ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE
The paper presents the results of studies on the optimization of culture media for joint cultivation of Bradyrhizobium japonicum and Azospirillum brasilense bacteria at periodic passages. It was established that cultivation of studied microorganisms in a mixed culture the number of nodule bacteria v...
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| Datum: | 2014 |
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| Sprache: | Englisch Ukrainisch |
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Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine
2014
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| Назва журналу: | Agriciltural microbiology |
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Agriciltural microbiology| _version_ | 1871465650251104256 |
|---|---|
| author | Козар, С. Ф. |
| author_facet | Козар, С. Ф. |
| author_institution_txt_mv | [
{
"author": "С. Ф. Козар",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
}
] |
| author_sort | Козар, С. Ф. |
| baseUrl_str | https://smic.in.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T10:10:43Z |
| description | The paper presents the results of studies on the optimization of culture media for joint cultivation of Bradyrhizobium japonicum and Azospirillum brasilense bacteria at periodic passages. It was established that cultivation of studied microorganisms in a mixed culture the number of nodule bacteria vary from 0.76 to 6.72 billion cells/cm3, depending on the concentration of medium components. It was shown that the effect of molasses, fodder yeast and phosphates on growth activity of B. japonicum significantly depends not only on the amount of chemical components in the media, but also on the strain of A. brasilense, used for joint cultivation. Based on the results of multivariate experiment an optimized media for joint cultivation of studied microorganisms was proposed. |
| doi_str_mv | 10.35868/1997-3004.19.27-32 |
| first_indexed | 2025-07-17T12:24:14Z |
| format | Article |
| fulltext |
OPTIMIZATION OF CULTURAL MEDIA FOR JOINT CULTIVATION OF
BRADYRHIZOBIUM JAPONICUM AND AZOSPIRILLUM BRASILENSE
S. F. Kozar
Perspective is the creation of microbial
preparations based on mixed cultures
diazotrophs particular which include
Bradyrhizobium japonicum and Azospirillum
brasilense bacteria [1; 2]. This is due to the
fact that at joint cultivation of these nitrogen
fixers their growth activity is increased, which
promotes the obtaining of high titre of
microorganisms in biological preparation. In
addition, at pre-plant soybean inoculation by
nodule bacteria together with azospirillums
more nodules appear on the roots of plants,
compared to monobacterization, and nitrogen
fixation activity increases, which in turn
contributes to obtaining additional yield of
this legume.
An important and difficult stage of
development of microbial preparations is the
optimization of the environment for the
cultivation of microorganisms, which are their
active basis. Especially difficult this task is at
simultaneous cultivation of two or more types
of bacteria that have different needs for
nutrition sources [3].
At this both the availability of specific
components of the environment for each type
of microorganisms and features of possible
competition for certain compounds of
nutrients should be ensured.
Based on the above, the goal of the
study was the optimization of liquid culture
medium content for joint growing of B.
japonicum and A. Brasilense in periodic
culture.
Materials and methods. Objects of the
study: B. japonicum M-8 [4], A. brasilense
18-2 [5], A. brasilense 410 [6]. All the
microorganisms were obtained from the
National Collection of useful soil
microorganisms of Institute of Agricultural
Microbiology and Agroindustrial
Manufacture of NAAS. We express our
sincere gratitude to the authors for courtesy
strains.
The number of B. japonicum was
determined by plate method through depth
seeding of suspensions in agar medium based
on pea broth [7]. The number of A. brasilense
was determined by plate method in agar
medium of such a composition, g/dm3: potato
broth – 1, malic acid – 3.6, sucrose – 2.5; pH
7.2-7.4 (diluted by NaOH solution), agar-agar
– 11 g.
Petri dishes were kept in an incubator
at the temperature of (28.0±2.0)°C. Counting
of B. japonicum colonies was performed on
the tenth day, A. brasilense – on the fourth
day.
The cultivation of bacteria was carried
out in the vessels of 500 cm3 on the shaker at
a frequency of 240 rpm. at the temperature of
(28.0±2.0)°C. B. japonicum and A. brasilense
seeding material were applied in the ratio 1: 1.
The duration of cultivation is 72 hours.
The optimization of the medium was
performed using an algorithm for determining
the effects of changing factors on the process
of joint cultivation of microorganisms
Bradyrhizobium and Azospirillum. To do this,
based on previously obtained data as for the
impact of the sources of nutrients on the
growth of B. japonicum and A. Brasilense, the
optimization was performed using
symmetrical plans all factors of which vary at
the same number of levels. The optimization
was performed using the method of
orthogonal Latin rectangles [8-10]. The
criterion of optimization was the number of
viable bacterial cells.
