Method of production of bacteriologically pure cultures of marine blue-green microalgae

FIELD: biotechnology.

SUBSTANCE: method of production of bacteriologically pure cultures of marine blue-green algae provides chemical sterilisation of microalgae cultures by treatment them in the solution of sterile sea water containing 0.1% phenol and 1.0% ethyl alcohol. Microalgae are kept in sterile sea water containing phenol and ethyl alcohol in a predetermined ratio for 4-5 hrs. Irradiation with UV light is carried out for 9-10 minutes. The treatment of microalgae with antibiotics and fungicide is carried out. At that the antibiotics are used as chloromycetin and ampicillin in the amount of 50 µg/ml each, and fungicide is used as nystatin in the amount of 25 µg/ml.

EFFECT: invention enables to improve the efficiency of extraction of bacteriologically pure culture of blue-green microalgae.

1 tbl, 20 ex

 

The invention relates to Algologie and Microbiology, and in particular to methods of allocation bacteriologically pure cultures of marine blue-green microalgae.

Currently, much attention is paid to the development of test systems for the evaluation of toxicity of the components of the marine ecosystems of water bodies, which requires the isolation of pure cultures of aquatic organisms, in particular microalgae. The standard procedure consists of isolating and growing algological pure cultures of algae, which then selects the most promising for biological testing. However, cultures of microalgae always be contaminated by bacterial microflora, spores and mycelium of fungi, which prevents further research, distorts the results. Purification from bacteria is a very time consuming process, because between algae and bacteria are dense biocenotic relationships. The mucous membranes of the algae serve as food source and shelter for microorganisms. Such a close relationship determines the difficulty of separating these communities for the isolated culture: algae and bacteria. Getting bacteriologically pure cultures of microalgae is a prerequisite for successful research, development, test systems for the evaluation of toxicity of the components of the ecosystem the marine waters and other scientific works.

There is a method of allocating axenically cultures of microalgae method agar plates (Wasser S. P., Kondratieff N. In., Masuk N. P. and other Algae. The Handbook. - Kiev: Naukova Dumka, 1989. - 608 C.) (1), in which microstrokes the suspension is introduced into the Petri dish and the microbial loop are distributed uniformly on the surface mycopathologia agar (MPA) followed by incubation in a thermostat. Then separate micro-colonies grown on the surface mycopathologia agar, remove sterile instruments together with pieces of dense nutrient medium and bring in a test tube with the appropriate sterile elective nutrient media for the accumulation of algae biomass. Exericise culture of algae get through repeated re-sieving on the surface of MPA in sterile conditions.

The known method has a limited range of application. For example, when selecting axenically cultures of the marine blue-green algae is not able to achieve their full freedom from bacteria, since the latter are strongly associated with strongly developed mucous membranes of these microalgae. The process of separating them tedious, time-consuming and sometimes inefficient, requires multiple re-seed.

In another method of purification of cultures of bacteria and other microorganisms carry out the irradiation of cultures of microalgae ultra olejowym light (K. Kvitko Century Obtaining cultures of individual cells of Chlorella // Research on genetics. - L.: Publishing house of Leningrad University, 1961. - Vol.1. - S. 50-54) (2). However, this method is not effective enough, because you cannot achieve full freedom of algae on bacteria and fungi. While the duration of UV exposure should not exceed 10 minutes, because the longer UV exposure to skin and eye irritant and can cause irreversible genetic changes irradiated culture.

Closest to the proposed method is chosen as the prototype of the method of purification of cultures of bacteria and other microorganisms (especially fungi) using chemical sterilization by treatment with 0.1% phenol and 1.0% ethyl alcohol. (Ecological physiology of marine planktonic algae. Kyiv: Publishing house "Naukova Dumka", 1971, S. 5-22) (3).

However, the chemical sterilization of microalgae also allows you to get a fully bacteriologically pure cultures of algae.

The inventive method solves the problem of increasing the efficiency of the allocation of bacteriologically pure cultures of marine blue-green microalgae.

