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The main stages in the production of biodiesel fuel from lipids of filamentous fungi belonging to the order Mucorales are described. Fungi of the family Cunninghamellaceae have been screened; the lipogenic activity of the examined strains has been assessed; and a producer generating up to 50% of lipids, represented by triacylglycerols, has been found. The substitution effect of a source of carbon and nitrogen with less expensive components (in particular, various industrial wastes) has been studied, as well as their influence on the quantity and major characteristics of the final product. An ecologically friendly method for extracting lipids from fungal mycelia, utilizing supercritical technologies, has been used. A correlation between the lipid content in the spore inoculum and the maximal lipid content in biomass has been discovered; this correlation is proposed for optimizing the biotechnology and increasing the yield of final products.

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking beta-carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene production results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on beta-carotene synthesis.

Biotransformation of beta-carotene with enzyme preparations isolated from the mycelium of Blakeslea trispora resulted in the formation of its hydroxylated metabolite and apocarotenals, products of oxidative degradation of this compound. By spectral, chromatographic, and chemical properties, the beta-carotene derivative was identified as 4-hydroxy-beta-carotene (isocryptoxanthine). One of the products of oxidative degradation of beta-carotene, beta-apo-13-carotenone, underwent modification in the presence of enzyme preparations from Blakeslea trispora with the formation of trisporic acid precursors. It should be emphasized that beta-apo-13-carotenone transformation proceeded more rapidly than beta-carotene oxidation by carbon in the 4-position. Our findings suggest that under conditions of oxidative stress, oxidative degradation of beta-carotene into beta-apo-13-carotenone leads to the formation of considerable amounts of trisporic acids.

The composition of fatty acids synthesized de novo by thirty strains of zygomycetes from various taxa was studied. The qualitative fatty acid compositions of the fungal lipids were found to be virtually identical, but there were significant differences in the contents of individual acids. Highly active producers of essential C18 fatty acids, with their content exceeding 30-40% of total fatty acids, were discovered among the fungi of the families Mucoraceae, Pilobolaceae, and Radiomycetaceae. Linoleic acid was found to predominate in the fungi of the genera Radiomyces, Mycotypha, and Circinella, and linolenic acid (identified as its gamma-isomer by gas-liquid chromatography), in the fungi of the genera Absidia, Circinella, Pilaira, and Hesseltinella. The total yield (mg/l) of bioactive acids (C18:3, C18:2, C18:1) varied from 761.4 in Pilaira anomala to 3477.9 in Syncephalastrum racemosum; the total yield of essential acids, from 520.7 in Pilaira anomala to 1154.5 in Hesseltinella vesiculosa; of linoleic acid, from 279.7 in Pilaira anomala to 836.3 in Mycotypha indica; and of linolenic acid, from 120.8 in Mycotypha indica to 708.0 in Hesseltinella vesiculosa. The data on the efficient synthesis of these acids make the actively producing strains promising for biotechnological synthesis of commercially valuable lipids. Linderina pennispora VKM F-1219, a zygomycete of the family Kickxellaceae, which was earlier singled out into the order Kickxellales [12], was shown to differ from zygomycetes of the order Mucorales in having a high content of cis-9-hexadecenoic (palmitoleic) acid, reaching 37.0% of the fatty acid total.

Changes in the composition of neutral lipids and phospholipids were studied during the growth of Cunninghamella japonica under the action of cycloheximide and actinomycin D. The data were used to discuss how the synthesis of enzymes catalysing the formation of individual lipid classes was regulated and whether it would be possible to control the composition of phospholipids and neutral lipids using inhibitors of RNA and protein synthesis. The results are also indicative of a certain correlation between growth phases of the fungus and changes in certain characteristics of membrane lipids.

