INDUSTRIAL WASTE BIOCONVERSION INTO SURFACTANTS BY Rhodococcus erythropolis ІMV Ас-5017, Acinetobacter calcoaceticus ІMV В-7241 and Nocardia vaccinii ІMV В-7405 T. P. Pirog, M. O. Shulyakova, L. V. Nikituk, S. I. Antonuk, I. V. Elperin

Until recently there has been no doubt that the environment, air, land and water, always effectively “recycle” domestic, industrial and agricultural waste. Now we know that it is not so. Humanity faces two fundamental problems: management of waste which are constantly generated in huge quantities, and degradation of toxic compounds accumulated for decades in landfills, water and soil. It should be noted that the danger lies not only in waste containing toxic substances (such as phenol and its derivatives) but also in waste that enter the environment in uncontrolled quantities, such as oil containing (waste from oil and fat production, fried oil used in catering, etc.). Biofuel, including biodiesel, is one of the most promising substitutes for fossil fuels. In the last decade, biodiesel production increased significantly [1, 2]. For example, the expected UDC 759.873.088.5:661.185 https://doi.org/10.15407/biotech10.02.022

annual increase in biodiesel production is 8-10% [2].However, due to rapidly growing demand for biodiesel, there is a problem of recycling its byproduct, glycerol.This glycerol fraction contains a wide variety of impurities, making impossible its use in many traditional areas of application, and storage, disposing of it is a serious environmental problem because of its increased alkalinity and the content of methanol [1,2].
As a result of increased industrial activity, mono-and polyaromatic hydrocarbons and their derivatives which are toxic, carcinogenic and resistant to external factors more and more pollute the environment that is a significant danger to human health and the biosphere as a whole [3][4][5].At the turn of XX-XXI centuries, biotechnological methods of environmental remediation from aromatic compounds attract the interest due to their safety, low cost and high capacity of bacterial destructors [6,7].
Progressive rates of oil pollution necessitate the development of environmentally friendly and economically reasonable purification methods aimed at intensifying processes of hydrocarbons decomposition.Such a method of soil and water treatment is bioremediation, based on natural potential of microorganisms.It is known that in presence of heavy metals, efficiency of oil degradation can be reduced, so today it is important to find environmental remediation methods for such complex environmental pollution [8].
In recent decades, microbial surfactants (MS) have become the subject of intense theoretical and applied researches driven by their possible practical use in various industries, as well as nature conservation technologies to clean the environment [9][10][11].
In our previous studies from oilcontaminated soil samples we isolated strains of oil-oxidizing bacteria identified as Rhodococcus erythropolis EK-1, Acinetobacter calcoaceticus K-4, and Nocardia vaccinii K-8 [12].The strains are registered in Depository of microorganisms of Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine under the numbers ІМV Ас-5017, ІМV B-7241, and ІМV B-7405 respectively.The strains R. erythropolis ІМV Ас-5017, A. calcoaceticus ІМV B-7241, and N. vaccinii ІМV B-7405 have been shown to synthesize metabolites with surface-active and emulsifying properties when grown on hydrophilic (glucose, ethanol, glycerol) and hydrophobic (liquid paraffins, n-hexadecane) substrates [13].
The purpose of this work is to realize alternative processing of toxic industrial waste into surfactants by R. erythropolis ІМV Ас-5017, A. calcoaceticus ІМV B-7241, and N. vaccinii ІМV B-7405 for environmental bioremediation.

Materials and Methods
Study objects.Study objects were strains of oil-oxidizing bacteria isolated from oil-contaminated soil and identified as Rhodococcus erythropolis ЕК-1, Acinetobacter calcoaceticus К-4, and Nocardia vaccinii K-8 [12].The strains are registered in Depository of microorganisms of Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine under the numbers ІМV Ас-5017, ІМV B-7241, and ІМV B-7405 respectively.
N. vaccinii IМV B-7405 strain was grown in liquid mineral medium (g/l): NaNO 3 -0.5, In one version of the experiment, the concentration of nitrogen nutrition source in the culture medium of studied strains was increased twice.
As the source of carbon and energy we used refined sunflower oil "Oleyna" (Dnepropetrovsk oil extraction plant), waste oil after frying potatoes and meat (the McDonald's network of fast food restaurants, Kyiv), and unrefined sunflower oil (4%, v/v).
