RHEOLOGICAL PROPERTIES OF THE MICROBIAL EXOPOLYSACCHARIDE ETHAPOLAN SYNTESIZED IN THE PRESENCE OF EXOGENOUS FATTY ACIDS IN THE CULTURE MEDIUM

 

under section № 2 (biotechnology) 

Olefirenko Y.Y., student

                                       Pirog T.P., Doctor of Biological Sciences, professor

National University of Food Technologies


RHEOLOGICAL PROPERTIES OF THE MICROBIAL EXOPOLYSACCHARIDE ETHAPOLAN SYNTESIZED IN THE PRESENCE OF EXOGENOUS FATTY ACIDS IN THE CULTURE MEDIUM

Ethapolan is a complex of microbial exopolysaccharides (EPS) synthesized by Acinetobacter sp. IMB B-7005. His practically valuable rheological properties are determined by the content of acetylated component and the concentration of fatty acids in its composition [1, p. 5]. 

The disadvantages of previously developed technologies of ethapolan synthesis on the mixture of energy deficient (acetat and molasses) [2, p. 34] and energy unequivalent (fumarate and molasses) substrates were the increase of the pH level (up to 9.0 – 9.8) during the cultivation process because of the assimilation of organic acid salts (acetate, fumarate) by the proton-driven symport. In such conditions the low acetylated ethapolan was synthesized which solutions are not appropriate for the practical use because of their rheological properties.

Basing on the literature material about different changes in physical and chemical properties of the polimer emulsan synthesized by Acinetobacter venetianus RAG-1 in the case of adding of exogenous fatty acids into the medium [3, p. 202] we made the assumption that the addition of such compounds into the medium of Acinetobacter sp. IMB B-7005 can influence on the rheological properties of ethapolan solutions. Thus, the aim of this work was to research the impact of the exogenous fatty acids on the rheological properties of ethapolan.

          Cultivation of Acinetobacter sp. IMB B-7005 was carried out in the medium with acetate and molasses, fumarate and molasses as the sourсe of carbon and energy.

The precursors (sunflower oil and oleic acid at the concentration of 0,1 – 0,5% (v/v)) were added into the medium at the beginning of the cultivation, in the exponential and stationary growth phase.         

Rheological properties of 0,05% ethapolan solutions were performed by the changes of the viscosity degree  in the presence of 0,1 M KCl and in the  Cu2+-glycine system, what is the individual property of this polymer.

         Sunflower oil contains stearic (C18), palmitic (C15), myristic (C13), peanut (C19), oleic (C18), linoleic (C18) acids. That is why we suggested that some of these acids can be included to the EPS composition as the side chains of the acetylated polysaccharide and change rheological properties of ethapolan as the result [3, p. 202].

           The cultivation of Acinetobacter sp. IMB B-7005 in the presence of 0,1 – 0,5% of sunflower oil in the medium with acetate and molasses was accompanied by the increase of the viscosity of 0,05% ethapolan solutions in 1,5 – 2,2 fold both in the presence of KCl and in the Cu2+-glycine system, comparing to the rheological properties of ethapolan synthesized in the medium without oil.

           The next step in our work was connected with the cultivation of Acinetobacter sp. IMB B-7005 in the medium with the mixture of fumarate and molasses as the source of the carbon and energy with the addition of exogenous sunflower oil on the different phases of growth. Fumarate is energy deficient substrate. It compensates the loss of glucose during its oxidation to CO2 to obtain energy for the constructive metabolism, increasing the efficiency of the transformation of both carbon substrates into EPS [4, p. 794]. 

           The cultivation of Acinetobacter sp. IMB B-7005 in the medium with a mixture of fumarate and molasses with addition of 0,1– 0,3% of sunflower oil in stationary growth phase was accompanied by the increasing of the viscosity of 0,05% solutions of culture broth  in 1,1 – 1,9 fold both  in the presence of KCl and in the Cu2+-glycine system, comparing to the rheological properties of ethapolan synthesized in the medium without precursors. The increase of the viscosity of 0,05% culture broth solution  in 2,1 times in the presence of KCl was  also observed  after the adding of 0,5% of sunflower oil at the beggining of the cultivation process. The viscosity in the Cu2+-glycine system of 0,05% ethapolan solution, synthesized in the presence of 0,1 – 0,3% of sunflower oil in a medium with fumarate and molasses, was in 1,1 – 1,2 fold higher than the viscosity of the EPS solutions, synthesized without adding oil.