The transformation of acetate from the Reichstein substance "S" by the culture of Tieghemella orchidis was studied upon a single and fractional addition of the substrate. The effect of streptomycin sulphate, Tween-80, ethanol (steroid solvent) and steroid solution on the respiratory activity of the culture was investigated upon a single and fractional addition of the steroid. During transformation the respiratory activity of the culture decreased--by the end of the process (18-20 hours) it fell 2-2.5-fold upon a single addition of the steroid (1 g/l) and by 20% upon a fractional addition of the steroid (2 g/l). In the latter case the respiratory activity dropped step-by-step if the concentration of the steroid was 1.4-1.6 g/l; simultaneously the transformation activity declined.

Transport of 14C-biotin was studied in cells of different biotin-prototrophous yeast and mold fungal strains. An inverse correlation was established between the capability of the fungi to synthesize biotin and the exogenous vitamin transport: 14C-biotin did not penetrate into the cells of strains which excreted great quantities of the vitamin. It is likely that a higher level of biotin synthesis in certain fungi is caused by a peculiar transport system, which results in a one-way permeability of their cell membrane for biotin. Biotin is eliminated from the cell and cannot repress its own synthesis. Active transport of biotin in the studied prototrophous organisms occurs against a concentration gradient and does not depend on the presence of glucose in the medium. There are apparently other energy sources for this process.

The growth, assimilation of organic nitrogen compounds, and production and distribution of free amino acids were studied during the growth of Cunninghamella elegans (--) 1204 on the defined medium 12 containing glucose and dodecane and without an additional carbon source (control). The culture utilized all leucine, glycine, and asparagine after three days of the growth, irrespective of the source of carbon in the medium. Glutamic acid was assimilated only in the presence of glucose. Some 15 free amino acids and 2 amides were detected in the cells, and also some correlations in the composition of the pool in the presence of different sources of carbon in the medium. The substitution of dodecane for glucose decelerates metabolism of free intracellular amino acids and stimulates their accumulation in the cells, especially if there is a deficiency of carbon sources: the overall content of amino acids and amides on the medium with glucose is from 2.2 to 4.1 mg/g; with dodecane, from 9.8 to 16.5 mg/g; in the control, from 16.4 to 25.8 mg/g.

Carotene synthesis by Blakeslea trispora is accompanied by a decrease in the rate of the main metabolic processes (protein synthesis, respiration) and changes in the bioenergetic pool of the mycelium. Considerable changes occur at the same time in the cytology of the microscopic fungus: the cell wall of the culture containing considerable amounts of carotene consists of many layers and peculiar thickened regions; partitions are formed; the nuclei are in the stage of interphase; lipid inclusions are larger and their number is higher.

Trisporic acids were found to increase the content of carotene, protein, and RNA in the (-)strain of Blakeslea trispora. Trisporic acids are inductors of the synthesis of carotenogenous enzymes as was found in experiments with the inhibitor of transcription (cycloheximide) and translation (actinomycin D). Trisporic acids stimulate the formation of neutral fractions of the sex hormone, the process being regulated presumably at the level of posttranscription control.

beta-Ionone is found to stimulate considerably carotinoids synthesis in Blakeslea trispora. The stabilization of carotene-synthesizing ability of B. trispora in the presence of beta-ionine under prolonged incubation time is observed. Stabilization of polyribosomes in the presence of beta-ionine is observed when studying polyribosome content in B. trispora. A hypothesis is expressed on the existence of biochemical "receptors" as a linkage between synthesized protein and destroying mRNA.

The ability to oxidize n-alkanes was studied with various species of fungi belonging to the Cunninghamella genus. These fungi are able to assimilate hydrocarbons and to accumulate up to 1.5 g/litre of biomass. The most active strain was Cunninghamella elegans (-) 1204. The amount of lipids formed, and their composition, depended on the length of the carbon chain of oxidized alkane. The content of fat in the cells increased with the length of the hydrocarbon chain. The following lipid fractions have been detected: phospholipids, monoglycerides, diglycerides, triglycerides, sterols, free fatty acids, sterol esters, and hydrocarbons. The qualitative composition of the fractions depended, to a considerable extent, on the n-alkane utilized. Investigation of the fatty-acid composition of intracellular lipids has shown that fatty acids with an even number of carbon atoms are formed from hydrocarbons with an even number of these atoms, while fatty acids both with an even and odd number of carbon atoms are synthesized from hydrocarbons with an odd number of these atoms. The relative content of the acids with the same number of carbon atoms as that of the alkane being utilized increased with the length of the carbon chain.

