Biological sciences / 6. Microbiology

Stud. Ivahniuk M.O., prof., d.b.s. Pirog T.P.

National university of food technology, Ukraine

Sunflower oil as the substrate for synthesis of microbial exopolysaccharide ethapolan by Acinetobacter sp. IМV B-7005

Microbial exopolysaccharides (EPS) due to the ability of their solutions to gelation, emulsification, suspending and changing rheological properties of aqueous systems are widely used in various industries, agriculture and medicine [1, 2].

The vast majority of known microbial EPS are obtained from carbohydrate substrates. Usually, products derived from sugar beet: molasses, sugar syrup, sucrose or corn: starch, hydrolyzed starch, glucose syrup, glucose, maltose are used as substrates in the industrial production of EPS [3]. But studies conducted in the 70-80s of the twentieth century demonstrated the possibility of expanding the resource base of microbiological production of EPS by using of non-food substrates (methane, methanol, ethanol, ethylene glycol, hydrocarbons) [3]. However, the concentration of polysaccharides synthesized on non- carbohydrate substrates remains low for today.

Last years the researches of using industrial waste have been activated to obtain a practically valuable microbial metabolites [4]. Replacing traditional substrates for microbial synthesis by industrial waste will allow to decrease the cost of technology in several times, and recycle unwanted waste. Oil-containing waste are promising for using in microbial technologies, as the world production of sunflower oil is about 2.5−3 million tons, so it is cheap and available in necessary quantities [5, 6].

Note, that the literature data about synthesis of microbial EPS on any industrial waste (not just oil-containing) is extremely limited. In recent years Cellulomonas flavigena UNP3 was described as the strain, which is able to synthesize kurdlan-like EPS in the medium with vegetable oil [7]. It is known that Xanthomonas campestris ATCC 13951 synthesized 28 g/l of xanthan under cultivation in reactor
(2 l) during 96 h in the medium containing partially hydrolyzed molasses (the concentration of lactose, galactose, glucose was 4.7; 17.8, 17.8, respectively) as the carbon source [9]. It was determined that Pseudomonas oleovorans NRRLB-14682 synthesized EPS (12.18 g/l) on the medium with crude glycerol (by-product of biodiesel production) [10]. Acinetobacter sp. DR1 under cultivation in the medium with diesel oil (2 %) synthesized about 5 g EPS/g biomass [11]. It should be noted, that until now in the available literature we couldn’t find information about the synthesis of microbial EPS on sunflower oil.

Previously, we have established the possibility to use sunflower oil as a source of carbon and energy for the synthesis of microbial polysaccharide ethapolan by Acinetobacter sp. IMV B-7005 [8]. However, in earlier studies, the concentration of oil in the cultivation medium was low (only 1% v/v). As for the synthesis of ethapolan we supposed to use fried oil as a substrate, volume of which is extremely large, so its content in the medium has to be more higher.

The purpose of this work − to research intensification of microbial polysaccharide ethapolan synthesis in medium with the maximum concentration of sunflower oil.

Our previous data [8] have shown that during Acinetobacter sp. IMV B-7005 growth in medium with 1 % of sunflower oil, 5 g/l of EPS were synthesized. Further studies demonstrated that increasing sunflower oil content in the medium of IMV
B-7005 strain to 2−3 % was accompanied by increasing of synthesized ethapolan concentration to 5,8−6,3 g/l, but the EPS-synthesizing ability was slightly decreased (Table 1). Indices of EPS synthesis decreased with the higher substrate concentration (4−5 %) and the highest EPS-synthesizing ability (5 g EPS/g ADB) was observed under Acinetobacter sp. IMV B-7005 cultivation in the medium with 1 % of sunflower oil (Table 1).

As in case of increasing of carbon’s concentration in the medium, C/N ratio changes, that significant impacts on synthesis of microbial polysaccharides [3], so on the next stage we increased concentration of nitrogen source simultaneously with enhancing of oil content (Table 2).

