IV Международная студенческая научная конференция Студенческий научный форум - 2012

The search of new sources of vitamins and essential oils production, possessing of valuable pharmacological effects, is a modern area of biotechnology development. The use of producers of biologically active substances requires a particular research of their cultural-morphological and physiological-biochemical features, basic biotechnological rates for prediction of the culture stability in a manufacturing and a subsequent introduction in pharmaceutical industry.

Micromycetes of Eremotheciaceae family are perspective producers of riboflavin and essential oil, which is similar to a rose oil compound. As it is known these substances are pharmacologically valuable [1,2]. The riboflavin (vitamin B₂) renders metabolic action, interacting ATP and forming flavoproteins coenzymes, supports a normal state of mucous membranes of digestive tract, lips and tongue coverings, is necessary for iron absorption, and plays an important role in vision preservation. The rose oil possesses spasmolytic and mild antibacterial (bacteriostatic) effects.

The aim of this research is to study morphological and physiological features of species of Eremothecium genus and an influence of cultivation conditions on a riboflavin and essential oil accumulation.

The strains Eremothecium ashbyi Guilliermond 1935 BKM F-124, ВКМ F-3009 (selected from BKMF-124 mutant) and Eremothecium gossypii (S.F.Ashby et W.Nowell 1926) Kurtzman 1995 (syn. Ashbya gossypii (S.F.Ashby et W.Nowell 1926) Guilliermond 1928) BKMF-1398, BKMF - 2627, BKMF-3276 (selected from BKMF-1398 mutant), BKMF-3296 served as objects for this research.

The cultures were supported at 4º С on a splayed agar medium, containing soya-bean flour (4%) and sucrose (1%), a wort agar, a Saburo agar, a potato glucose agar, a meat peptone agar, a Czapek`s medium, a nutrient  agar [1]. Their morphology was studied with a microscope BIOMED-3 (zoom factor 10, 40, 100) in mounts stained a methylene blue and a black ink.

The fermentation for the study of "over synthesis" (it`s 2 words) products and their accumulation dynamics corresponded with methods [1,2] was carried out during 18...84 h in a fluid medium at continuous shaking. An inoculating material was cultivated on the fluid medium of various composition (g/l): soya-saccharose ( soya-bean flour - 20; sucrose - 20, рН 7,0) and glucose-peptone (glucose - 7,5; peptone - 4,0; sodium succinate - 2,0; K₂HPO₄ - 0,5; inositol - 0,14, рН 6,5) at continuous shaking within 24-72 hours. The soya-bean sucrose medium served as a fermentation medium. The volume of inoculants was from 1 up to 5 % (v/v).

Micromorphology. E. ashbyi has a dichotomous branching mycelium, consisting from multinuclear cells, yellowing with age. The mycelium color is stipulated by the riboflavin presence which accumulates in such quantities that can precipitate in vacuoles like crystals. The hyphae diameter varies within 2, 5...16, 5 µm. In the time of senescence some cells become sporogenous. Conidia are spindle-shaped. Sporangia are oblong, polysporous, in chains, but sometimes single, 65-90x14-20 µm. Ascospores are free located in them, release and germinate after a rupture of the ascus membrane. Spores are square-headed and needle-shaped, straight or often bent, simple, a part of the spore coarct to the end, and granules are absent. The sizes of the ascospores are following: length - 20, 2...26, 7 µm, diameter - 2, 5...2, 8 µm.

E. gossypii forms hyphae, often vacuolated and containing numerous inclusions, firstly non-septate, in the time of senescence septate, dichotomous branching, diameter 1,87...12,50 µm. The vegetative reproduction is carried out by lateral conidia or transverse hyphae splitting. The hyphae parts of some strains become inflated and thick-walled. Asci (sporefirous sacs, sporangia) are numerous, single, in groups or in chains, 100-200 µmx10-20 µm, vacuolated or containing a granulated protoplasm. Their shape is from square-headed till cylindrical or more often sigmoid. Ascospores are parallel grouped in 2 or more clusters from 2 or 6, laid along the ascus length.  Their number is from 4 till 32 per the ascus. The asci sizes are 17,5-39,3x1,31-4,6 µm. Their shape is needle-shaped or spindle-shaped, often with a thin septum in the middle.