Also as a source of carbon and energy, refined (> 99.5%) glycerol was used in concentration 1.0-1.5% (v/v).To modify the average composition of technical glycerol, NaCl or KCl at concentration of 2.5%, and 0.3% methanol or ethanol (v/v) were added into a mineral medium with refined glycerol.This substrate is hereinafter called "modified glycerol".Also as a source of carbon and energy we used technical glycerol which is a waste of biodiesel production (Zaporizhzhia biofuel plant, Zaporizhzhia, Ukraine).Concentration of technical glycerol in culture medium was 2-10% (v/v).When using technical glycerol as substrate, its content in the medium was calculated as equimolar by carbon concentration to the purified glycerol, taking into account the average content in the glycerol fraction (70%).
As the sole source of carbon and energy in a medium, phenol, hexachlorobenzene, naphthalene, benzoic, sulfanilic and N-fenylantranilic acids were also used at a concentration of 0.3-1.5% (w/w), and benzene and toluene at a concentration of 0.3-1.5% (v/v).Phenol and sulfanilic acid were dissolved in distilled water and sterilized in autoclave for 40 min at 120 С, and weights of hexachlorobenzene, naphthalene, benzoic and N-fenylantranilic acids were sterilized with UV light for 30 min.
As inoculum we used the cultures from the exponential growth phase, grown on the respective liquid medium containing 0.5-1% (v/v) of substrate.The quantity of inoculation material (10 4 -10 5 cells/ml) was 5-10% of the volume of the culture medium.The bacteria were cultivated in 750 ml flasks containing 100 ml of medium on the shaker (320 oscillations per min) at 28-30 С for 120 h.
Indexes of growth and surfactants synthesis.Biomass was determined by optical density of the cell suspension with the following determination of dry biomass by calibration curve.
The ability to synthesize MS was evaluated on the following parameters: surface tension ( s ) of the cell-less culture liquid, measured with semi-automatic tensiometer (LAUDA TD1C, Germany); conditional surfactant concentration (MS*, dimensionless); the amount of extracellular synthesized surfactants (g/l), and emulsification index (Е 24 ).
For rapid determination of the quantitative content of surfactants in the culture liquid the index of conditional MS concentration (MS*) was used, defined as the dilution rate of cellless culture liquid (supernatant) to the point of CMC (critical micelle concentration).Then, plot of surface tension  s against the logarithm of supernatant dilution was made.Abscissa of the inflection point of the curve corresponds to the MS*.
The amount of extracellular synthesized MS (g/l) was determined gravimetrically after extraction from supernatant of culture liquid using modified Folch mixture.To obtain supernatant, culture liquid was centrifuged at 5,000 g for 20 min.Isolation of extracellular MS was performed as described below.
In a cylindrical separatory funnel (500 ml), 100 ml of the supernatant and 20 ml of 1 M HCl solution were added, funnel was then closed with polished stopper and shaken for 3 min, then another 15 ml of 1 M HCl solution and 65 ml of chloroform and methanol (2:1) mixture were added and stirred for 5 min (lipid extraction).The extracted liquid was left in separating funnel to separate phases and subsequently the lower fraction (organic extract 1) was removed and the water phase was reextracted.The second extraction was carried out adding 35 ml of 1 M HCl solution and 65 ml of chloroform and methanol (2:1) mixture to the water phase, extracting lipids for 5 min.After phase separation, the lower fraction was isolated as organic extract 2. At the third cycle of extraction, 100 ml of chloroform and methanol (2:1) mixture was added to the water phase, and organic extract 3 was obtained as described above.Then extracts 1-3 were combined and evaporated on a rotary evaporator IP-1M2 (Russia) at 50 С and 0.4 bar absolute pressure to constant weight.
Emulsification index for the 50-fold diluted culture liquid was determined as follows: to 2 ml of culture liquid diluted with distilled water, 2 ml sunflower oil (a substrate for emulsification) were added and shaken for 2 min.Definition of emulsification index (Е 24 ) was carried out after 24 h, as the ratio of height of emulsion layer to the total height of liquid in a test tube, expressed as a percentage.