Mathematical processing of
experimental data was performed by
calculating the influence effect on the number
of viable cells for all levels of the studied
factors. The effect of different levels
influence was calculated by the difference of
arithmetic average of process output (number
of cells) in all versions, where the factor was
at this level and the average value of each
series of experiments by the formulas:
푏 = , 푏 = − 푏
where b0 - arithmetic average of the number
of bacteria in all variants;
∑ 푦 - total number of bacteria in all
variants of the experiment;
bik - effect of each level;
yi – output in the i-th variant of
planning;
N – total number of variants in the
plan;
m – number of levels of each factor;
푦 - the amount of outputs in
those variants of the plan, where i-th
factor was on the k-th level.
Planning matrix was constituted
according to the scheme 3i3k (Table 1),
which allowed to study mutual influence of
three factors on the growth of B. japonicum
and A. brasilense on three levels in the
conditions of one experiment. Medium after
the first phase of optimization was chosen for
the basis, which includes: molasses, fodder
yeast (dry), glucose, (NH4)2SO4, KH2PO4,
K2HPO4 ·3H2O, MgSO4 7H2O, CaCO3.
Table 1. Scheme of experiment
planning with 3 factors and 3 levels of
variation
Variant
number
j
Factors levels k
Factor
i1
Factor
i2
Factor
i3
1 1 1 1
2 2 1 2
3 3 1 3
4 1 2 2
5 2 2 3
6 3 2 1
7 1 3 3
8 2 3 1
9 3 3 2
Statistical analysis of experiment data
was performed using the computer program
Statistica 6.0.
Results and discussion.
The study of growth activity of B.
japonicum and J. brasilense while cultivating
in mixed culture was performed in the
medium which included the sources of
various nutrients, the impact of which we
studied at the first phase of its optimization
[11]. At the second stage the following levels
of components of the medium were
established:
- molasses: 10.0; 15.0; 20.0 g/dm3
(step – 5 g/dm3);
- fodder yeast: 3.0; 4.0; 5.0 g/dm3
(step – 1 g/dm3);
- phosphates (1:1) (KH2PO4 +
K2HPO4· 3H2O): 0.3; 0.4; 0.5 g/dm3 (step –
0.1 g/dm3).
Analyzing the obtained data of
multifactor experiment (Table 2) it should be
noted that the number of B. japonicum M-8
bacteria at the joint cultivation with A.
brasilense 18-2 ranged from 1.68 to 6.61
billion cells/cm3. At this, the number of
azospirillums was varying in a narrow range
from 0.83 to 2.91 billion cells/cm3. While
obtaining the inoculant for soybeans the focus
should primarily be on the rhizobia titre,
that’s why optimal in this case, in our
opinion, should be considered the medium,
which is provided in version 6.
The effects of all factors influence on
the number of B. japonicum M-8, which was
cultivated together with A. brasilense 18-2
were deducted at their levels. It was found
that the maximum effect for molasses in
relation to the rhizobia was received at its
concentration of 20 g/dm3 (table 3), in
relation to azospirillums – at the
concentration of 15 g/dm3 (table 4).
Maximum effects as for the impact of fodder
yeast in relation to both studied
microorganisms were observed at the use of
component concentration of 4.0 g/dm3. At the
study of influence of different content of
phosphate salts on the growth of rhizobia the
greatest effect was obtained at the use of the
minimum concentration of this component,
and on the growth of A. brasilense 18-2 – at
the use of the concentration of 0.5 g/dm3.
Table 3. Effects of multifactor
experiment on optimization of nutrient
medium for B. japonicum M-8
Medium
components
Concentration,
g/dm3
Effect
Molasses 10.0 -0.82
15.0 0.13
20.0 0.69
Fodder yeast 3.0 -0.49
4.0 1.16
5.0 -0.67
K2HPO4· 3H2O
+ KH2PO4 (1:1)
0.3 1.22
0.4 1.00
0.5 -0.22
Table 2. The number of B. japonicum M-8 and A. brasilense 18-2 bacteria in the versions
of multifactor experiment on optimization of nutrient medium, billion cells/cm3
Experiment
variants
Medium components concentration, g/dm3 Number of B.
japonicum M-8
bacteria
Number of A.
