Biological sciences/6. Microbiology

Stud. Voronenko A.A., post grad. stud. Ivakhniuk M.O.

National university of food technology, Ukraine

The biosynthesis of microbial exopolysaccharide ethapolan under Acinetobacter sp. IMV B-7005 cultivation on molasses and sunflower oil mixture

Microbial exopolysaccharides (EPS) – high molecular polymers, which due to the ability of their solutions to gelation, emulsification, suspending and changing rheological properties of aqueous systems are widely used in various industries [3, 7, 10]. Note, that carbohydrate substrates are usually used for the microbial EPS production today.

The important advantage of microbial polysaccharide ethapolan (produced by Acinetobacter sp. IMV B-7005) compared to the world well known EPSs is the possibility of its obtaining on a wide variety of C₂–C₆-substrates (carbohydrates, ethanol, acetate, organic acids) [10]. Recently the suitability of oil-containing substrates for ethapolan production was established [2].

One of the approaches to intensify microbial synthesis technologies is using mixture of growth substrates [10]. Increasing biomass concentration, growth rate, decrease of lag phase duration are often observed, while microorganisms were cultivated on mixed substrates [10].

Previously [9], the possibility of ethapolan production intensification on mixture of energetically unequal (ethanol and glucose, fumarate and glucose) and energy-deficient (acetate and glucose) growth substrates was established. In the next studies glucose in C₂–C₆-substrates mixture was replaced by molasses (by-product of sugar production) for EPS costs reduction [9].

Thus, under IMV B-7005 strain cultivation on molasses (0,75 % by carbohydrates) and ethanol (0,75 % v/v) mixture the amount of EPS was increased in 1,3 times compared to indices on monosubstrate molasses [9].

The purpose of this work – to research the possibility of ethapolan synthesis on molasses and sunflower oil mixture.

At the first stage principle possibility using molasses and sunflower oil mixture for ethapolan synthesis was analyzed (Table 1). Despite of different carbon sources (molasses, oil or their mixture) in inoculum preparation medium the amount of EPS and EPS-synthesizing ability were 2,1-2,5 g/l and 0,6-0,8 g EPS/ g biomass respectively. Note, that highest ethapolan production was observed when inoculum was grown on molasses. Therefore molasses was used for inoculum preparation in the next experiments.

However, indices of ethapolan synthesis on molasses (0,5 %) and oil (0,5 %) mixture were lower compared with those on C₂–C₆-substrates mixture [10]. On the next step the concentration of monosubstrates mixture was increased.

Table 1

Indices of ethapolan synthesis on molasses and sunflower oil mixture depending on inoculum preparation method

Concentration of substrate for		Synthesis indices
EPS biosynthesis, %	inoculum preparation, %	EPS, g/l	EPS-synthesizing ability, g EPS/ g biomass
Molasses, 0,5 + oil, 0,5	Molasses, 0,5	2,5±0,13	0,8±0,04
	Oil, 0,5	2,0±0,10	0,7±0,04
	Molasses, 0,25 + oil, 0,25	2,1±0,11	0,6±0,03

Note. Table 1 and 2: NH₄NO₃ concentration in cultivation medium was 0,4 g/l.

Results presented in Table 2 show that enhancing molasses and oil concentrations to 1,5 % were accompanied by increasing EPS amount in more than 5 times (to 13,3 g/l) and EPS-synthesizing ability – almost 2,8 times (to 2,2 g EPS/ g biomass) in comparison with indices on lower substrates concentrations (0,5 %).

It is known that carbon/nitrogen ratio plays significant role in EPS synthesis [10]. Note, that molasses can be additional nitrogen source because it contains about 1 % of available nitrogen for microorganisms [9]. On the next stage our research was directed on determining optimal concentration of mineral nitrogen source in medium for inoculum preparation and biosynthesis.

Table 2

Ethapolan synthesis depending on the concentration of molasses and sunflower oil in mixture

Concentration of substrate for EPS synthesis, %	Synthesis indices
	EPS, g/l	EPS-synthesizing ability, g EPS/ g biomass
Molasses, 0,5 + oil, 0,5	2,5±0,13	0,8±0,04
Molasses, 1,0 + oil, 1,0	7,5±0,38	1,8±0,09
Molasses, 1,5 + oil, 1,5	13,3±0,67	2,2±0,11

Experimental data (see Table 3) show that lowering ammonium nitrate concentration from 0,4 g/l (basic) to 0 and 0,2 g/l in medium for EPS biosynthesis and inoculum preparation respectively accompanied by increasing ethapolan synthesis (amount of EPS – 14,4 g/l, EPS-synthesizing ability – 3,0 g EPS /g biomass).

Table 3

Effect of the nitrogen source concentration on ethapolan synthesis

NH4NO3 concentration in medium for		Synthesis indices
biosynthesis, g/l	inoculum preparation, g/l	EPS, g/l	EPS-synthesizing ability, g EPS/g biomass
0	0	13,3±0,66	2,2±0,11
	0,2	14,4±0,72	3,0±0,15
	0,4	14,1±0,71	2,9±0,14
0,2	0	14,1±0,70	2,8±0,14
	0,2	13,9±0,69	2,7±0,13
	0,4	13,7±0,69	2,4±0,12
0,4	0	13,3±0,66	2,4±0,12
	0,2	13,4±0,67	2,4±0,12
	0,4	13,3±0,66	2,2±0,11

Note, that in modern literature there are many reports about biosynthesis of microbial EPS on carbohydrate substrates [1, 4, 10]. Despite this, information about polysaccharide synthesis on oil-containing substrates is limited. So, it is known that fungus Pleurotus ostreatus FPO-1001 synthesized 0,8 g/l of polysaccharide under cultivation on waste sunflower oil (10 g/l) [8]. In the review [6] shown that Xanthomonas campestris NRRL B-1459 S4LII produced 7 g/l of xanthan during growth on olive mill wastewater (20 % v/v).

At present information about using mixed substrates for microbial EPS synthesis is practically absent. At the same time these substrates successfully used for microbial synthesis intensification of fermentation products (ethanol, lactic acid, butanediol), primary (amino acids, n-hydroxybenzoate, triglycerides) and secondary (lovastatin, surfactants) metabolites [9].

Thus, our previous researches about ethapolan synthesis on growth substrates mixture [9, 10] and results presented in this paper are one of the first that showed the possibility of mixed substrates using to improve the technology of practically viable microbial EPS.

As a result of this work, the ability of ethapolan synthesis on molasses and sunflower oil mixture was established. The highest synthesis indices (the amount of EPS 14,4 g/l, EPS-synthesizing ability 3,0 g EPS/ g biomass) were observed during growth of IMV B-7005 strain under such conditions: using molasses (1,5 %) and sunflower oil (1,5 %) mixture; lowering NH₄NO₃ concentration to 0,2 g/l in medium for inoculum preparation; exclusion ammonium nitrate from biosynthesis medium.

These results are the basic for development of ethapolan production technology on molasses and waste (fried) sunflower oil.

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