           The following step was connected with the cultivation of Acinetobacter sp. IMB B-7005 in the presence of oleic acid in the growth medium.

           The cultivation of the strain IMB B-7005 in the medium with acetate and molasses with the adding of oleic acid independently of  the phase of growth and the acid concentration was accompained with the increase in rheological characteristics in 1,2–3,4 fold both in the presence of KCl and in the Cu2+-glycine system in comparison with the rheological properties of ethapolan synthesized in the growth medium without acid.

            Adding of 0,1 – 0,5% of oleic acid into the medium with acetate and molasses at the beginning of the process was accompanied by the increase of the viscosity of 0,05% ethapolan solutions in 9,5 – 17,7 fold in the Cu2+-glycine  system comparing to the rheological characteristics of EPS synthesized in the medium without fatty acids.

           Independently of the concentration (0,1 – 0,5%) and the moment of adding of  oleic acid into the medium with fumarate and molasses it was observed the increase of the viscosity of the culture broth solutions in 1,4 – 3,9 fold in the presence of KCl was observed, in comparison to the viscosity after cultivation without precursors. 

           The increase of the rheological properties of 0,05% ethapolan solutions in 1,1 – 2,2 fold both  in the presence of KCl and in the Cu2+-glicine system was observed independently of the moment of adding to the medium with fumarate and molasses of oleic acid in concentration of 0,3%.

           The highest indexes of the rheological properties of 0,05% culture broth solutions and the conditions of cultivation are listed in the table below. 

Table

The influence of exogenous fatty acids on the rheological properties of 0,05% solutions of culture broth of Acinetobacter sp. IMB B-7005

The source of carbon and energy, % (v/v)

Fatty acid

Fatty acids concent-ration, % (v/v)

Moment of adding, growth phase

Change of the viscosity degree, % to the control

in the presence of KCl

in the  Cu2+-glycine system

Acetate, 1,1%

+

molasses, 0,75%

Sunflower oil

0,1

Lag-phase

221,2±11

152,2±7,6

0,3

Lag-phase

228,2±11,4

62±3,1

0,5

Lag-phase

173,5±8,7

142,6±7

Oleic acid

0,1

Stationary

200±10

200±10

0,3

Stationary

240±12

176,9±8,8

0,5

Lag-phase

80,8±4

343±17,2

Fumarate, 0,18%

+

molasses, 0,5%

Sunflower oil

0,1

Stationary

192±10

124±7

0,3

Stationary

112±6

126±6

0,5

Lag-phase

210±10

101±5

Oleic acid

0,1

Stationary

151±8

52±3

0,3

Exponential

337±17

61±3

0,5

Lag-phase

390±20

246±12

Remark. The control (100%) – the viscosity of the culture broth after cultivation on the mixed substrates without adding of the corresponding fatty acids.

It should be mentioned that adding of exogenous fatty acids into the growth medium of  Acinetobacter sp. IMB B-7005  with mixed substrates is accompanied by the increase of not only rheological characteristics of solutions of purified ethapolan preparations  but also of culture broth solutions containing ethapolan. As for enhanced oil recovery and in technical detergents etapolan is usually used as postfermentation culture broth, the implementation of technology of its producing using a mixture of growth substrates with adding of exogenous fatty acids can significantly reduce the cost of the desired product by utilization of cheap substrates and  improving its the rheological characteristics.


References

  1. Pidhorskyy V.S., Iutynska G.O., Pirog T.P. The intensification of the technology of the microbial synthesis. – K.: “Science thought”, 2010. – 327 p. 
  2. Pirog T.P., Vysyatetskaya N.V., Korzh Y.V. Features of the synthesis of the exopolysaccharide ethapolan on the mixture of energy deficient growth substrates // Microbiology. – 2007. – Vol 76, N 1. – P. 32 – 38.
  3. Dams-Kozlowska H., Mercaldi M., Kaplan D. Modification and application of the Acinetobacter venetianus RAG-1 exopolysaccharide, the emulsan complex and its components // Appl microbiol biotechnol. – 2008. – P. 201 – 210.
  4. Pirog T.P., Vysyatetskaya N.V., Korzh Y.V. Formation of the exopolysaccharide ethapolan when the grown of Acinetobacter sp. B-7005 was carried out on the mixture of fumarate and glucose // Microbiology. – 2007. – Vol 76, N 6. – P. 790 – 796.