brasilense 18-
2 bacteria
Molasses Fodder yeast K2HPO4· 3H2O
+ KH2PO4 (1:1)
1 10 3 0.3 2.57 1.37
2 15 3 0.4 2.27 1.03
3 20 3 0.5 5.91 2.67
4 10 4 0.4 5.25 2.77
5 15 4 0.5 3.73 2.91
6 20 4 0.3 6.72 2.63
7 10 5 0.5 1.92 1.25
8 15 5 0.3 6.61 2.45
9 20 5 0.4 1.68 0.83
Table 4. Effects of multifactor
experiment on optimization of nutrient
medium for A. brasilense 18-2
Medium
components
Concentration,
g/dm3
Effect
Molasses 10.0 -0.19
15.0 0.14
20.0 0.05
Fodder yeast 3.0 -0.30
4.0 0.78
5.0 -0.48
K2HPO4· 3H2O
+ KH2PO4 (1:1)
0.3 0.16
0.4 -0.45
0.5 0.28
It should be noted that B. japonicum
M-8 bacteria were more sensitive to changes
in the concentrations of studied medium
components in comparison with A. brasilense
18-2 at the cultivation of these
microorganisms in a mixed culture.
The number of B. japonicum M-8
bacteria at the joint cultivation with A.
brasilense 410 diazotrophs ranged from 0.76
to 3.20 billion cells/cm3. The number of
azospirillums was varying in the range of 0.70
to 3.34 billion cells/cm3 at this. Optimum in
this case to be considered the medium
comprising of molasses at the concentration
of 20 g/dm3, fodder yeast – 5 g/dm3,
phosphates – 0.4 g/dm3 (table 5).
In the studies of the impact of all
factors on the number of B. japonicum M-8
and A. brasilense 410 cultivated together, it
was found that the maximum effect of
molasses in relation to the studied
microorganisms was observed at its
concentration in the culture medium at the
rate of 20 g/dm3, which represents its
maximum studied value. In the study of the
effects of other investigated components it
was found (table 6-7) that the greatest impact
on the number of grown bacteria was
observed at maximum values of
concentrations of these components - 5 g/dm3
of fodder yeast and 0.5 g/dm3 of the mixture
of phosphate salts.
Table 6. Effects of multifactor
experiment on optimization of nutrient
medium for B. japonicum M-8, billion
cells/cm3
Medium
components
Concentration,
g/dm3
Effect
Molasses 10.0 -0.39
15.0 0.18
20.0 0.63
Fodder yeast 3.0 -0.16
4.0 -0.11
5.0 0.69
K2HPO4· 3H2O
+ KH2PO4 (1:1)
0.3 -0.11
0.4 0.01
0.5 0.52
Table 5. The number of B. japonicum M-8 and A. brasilense 410 bacteria in the versions of
multifactor experiment on optimization of nutrient medium, billion cells/cm3
Experiment
variants
Medium components concentration, g/dm3 Number of B.
japonicum M-8
bacteria
Number of A.
brasilense 410
bacteria
Molasses Fodder yeast K2HPO4· 3H2O
+ KH2PO4 (1:1)
1 10 3 0.3 1.20 1.90
2 15 3 0.4 1.83 2.20
3 20 3 0.5 2.24 2.56
4 10 4 0.4 0.76 0.70
5 15 4 0.5 2.46 3.00
6 20 4 0.3 2.21 1.33
7 10 5 0.5 2.62 3.08
8 15 5 0.3 2.01 1.23
9 20 5 0.4 3.20 3.34
Table 7. Effects of multifactor
experiment on optimization of nutrient
medium for A. brasilense 410, billion
cells/cm3
Medium
components
Concentration,
g/dm3
Effect
Molasses 10.0 -0.11
15.0 0.14
20.0 0.41
Fodder yeast 3.0 0.22
4.0 -0.32
5.0 0.55
K2HPO4· 3H2O
+ KH2PO4 (1:1)
0.3 -0.51
0.4 0.08
0.5 0.88
Based on these data, we can note that
to achieve maximum impact on the growth of
B. japonicum M-8 and J. brasilense 410
bacteria high concentrations of fodder yeast
and phosphates are required.
Thus, by the results of multifactor
experiment an optimized medium for joint
growing of rhizobia and different strains of
azospirillums was offered. It is shown that the
effect of molasses, fodder yeast and
phosphates influence on growth activity of B.
japonicum depends not only on the amount of
chemicals in the medium, but also on A.
