledonous and dicotyledonous plants and it is an effective pre-emergent and post-emergent herbicide (6).

Some microorganism species are capable of degrading herbicides, specifically chiral forms of mecoprop ((RS)-2-(4-chloro-2-methylphenoxy)propionic acid) with different degradation rates. Previous investigations indicated that the use of a certain form of propionic acid-based herbicides decreases the degradation of mecoprop by these organisms, which, consequently, increases the effectiveness of the mentioned agents (125). However, after the application of herbicides, it is important to remove them from the applied region since they may be considered as a potential health hazard. In order to eliminate these agents, many microorganism species may be used efficiently (126).

Preservative and safe food additive

Regarding the unstable physical conditions such as heat, excessive moisture, unpredictable rainfall, and also poor drying conditions, the addition of preservatives into food is very important since they tend to prevent the possible spoilage that could lead to food poisoning (127).

Propionic acid and its Ca, K and Na salts are common food additives used for food preservation. Wheat is usually cross-contaminated during harvest and especially in unfavourable storage conditions by fungi resulting in quality and economic losses. The use of PA and its salts may eliminate these contaminations during storage of crops (20).

Another method to increase the effect of PA as a food preservative is introducing this acid by specific carrier substances (e.g. vermiculite). The vermiculite pores of a certain diameter allow PA to penetrate particularly inside the grains.

Propionic acid is a generally recognised as safe (GRAS) food preservative. Some studies have reported that PA can exacerbate autism spectrum disorder (ASD) symptoms in humans. Besides Propionibacterium, gut bacteria produce PA by fermentation. As a result of in vivo production, PA can pass through the blood-brain and gut-blood barriers. Thereby, PA can cause neuroactive effects similar to ASD. Several cases of provoked ASD symptoms in children as a result of consumption of processed wheat or dairy products containing PA as food preservative have been reported (128, 129).