Svetlana V. Kamzolova

Organic Acid Production by the Yeast Yarrowia lipolytica: A Review of Prospects

The review sums up the results of studies of (1) physiological growth characteristics of the yeast Yarrowia lipolytica cultured in the presence of diverse carbon sources (n-alkanes, glucose, and glycerol) and (2) superhigh synthesis of organic acids, which was performed at the Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. Microbiological processes of obtaining α-ketoglutaric, pyruvic, isocitric, and citric acids are discussed.

Oxygen requirements for growth and citric acid production of Yarrowia lipolytica

During continuous cultivation of Yarrowia lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y. lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 were proposed. At relatively low pO(2) value and a high iron concentration, citric acid accumulation was as high as 120 g l(-1); the specific rate of citric acid synthesis reached 120 mg citric acid (g cells h)(-1). The mass yield coefficient was 0.87 and the energy yield coefficient was 0.31.

Citric acid production patent review.

Current Review article summarizes the developments in citric acid production technologies in East and West last 100 years. Citric acid is commercially produced by large scale fermentation mostly using selected fungal or yeast strains in aerobe bioreactors and still remains one of the runners in industrial production of biotechnological bulk metabolites obtained by microbial fermentation since about 100 years, reflecting the historical development of modern biotechnology and fermentation process technology in East and West. Citric acid fermentation was first found as a fungal product in cultures of Penicillium glaucum on sugar medium by Wehmer in 1893. Citric acid is an important multifunctional organic acid with a broad range of versatile uses in household and industrial applications that has been produced industrially since the beginning of 20(th) century. There is a great worldwide demand for citric acid consumption due to its low toxicity, mainly being used as acidulant in pharmaceutical and food industries. Global citric acid production has reached 1.4 million tones, increasing annually at 3.5-4.0% in demand and consumption. Citric acid production by fungal submerged fermentation is still dominating, however new perspectives like solid-state processes or continuous yeast processes can be attractive for producers to stand in todays strong competition in industry. Further perspectives aiming in the improvement of citric acid production are the improvement of citric acid producing strains by classical and modern mutagenesis and selection as well as downstream processes. Many inexpensive by-products and residues of the agro-industry (e.g. molasses, glycerin etc.) can be economically utilized as substrates in the production of citric acid, especially in solid-state fermentation, enormously reducing production costs and minimizing environmental problems. Alternatively, continuous processes utilizing yeasts which reach 200-250 g/l citric acid can stand in todays strong competition in citric acid industry and replace the traditional discontinuous fungi processes.

Characteristics of the growth on rapeseed oil and synthesis of citric and isocitric acids by Yarrowia lipolytica yeasts

The native strain Yarrowia lipolytica VKMY-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapesseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.

Production of technical-grade sodium citrate from glycerol-containing biodiesel waste by Yarrowia lipolytica.

The production of technical-grade sodium citrate from the glycerol-containing biodiesel waste by Yarrowia lipolytica was studied. Batch experiments showed that citrate was actively produced within 144 h, then citrate formation decreased presumably due to inhibition of enzymes involved in this process. In contrast, when the method of repeated batch cultivation was used, the formation of citrate continued for more than 500 h. In this case, the final concentration of citrate in the culture liquid reached 79-82 g/L. Trisodium citrate was isolated from the culture liquid filtrate by the addition of a small amount of NaOH, so that the pH of the filtrate increased to 7-8. This simple and economic isolation procedure gave the yield of crude preparation containing trisodium citrate 5.5-hydrate up to 82-86%.

Succinic acid production from n-alkanes

A novel process of production of succinic acid (SA) has been developed, which includes the synthesis of alpha‐ketoglutaric acid by a thiamine‐auxotrophic yeast strain Yarrowia lipolytica VKM Y‐2412 from n‐alkanes and subsequent oxidation of the acid by hydrogen peroxide to SA. The concentration of SA in the culture broth and its yield were found to be 38.8 g/L and 82.45% of n‐alkane consumed, respectively. The isolation procedure involved the extraction of the residual alkanes with the mixture of ethyl acetate and hexane, the decomposition of H2O2 in the filtrate followed by filtrate bleaching and acidification with a mineral acid; the evaporation of filtrate and the ethanol extraction of SA from lyophilized residue. The purity of the SA isolated from the culture liquid filtrate reached 99.5%.