Table 1

Depending ethapolan synthesis on the concentration of sunflower oil in the cultivation medium of Acinetobacter sp. IMV B-7005

Concentration of sunflower oil in the medium, %	EPS, g/l	EPS-synthesizing ability, g ЕPS/g ADB
1	5,0±0,25	5,0±0,25
2	5,8±0,29	4,7±0,23
3	6,3±0,31	4,0±0,20
4	5,0±0,25	3,7±0,19
5	4,9±0,24	3,6±0,18

Note. The concentration of pantothenate in the medium was 0.00085 %, ammonium nitrate − 0.4 g/l.

As in case of increasing of carbon’s concentration in the medium, C/N ratio changes, that significant impacts on synthesis of microbial polysaccharides [3], so on the next stage we increased concentration of nitrogen source simultaneously with enhancing of oil content (Table 2).

Results presented in Table 2 show that increasing ammonium nitrate concentration to 0.8 g/l in a medium containing 3−5 % of sunflower oil promotes degrease of synthesized ethapolan concentration and EPS-synthesizing ability compared with those in the medium with lower (0.4 g/l) concentration of nitrogen sources (see Table 1 and 2). However, concentration of synthesized ethapolan in the medium with 4 and 5 % of sunflower oil and 0.6 g/l of NH₄NO₃ was 5.6 and 6.4 g/l, respectively. That is higher than in medium with 0.4 g/l of ammonium nitrate (5.0 and 4.9 g / l, see. Table. 1 and 2). EPS-synthesizing ability also increased under such cultivation conditions of IMV B-7005 strain. Thus, parameters of ethapolan synthesis were improved by increasing NH₄NO₃ concentration to 0.6 g/l with increase of oil content to 4−5 % in the medium.

Table 2

The influence of the nitrogen source concentration on the synthesis of ethapolan under Acinetobacter sp. IMV B-7005 cultivation on sunflower oil

Concentration of ammonium nitrate, g/l	Concentration of sunflower oil in the medium, %	EPS, g/l	EPS-synthesizing ability, g ЕPS/g ADB
0,6	3	4,6±0,23	4,1±0,21
	4	5,6±0,28	4,2±0,21
	5	6,4±0,32	3,9±0,19
0,8	3	3,2±0,16	3,0±0,15
	4	3,4±0,17	2,9±0,14
	5	3,6±0,18	2,7±0,13

Note. The concentration of pantothenate in the medium was 0.00085 %.

The concentration of pantothenate in the medium is another factor that may affect on synthesis of ethapolan, as Acinetobacter sp. IMV B-7005 is auxotroph for calcium pantothenate [3]. Therefore, on the next stage concentration of pantothenate in the cultivation medium of IMV B-7005 strain was increased with enhancing sunflower oil and nitrogen source content (Table 3).

Table 3

Synthesis of ethapolan depending on the concentration of pantothenate in Acinetobacter sp. IMV B-7005 medium with sunflower oil

Concentration in the medium			ЕPS, g/l
of ammonium nitrate, g/l	of pantothenate, %	of sunflower oil, %
0,4	0,00085	4	4,8±0,24
		5	4,9±0,24
	0,00095	4	5,6±0,28
		5	6,7±0,33
0,6	0,00085	4	5,6±0,28
		5	6,4±0,32
	0,00095	4	5,5±0,27
		5	6,6±0,33

Thus, increasing of pantothenate content to 0.00095 % in medium with 0.4 g/l of ammonium nitrate and 5 % of sunflower oil allowed to enhance the concentration of EPS in 1.4 times, comparing with results in the medium with lower amount of pantothenate.

However, no positive effect on the synthesis of ethapolan with higher concentrations of pantothenate and NH₄NO₃ (0,6 g/l) in the medium was observed (Table 3).

As a result of this work cultivation’s conditions were established for producer of microbial exopolysaccharide ethapolan. They provide synthesis of 6,6−6,7 g/l of EPS in the medium with a high content of sunflower oil (4−5 %). These results were achieved in the case of both increasing of nitrogen sources content to 0.6 g/l and/or pantothenate − up to 0.00095 % with increasing of the substrate concentration for ethapolan synthesis. The experimental data are basic for the development of this polysaccharide technology in the medium with fried sunflower oil or other oil-containing industrial waste.

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