Lipid drops has already been present at the vegetative hyphae of a daily culture (exponential growth phase). The mycelium vacuolization is marked in the period of 36...48 h of cultivation. The sporogenesis begins in the time of culture senescence, not earlier stationary phase (48...60 h of the growth). The asci with ascospores are intercalary and pullulated cells (conidia) - terminal or lateral on the mycelium hyphae. The sacs break and autolysate to release the mature ascospores, beginning from the stationary culture growth phase.

The essential differences regarding to micromorphological  characteristics  between Eremothecium ashbyi, Eremothecium gossypii (Ashbya gossypii, Nematospora gossypii) are not revealed during the carried microscopic analyze which confirms the taxonomic classification, offered by Kurtzman and based on the results of rDNA and rRNA sequence divergence and permits to attribute them to one genus of Eremotheciaceae family. The following morphological features are representative for all studied species: a possible presence of pullulated, multilateral on thin base spherical, egg-shaped, ellipsoid or cylindrical cells at certain cultivation conditions; a formation of conidia and a presence of pseudohyphae and true hyphae; asci contains 4-32 spindle-shaped or needle-shaped ascospores which are bent, have a central septum and often wedge-shaped, terminal thickening of cell wall. It corresponds with the data, adduced in scientific papers [1, 3, 4].

Physiological and biochemical properties. The features of Eremothecium species physiology were studied in different conditions of cultivation. Temperature rates, values of initial pH and aeration regimes were varied. The examined microorganisms grow up in the temperature range of 20...35º С, the optimal condition is 26...28º С (do not grow up at 37º С). The pH range for growth is 3, 2...7, 5, the optimal pH - 5, 5...6, 5. The microorganisms are aerobes in the conditions of surficial and submerged cultivation.

The use (utilization) of solitary carbon (glucose, fructose, galactose, xylose, arabinose, ramnose, maltose, saccharose, cellobiose, rafinose, inositol, dulcitole, mannitol, sorbitol,  glycerine, ethanol, glucosamine, acetate, citrate, succinate, starch, cellulose) and nitrogen (casein, gelatin, nitrate, peptone) sources by species Eremothecium genus was investigated on Czapek`s medium modifications. The consumption of solitary sources of carbon, energy, and nitrogen by examined strains occurs with a different intensity.

The study of physiological and biochemical properties gives a possibility to indentify  in a varying degree the activity of different microorganisms fermentative systems in relation to solitary sources of carbon and energy of various chemical origin. The received data shows the enzymes set which is produced by the studied micromycetes considerably differs and shows to be species typical (Table 1).

Table 1. The differential physiological and biochemical features of Eremothecium genus species

This estimation of Eremothecium biosynthetic activity revealed, geraniol, nerol, citronellol, and β-phenylethanol are basic aroma building compounds. However, the part of these substances in synthesized by E. gossypii mycelium lipids compound is considerably higher in comparison with E. ashbyi, and the monoterpene alcohols ratio is more similar to their content in a rose essential oil. The basic components and their percentage in the composition of aroma building compounds, synthesized by E. ashbyi and E. gosypii, are presented in Tables 2 and 3.

Table 2. The composition of E. аshbyi and E. gossypii aromatic products

The compounds of minor fragrant substances composition are neral, geranial, and ethers of monoterpene alcohols, linalool, and peculiar to rose oils. The certain variation of showed rates should be marked which depends on the different medium compound.