Mutant Yarrowia lipolytica strains producing citric acid from glucose

The possibility of obtaining mutant yeasts Yarrowia lipolytica VKM Y-2373 with increased ability to synthesize citric acid from glucose by using UV irradiation and N-methyl-N’-nitro-N-nitrosoguanidine was studied. Of 1500 colonies of the Y. lipolytica treated with either UV or N-methyl-N’-nitro-N-nitrosoguanidine, three mutants were selected that displayed higher (by 23%) biosynthetic ability as compared with the initial strain. Additionally, three mutants were selected from 1000 colonies of the Y. lipolytica exposed to a combined action of UV and N-methyl-N’-nitro-N-nitrosoguanidine; their biosynthetic activity exceeded that of the initial strain by 43.9%. The selective media with citrate and acetate were developed for a rapid selection of mutants as well as the express methods for the detection of active citric acid producers on the solid media with chalk and bromocresol containing a limiting concentration of amine nitrogen and an excess of glucose.

Arachidonic acid as an elicitor of the plant defense response to phytopathogens

Review summarizes both literature and own experimental data on the application of arachidonic acid (AA, C20:4 omega-6) as an elicitor of the plant defense reactions to phytopathogens. The elicitation activity of AA was shown to depend considerably on its concentration. High AA concentrations (over 10-5 M) induce necrosis of plant tissues and accumulation of antimicrobial compounds (phytoalexins), whereas low AA concentrations (10-7–10-7 M) elicit systemic and prolonged resistance to the phytopathogen infections that is similar to immunization process. Biochemical mechanisms responsible for the elicitor activity of AA involve the whole complex of reactions including reconstruction of the cell ultrastructure, an increase in the amounts of certain enzymes and protective substances, a decrease in sterol content, redirection of isoprenoid biosynthesis from sterol derivatives toward sesquiterpenoid phytoalexins, and appearance of signal molecules, which are spread all over the plant tissues making them immunized. Field experiments revealed that the treatment of potato, tomato, sugar beet, and vine plants with low concentrations of pure AA or AA-containing preparations isolated from Phytophtora and Mortierella fungi increased plant resistance to diseases (late blight, common scrab, rhizoctonoise, cercosporose, powdery mildew, etc.) and thus enhanced the harvest yield. The application of AA-containing preparations is an alternative to the use of environmentally dangerous chemical fungicides. The processes of microbiological AA production from renewable inexpensive raw substrates are considered.

Application of organic acids for plant protection against phytopathogens

The basic tendency in the field of plant protection concerns with reducing the use of pesticides and their replacement by environmentally acceptable biological preparations. The most promising approach to plant protection is application of microbial metabolites. In the last years, bactericidal, fungicidal, and nematodocidal activities were revealed for citric, succinic, α-ketoglutaric, palmitoleic, and other organic acids. It was shown that application of carboxylic acids resulted in acceleration of plant development and the yield increase. Of special interest is the use of arachidonic acid in very low concentrations as an inductor (elicitor) of protective functions in plants. The bottleneck in practical applications of these simple, nontoxic, and moderately priced preparations is the absence of industrial production of the mentioned organic acids of required quality since even small contaminations of synthetic preparations decrease their quality and make them dangerous for ecology and toxic for plants, animals, and human. This review gives a general conception on the use of organic acids for plant protection against the most dangerous pathogens and pests, as well as focuses on microbiological processes for production of these microbial metabolites of high quality from available, inexpensive, and renewable substrates.

[The isolation, purification, and some properties of NAD-dependent isocitrate dehydrogenase from the organic acid-producing yeast Yarrowia lipolytica].

The NAD+-dependent isocitrate dehydrogenase of the organic acid–producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 μM, and that for isocitrate is 581 μM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.