Brasilense strain which is jointly cultivated
with.
|
| id | oai:ojs2.smic.in.ua:article-167 |
| institution | Agriciltural microbiology |
| keywords_txt_mv | keywords |
| language | English Ukrainian |
| last_indexed | 2026-07-23T01:05:24Z |
| publishDate | 2014 |
| publisher | Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine |
| record_format | ojs |
| resource_txt_mv | smicinua/0e/273df87668e5df571b88932ac7bb000e.pdf |
| spelling | oai:ojs2.smic.in.ua:article-1672026-07-22T10:10:43Z OPTIMIZATION OF CULTURAL MEDIA FOR JOINT CULTIVATION OF BRADYRHIZOBIUM JAPONICUM AND AZOSPIRILLUM BRASILENSE ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE Козар, С. Ф. Bradyrhizobium japonicum, Azospirillum brasilense, cultivation, medium, optimization Bradyrhizobium japonicum, Azospirillum brasilense, культивування, середовище, оптимізація The paper presents the results of studies on the optimization of culture media for joint cultivation of Bradyrhizobium japonicum and Azospirillum brasilense bacteria at periodic passages. It was established that cultivation of studied microorganisms in a mixed culture the number of nodule bacteria vary from 0.76 to 6.72 billion cells/cm3, depending on the concentration of medium components. It was shown that the effect of molasses, fodder yeast and phosphates on growth activity of B. japonicum significantly depends not only on the amount of chemical components in the media, but also on the strain of A. brasilense, used for joint cultivation. Based on the results of multivariate experiment an optimized media for joint cultivation of studied microorganisms was proposed. Представлено результати досліджень з оптимізації живильного середовища для сумісного культивування діазотрофів Bradyrhizobium japonicum і Azospirillum brasilense за періодичного культивування. Виявлено, що при вирощуванні цих мікроорганізмів у змішаній культурі чисельність бульбочкових бактерій може коливатися від 0,76 до 6,72 млрд. кл./см3 залежно від концентрації компонентів середовища. Показано, що ефект впливу меляси, кормових дріжджів і фосфатів на ростову активність B. japonicum залежить не лише від кількості хімічних сполук у середовищі, але й від штаму A. brasilense, з яким здійснюється культивування. За результатами багатофакторного експерименту запропоновано оптимізоване середовище для сумісного вирощування ризобій і азоспірил. Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine 2014-06-26 Article Article Рецензована Стаття application/pdf application/pdf https://smic.in.ua/index.php/journal/article/view/167 10.35868/1997-3004.19.27-32 Agricultural microbiology; Vol. 19 (2014): Agriciltural microbiology; 27-32 Сільськогосподарська мікробіологія; Том 19 (2014): Сільськогосподарська мікробіологія; 27-32 1997-3004 10.35868/1997-3004.19 en uk https://smic.in.ua/index.php/journal/article/view/167/217 https://smic.in.ua/index.php/journal/article/view/167/218 Авторське право (c) 2014 S. F. Kozar https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | Bradyrhizobium japonicum Azospirillum brasilense культивування середовище оптимізація Козар, С. Ф. ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE |
| title | ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE |
| title_alt | OPTIMIZATION OF CULTURAL MEDIA FOR JOINT CULTIVATION OF BRADYRHIZOBIUM JAPONICUM AND AZOSPIRILLUM BRASILENSE |
| title_full | ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE |
| title_fullStr | ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE |
| title_full_unstemmed | ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE |
| title_short | ОПТИМІЗАЦІЯ СЕРЕДОВИЩА ДЛЯ СУМІСНОГО КУЛЬТИВУВАННЯ BRADYRHIZOBIUM JAPONICUM І AZOSPIRILLUM BRASILENSE |
| title_sort | оптимізація середовища для сумісного культивування bradyrhizobium japonicum і azospirillum brasilense |
| topic | Bradyrhizobium japonicum Azospirillum brasilense культивування середовище оптимізація |
| topic_facet | Bradyrhizobium japonicum Azospirillum brasilense cultivation medium optimization Bradyrhizobium japonicum Azospirillum brasilense культивування середовище оптимізація |
| url | https://smic.in.ua/index.php/journal/article/view/167 |
| work_keys_str_mv | AT kozarsf optimizationofculturalmediaforjointcultivationofbradyrhizobiumjaponicumandazospirillumbrasilense AT kozarsf optimízacíâseredoviŝadlâsumísnogokulʹtivuvannâbradyrhizobiumjaponicumíazospirillumbrasilense |