Table 3. The essential oil compound of E. ashbyi and E. gossypii strains

Dynamics of essential oil and riboflavin accumulation. The dynamics of an E.ashbyi biomass cumulation during the cultivation in the fluid medium is subordinated by known laws for periodic cultures. The growth goes exponentially till 36 h and reaches 2, 0 g of dry biomass per 1 l of cultural liquid. Then the growth speed deceleration is observed which is typical for the stationary phase and the beginning of culture autolysis till the end of fermentation. It occurs a pH shift. pH reaches to 5,5 in the period of the active growth and increases till 6,2 in the stationary phase  and the lysis phase. The synthesis and the accumulation of the riboflavin begin in the stationary growth phase and increase gradually according with culture lysis till 30 µg/g of dry biomass (Pic. 1). The maximal accumulation of a basic monoterpene alcohol of the essential oil composition in fungus (geraniol) occurs in the period between 36 and 48 h of cultivation and compounds 25 µg/g of dry biomass. It correlates with periods of the maximal synthesis of intracellular neutral lipids. The growth dynamics of E.gossypii strains is subordinated by the same laws which is confirmed by Stahmann`s and Ertrtk`s researches [5,6]. The productivity of E.ashbyi concerning the essential oil by submerged cultivation on a soya-bean fermentation medium is 99,8...141,1 µg/l, at the same time the productivity of E.gossypii reaches to 565,5 µg/l of aromatic product.

The strain BKM F-3009 is the most productive essential oil strain of E.ashbyi species, exceeded by biosynthetic activity BKM F- 124 in 1,4 times, and the strain BKM F-3276 - of Eremothecium genus, which activity is higher on 29...32 % than BKM F-3009. Therefore E.gossypii BKM F-3276 could be recommended as a producer for biotechnological production of natural aromatic product which is similar to a rose essential oil.

Influence of antibiotics on E.ashbyi and E. gossypii growth. The relation of producers cultures to antibiotics is an important rate for biotechnological production. The influence of chemically different antibiotics with the wide range of antibacterial action on E. asbyi and E.gossypii growth is presented in Table 4.

Table 4. The antibiotics influence on the growth of E.ashbyi and E.gossypii

Penicillin relates to β-lactames and has a lactam ring in the base of chemical structure, and tetracycline - a condensed system. This difference in antibiotics chemical structure set conditions for different mechanisms of their action. The penicillin disturbs a cell wall synthesis at the expense of an inhibition of peptidoglycane formation process. The tetracycline depresses a protein synthesis on ribosomal level. As it is known there are some differences in chemical compound of cell walls of bacteria and yeast-like fungi, namely the peptidoglycane is not synthesized in micromycetes cells, and accordingly there are no enzymes, regulating the last step of this process and being the target for the penicillin action. The ribosome presence which takes part in the protein synthesis process is typical for bacteria and fungi therefore the tetracycline action on E.ashbyi and E.gossypii growth is more intense in comparison with the penicillin.

Thus, the comparative biotechnological characteristic of producers permits to recommend for the production of basic end product (riboflavin) E.ashbyi  and (essential oil) E. gossypii; accordingly the essential oil can be a side additional product for the first case and the riboflavin - for the second case.

References:

1. Семенова Е.Ф. Биосинтетическая активность и антимикробные свойства Eremothecium ashbyi Guill.// Известия вузов. Поволжский регион, 2007.-Серия «Медицинские науки», № 4.-С. 44 - 50.
2. Семенова Е.Ф., Бугорский П.С. Некоторые итоги поиска биотехнологически перспективных ароматобразующих культур// Труды / ВНИИ эфиромасличных культур. Симферополь, 1989. - Т. 20. - С. 14-16.
3. Kurtzman C.P. Relationships among the genera Ashbya, Eremothecium, Holleya and Nematospora determined from rDNA sequence divergence// Journal of Industrial Microbiology, 1995. - Vol.14. - P. 523-530.
4. The yeast, a taxonomic study. Ed. by Кurtzman С.Р. Fell J. W. Fourth Edition. Elsevier Science, 1998. - 1055 p.
5. Formation and degradation of lipid bodies found in the riboflavin/producing fungus Ashbia gossipii / K.-P. Stahmann , C. Kupp, S. D. Feldmann, S. Sahm// Appl. Microbiol. Biotechnol. - 1994. - № 42. - Р.121-127.
6. E. Ertrk, O. Erkmen, M.Oner. Effects of various supplements on riboflavin production by Ashbya gossypii // Tr. J. of Engineering and Environmental Science - 1998. - № 22. - Р.371-376.

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