Dimilin, an insecticide, was added together with a number of surfactants; it was found to penetrate into the cell of microscopic fungi where it slightly inhibited biomass production but had no effect on chitin biosynthesis. The insecticide was shown to be capable of inhibiting the secondary processes of biosynthesis, namely, carotene synthesis in the fungus. A possible mechanism of dimilin action in insects and fungi is discussed on the basis of the results.

The rate of growth of Cunninghamella elegans (--) 1204 is higher on a mineral medium with glucose (6.56 g/litre) than on a mineral medium containing undecane, tridecane, and pentadecane (0.72--0.87 g/litre); all glutamic acid is consumed only from the medium with glucose. The cells contain 15--16 free amino acids and 1--2 amides, glutamic and aspartic acids and alanine prevailing. The culture grown on the medium with glucose contains asparagine, and the cells cultivated on the medium with alkanes contain histidine. Non-proteinogenous aminobutyric acids were found in the pool of the cells grown on all tested media with an exception of the medium containing undecane.

Retinol stimulates carotene synthesis in the microscopic fungus Blakeslea trispora; the stimulating effect of vitamin A is not manifested in the presence of cycloheximide. Vitamin A and beta-ionone are presumed to affect carotene synthesis in Blakeslea trispora by imitating the action of natural compounds stimulating isoprenoid synthesis--trisporic acids.

The effect of exogenous lipid sources on the composition of fatty acids was studied in actinomycetes of the Streptomyces genus and in fungi belonging to the genera Blakeslea, Cunninghamella and Penicillium. The following sources of exogenous lipids were used: soybean and maize flour, sunflower by-products, chicken droppings, maize extract, yeast extract, peptone, sperm whale fat, sunflower and palm oil. The composition of fatty acids in total extracted lipids of the studied mycelial microorganisms was shown to reflect two processes: lipid synthesis de novo and assimilation of exogenous fatty acids. This fact ought to be taken into account both in the chemotaxonomic interpretation of fatty acid composition and in practical recommendations for the utilization of microbial lipids. It is of particular interest to study the physiological role of exogenous lipid metabolism in the cells of microorganisms.

By-products of the transformation were studied, which were formed in the process of 11alpha and 11beta-hydroxylation of 21-acetate of the Reichstein substance "S" by the culture of Tieghemella orchidis. The following steroid derivatives, known before, were isolated: epihydrocortisone, 6beta-hydroxy derivative of the Reichstein substance "S", hydrocortisone, cortisone. The following steroid derivatives were isolated from the initial chloroform extract of the fermentation medium, and identified: prednisolone, androstendione, testololactone, and 1,2-dehydro derivatives of the Reichstein substance S" and its 21-acetate. The quantitative ratio between the products of transformation depended on the conditions of growth and transformation. These compounds seem to be intermediate metabolites in the course of the steroid destruction via the pathway common for fungi and involving the destruction of D ring of the steroid molecule.

Three groups of Blakeslea trispora (+) and (-) mutants were obtained and their phenotypical characteristics were studied as well as biochemical changes in the course of mating and the ability to synthesize carotenoids when the sexual process of these mutants was disordered. The first group of mutants synthesized carotenoids at the control level, the second group produced them below the control level, and the third group accumulated less than 1% of carotenoids as compared to the control. The difference in the yields of carotenoids among the three groups of mutants in the mated culture is attributed to the presence (or absence) of the ability to synthesize trisporic acids in them.

The cell wall of Blakeslea trispora was found to contain chitin similar to crustaceaous chitin in physico-chemical properties; this was confirmed by the data of IR spectroscopy and X-ray diffraction analysis. Trisporic acids, hormonal regulators of reproduction, hardly affected the content of chitin, xylan, and other polysaccharides of the cell wall of Blakeslea trispora, but increased the content of protein.