This object is achieved in that a method of obtaining a bacteriologically pure cultures of marine blue-green algae consists of chemical sterilization Kul is ur microalgae by treatment in a solution of sterile sea water, containing 0.1% phenol and 1.0% ethanol, and additional irradiation with ultraviolet light for 9-10 minutes, and then treatment with antibiotics and fungicides, and the use of antibiotics chloramphenicol and ampicillin in the amount of 50 µg/ml each, and as a fungicide use nystatin in the amount of 25 μg/ml

Additional processing of microalgae after chemical sterilization first irradiation with ultraviolet light, and then antibiotics and fungicide, allows to obtain bacteriologically pure cultures of algae.

While the use of the antibiotic chloramphenicol in the amount of 50 μg/ml and ampicillin at 50 μg/ml, and as a fungicide - nystatin in the amount of 25 μg/ml allows to obtain, on the one hand, the almost complete absence of bacteria in the culture of marine blue-green microalgae, on the other hand, to avoid a decrease in growth rate and inhibition division of microalgae.

As a result of the analysis of the prior art is not detected similar, characterized by signs, identical with all the essential features of the claimed invention, and the definition of the prototype of the identified analogues revealed a set of essential towards the technical result of the distinctive features.

Therefore, the claimed invention meets the condition of "novelty."

With additional search other solutions related to the proposed method, these distinctive characteristics are not detected. Thus, the claimed invention meets the condition of "inventive step".

The method is as follows.

Cleaning algological pure cultures of marine blue-green microalgae from bacteria is carried out in 3 stages. stage 1 - this chemical sterilization. For this purpose apply antiseptics such as phenol and ethyl alcohol at a concentration of 0.1 and 1.0%, respectively. Algae incubated in sterile seawater with the above antiseptics for 4-5 hours On the 2nd stage, hold 9-10-minute exposure of the cultures of algae with UV light (bactericidal irradiator OBN - 150,2 lamp 30 W) in Petri dishes at a distance from the radiation source 50, see the 3rd stage spend processing algae antibiotics and fungicide in the following optimal combinations of chloramphenicol at a concentration of 50 μg/ml ampicillin at a concentration of 50 μg/ml and nystatin at a concentration of 25 μg/ml

The presence of bacterial microflora controlled by reseeding from liquid cultures of the marine blue-green microalgae on agar or liquid culture of Abijah. Controlled growth IU filnik aerobic and facultative anaerobic microorganisms, fungi and yeast.

In the determination of mesophilic aerobic and facultative anaerobic microorganisms in liquid culture of microalgae the study was performed according to GOST 10444.15-94.

In the determination of mesophilic aerobic and facultative anaerobic microorganisms on agar nutrient medium, and 1 CC of culture of blue-green algae were sown in two parallel Petri dishes. Crops were flooded by GOST 26670 one of the nutrient agar media and incubation at 30°C.

In the determination of mesophilic aerobic and facultative anaerobic microorganisms in a liquid nutrient medium and 1 CC of culture of blue-green algae were sown in two flasks or test tubes with one of the liquid nutrient media according to GOST 26670. The ratio between the number of planted crops of algae and nutrient medium from 1:5 to 1:7. Crops were incubated at a temperature of 30±1°C for 72±3 h under aerobic conditions.

After incubation on agar nutrient media in Petri dishes take into account the presence or absence of colonies of microorganisms. In liquid nutrient media noted the presence or absence of visible signs of growth (flatulence, the appearance of turbidity, sediment).

If the apparent bacterial growth after three days of incubation was not considered unlikely that the sample of algae, dobavlenny into the culture medium, contains bacteria. However, the culture continued to watch for 7-10 days, as it could be slow-growing species. Conclusions about the purity of the cultures was done in the absence of bacterial colonies or visible signs of their growth.

When determining fungi and yeast in liquid culture of microalgae the study was performed according to GOST 10.444.88.

1 CC of culture of algae were sown in two Petri dishes with medium Saburo with antibiotics. Crops thermostatically at a temperature of 24±10C for 5 days. After incubation noted the presence or absence of visible signs of growth. Colonies of yeast and fungi were separated visually (yeast form large shiny convex colonies with smooth surface and straight edge, fungi form a mycelium of different colors).

In the absence of mesophilic aerobic and facultative anaerobic microorganisms, fungi and yeasts considered liquid culture of microalgae bacteriologically sterile.

The method is illustrated by the following examples.

Example 1 (control). Water samples and scrapings of microscopic algae from coastal rocks for the subsequent allocation of these cultures of algae were collected in the period of active vegetation period (may-June) of the main species of microalgae in the Azov and Black seas were First grown cumulative mixed culture of algae, for which sampling was added to the environment of Bristol in the modification of Hollerbach, a mixture of trace elements and soil extract in certain proportions. The cumulative culture of microalgae were obtained by culture mixed culture in flasks with liquid nutrient media intended for laboratory cultivation of different algae (green, nitrogen-fixing and pattricia blue-green). Time of cultivation of algae were averaged 3-4 weeks.

Of cumulative culture, using microbiological methods of re-seeding and dilution, got algological pure culture. Cumulative sterile culture were inoculated in a Petri dish with agar nutrient medium. The Cup was placed on the light for algal growth. From colonies grown algae loop carried part of the culture again in liquid medium or in a test tube with a beveled agar medium. Cultivation of algae was carried out on nutrient media Tamiya, Aguinaga, Guseva and Walna. Environment are universal for all types of algae and allow you to get a shared culture of microalgae physiologically active. For the separation of microalgae used a glass spatula and sieving by rubbing the surface of the medium. This allows you to split the mixture MICR is Oracle on a single cell, that preserves their integrity and, therefore, greatly facilitates and accelerates the process to obtain pure cultures.

The works were selected algological pure cultures of the following blue-green microalgae: Oscillatoria laetevirens, Spirulina tenussima and Snowella rosea. Next conducted clearing each of the obtained cultures of algae and bacteria by chemical sterilization (1). With this purpose used antiseptics such as phenol and ethyl alcohol at a concentration of 0.1 and 1.0%, respectively. The algae were kept in sterile seawater with the above antiseptics for 4-5 h (table 1, experiment 1).

Check the purity of the obtained cultures of algae had a control crops their liquid cultures to that of the above-mentioned agar nutrient media that has seen the best growth of this culture. When this was recorded the growth of microorganisms. Thus, chemical sterilization is not possible to obtain absolutely pure cultures of algae. The data are shown in table 1, experiment 1.

Table 1
Growth of microorganisms in cultures of the marine blue-green microalgae in experiments with different methods of sterilization
No. Chem. sterilizationUV
irradiation
Antibiotics mg/mlFungicide,
nystatin, ug/ml
Bacteria-
local and fungal growth
chloramphenicoltetracyclineerythromycinampicillin
1+------+
2++-----+
3++50----+
4++50 50---+
5++505050--+
6++50505050-+
7++----25+
8--5050505025+
9+-5050 505025+
10-+5050505025+
11++5050--25+
12++-5050-25+
13++-50-5025+
14++50-- 5025-
15++5050-5025-
16++5050505025-
17++9050505025-
18++5050509025-
19++90505090 25-
20++9090509025-

Example 2. Culture dedicated marine blue-green algae were subjected to a first chemical sterilization (procedure described above), and then spent ten minutes exposure to the cultures of algae with UV light (bactericidal irradiator OBN - 150) in Petri dishes at a distance from the radiation source 50 cm (experiment 2). Check purity obtained after chemical sterilization and UV exposure of the cultures of microalgae had a control cultures liquid cultures on the above agar nutrient medium. When this was recorded the growth of microorganisms. The data are shown in table 1, experiment 2.

Thus, chemical sterilization, and radiation cultures of microalgae UV light is not allowed to get bacteriologically pure cultures of algae.

Example 3. Culture dedicated marine blue-green algae were subjected to a first chemical sterilization, then there was a ten-minute exposure of the cultures of algae ultraviolet with the etoy, as described in examples 1-2, and then subjected to treatment with the antibiotic chloramphenicol at a concentration of 50 μg/ml in an appropriate nutrient liquid medium. The results are shown in table 1, experiment 3.

Examples 4-20. Experiments were performed similar to that described above, i.e., the culture of selected microalgae were subjected to a first chemical sterilization, then there was a ten-minute exposure of the cultures of algae to ultraviolet light, and then subjected to treatment by different concentrations of antibiotics and fungicide in an appropriate nutrient liquid medium. The results are shown in table 1, experiments 4-20.

Were tested in different concentrations and different types of antibiotics and fungicides (table 1). After each treatment have been checking the purity of the obtained cultures of algae control crops from liquid cultures on agar nutrient medium. Thus, were selected antibiotics and fungicides, as well as their concentration, after processing which, when the control crops on agar nutrient medium was not recorded growth of microflora. Used the antibiotic chloramphenicol (50 and 90 μg/ml), tetracycline (50 and 90 μg/ml), erythromycin (50 mg/ml), ampicillin (50 and 90 μg/ml), and the fungicide - nystatin in the amount of 25 μg/ml were also conducted experiments with a series of samples of the marine blue-green MICR is Oracle, treated with antibiotics and fungicide, but not subjected to a preliminary chemical sterilization and/or treatment with ultraviolet light (experiments 8-10). Only treatment with chloramphenicol at a concentration of 50 μg/ml in combination with ampicillin 50 μg/ml and nystatin 25 μg/ml, performed after chemical sterilization, and radiation cultures UV light, have resulted in the almost complete absence of bacterial growth in several subcultures.

Were tested and the higher concentrations used antibiotics, which allowed to achieve 100% absence of bacterial microflora, however, such concentrations reduced the growth rate and inhibited division of microalgae. The research results are summarized in table 1.

The table shows that the effect associated with the full release of the marine blue-green microalgae from concomitant microflora preserving life mikrovodoroslevykh cells, is achieved by performing chemical sterilization, irradiation of cultures UV light, and then the processing of microalgae antibiotics and fungicide at the following minimum ratio of components: chloramphenicol and 50 μg/ml, ampicillin 50 μg/ml, and the fungicide, nystatin in the amount of 25 μg/ml

Thus, a stepwise method of chemical sterilization al is logicheskie pure cultures of marine blue-green microalgae with ultraviolet irradiation and further addition of certain concentrations and compositions of antibiotics and fungicides, allowed to get bacteriologically pure cultures of microalgae that can be used when conducting physiological-biochemical, cytological and genetic studies of algae and other scientific research. In particular, bacteriologically pure cultures of marine blue-green microalgae can be used to develop test systems based on fluorescence of algae to assess the toxicity of the components of the marine ecosystems of water bodies.

Using the proposed method improves the efficiency of the allocation of bacteriologically pure cultures of microalgae. This method is reliable, because it allows one to get bacteriologically pure cultures of marine blue-green microalgae regardless of the source separation and the degree of bacterial contamination.

The method of obtaining bacteriologically pure cultures of marine blue-green microalgae, including chemical sterilization cultures of microalgae by treatment in a solution of sterile seawater containing 0.1% phenol and 1.0% ethanol, characterized in that it further carry out the irradiation with ultraviolet light for 9-10 minutes, and then treatment with antibiotics and fungicides, and the use of antibiotics chloramphenicol and ampicillin in the amount of 50 µg/ml each, and in which the quality of fungicide use nystatin in the amount of 25 μg/ml



 

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FIELD: biotechnologies.

SUBSTANCE: inoculum of microalgae Desmodesmus sp. strain 2C166E is introduced into mineral medium BG-11 till final concentration of chlorophyll in mixture of 4-6 mcg/ml. Incubation is performed under constant lighting and environment barbotage by atmospheric air during 12-16 days under temperature of 25-27°C with consequent separation of microalgae biomass from nutritional medium with obtainment of microalgae biomass containing 33-35% of fatty acids from dry weight of cells.

EFFECT: invention allows increasing content of fatty acids in microalgae biomass.

1 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention can be used for processing sewage waters of the production of nitroaromatic or nitrohydroxyaromatic compounds, for instance, nitrobenzene or dinitrotoluene. To realise the method two-stage processing, including a stage of preliminary reduction and a stage of wet oxidation, is carried out. At the first stage alkaline sewage water is mixed with an organic reducing agent, which does not form salts in the sewage water, selected from peat, brown coal and/or hard coal. Processing in reducing conditions is carried out with heating to a temperature from 80 to 200°C and exposure at the said temperature for the time period from 5 min to 5 hours. At the second stage the sewage water obtained at the first stage is acidified and subjected to oxidation with oxygen-containing gas, for instance oxygen.

EFFECT: method suggests a technically safe, simple and economic technology of processing and purification of sewage waters, providing the reduction of harmful admixture to the level, acceptable for the supply of processed sewage waters to biological purification.

4 cl, 5 tbl, 6 ex

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