Study of oil biodegradation in water and soil.To simulate soil contamination with oil and metal cations, 1 kg of soil, 25 ml oil, surfactant preparations as post-fermented culture liquid (100-200 ml), and 0.01% diammonium phosphate as a source of nutrients were placed in a plastic container and mixed.In simulation of complex pollution with oil and metal cations, such substances were added to soil (singly or combined): 0.01-0.1 mmol Сu 2+ , Cd 2+ , Pb 2+ as 1М solutions of CuSO 4 •5H 2 O, CdSO 4 •8H 2 O, and Pb(СН 3 COOH) 4 salts respectively.Samples were stirred every 3 days to improve aeration, and were moisturized with sterile water.The duration of the experiment was 20 days.
To simulate water contamination with oil and metals, 2 l of pumped water covered with 6-15 ml of oil in a plastic container were treated with surfactant preparations in concentration of 5-10% (v/v) and 0.01-0.5 mmol Сu 2+ , Cd 2+ , and Pb 2+ separately and in various combinations.Diammonium phosphate (0.01%) was used as a source of nutrients.During the experiment (20 days) total viable cell counts in pumped water were performed by Koch method on MPA.
The amount of oil was evaluated gravimetrically.Oil extraction with hexane (1:1) was performed 3 times.The organic extract was evaporated to constant weight on a rotary evaporator IP-1M2 (Russia) at 55 C and 0.4 bar absolute pressure.
All experiments were thrice replicated; the number of parallel determinations in the experiments was 3 to 5. The statistical treatment of the experimental data was carried out as described previously [6,16].The differences between the means were considered significant at P < 0.05.

Surfactants synthesis on biodiesel waste.
At the first phase we studied the influence of technical glycerol components (methanol, ethanol, potassium and sodium) on surfactants synthesis by strains A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017, and N. vaccinii IMV B-7405, and the possibility of using biodiesel waste for the synthesis of surfactants.
The study showed that in medium containing pure glycerol (1%, v/v) and glycerol fraction components (K and Na salts -2.5%, methanol and ethanol -0.3%) the conditional concentration of MS increased by 11-68% for A. calcoaceticus ІМV B-7241, R. erythropolis ІМV Ас-5017, and N. vaccinii ІМV B-7405 compared to the values on medium without these salts and alcohols.On medium with technical glycerol (2.2%) obtained directly from the producer of biodiesel (Zaporizhzhia biofuel plant), strains synthesized extracellular MS with concentration twice higher than that for the purified substrate (Table 1).Note that the used concentrations of modified and technical glycerol were equimolar by carbon to 1% purified glycerol, and that modified glycerol was obtained by adding 2.5% NaCl and 0.3% methanol to purified substrate.
Taking into account the volume of biodiesel production in the world, and the amounts of technical glycerol obtained as a byproduct [1,2], it is clear that the effective use of this waste as a substrate in biotechnological processes needs as high as possible its content in the culture medium for producers of economically valuable microbial metabolites.
Thus, our next step was to find the cultivation conditions for R. erythropolis IMVAc-5017, A. calcoaceticus IMV B-7241, and N. vaccinii IMV B-7405 in medium with the highest possible concentration of technical glycerol that ensures high rates of surfactants synthesis.
Increased concentrations of inoculum to 10-15% and twice increased (compared to the basal medium) of nitrogen source content allowed to carry out the processes of synthesis of surfactants by strains IMV Ac-5017, IMV B-7241 and IMV B-7405 on medium with 7-8% technical glycerol (v/v).Under these conditions, the concentration of extracellular MS synthesized by studied strains amounted to 3.4-5.3g/l, which is 1.4-3,0-fold higher than at the basal medium with same concentration of substrate (Table 2).
Published data indicate that concentrations of MS synthesized by different producers on technical glycerol usually are low [14][15][16][17][18].For example, the strain of Bacillus subtilis LSFM-05 in the culture medium with 5% technical glycerol synthesized 1.36 g/l of surfactin [14].P. aeruginosa MSIC02 when grown on the medium with pre-hydrolyzed (treatment with sulfuric acid) technical glycerol (5% v/v) synthesized 1.27 g/l of rhamnolipidis, while on medium containing not-hydrolyzed substrate the synthesis level was several times lower [15].
The strain Starmerella bombicola ATCC 22214 synthesized up to 6.6 g/l of sophorolipids on medium containing 15% technical glycerol (v/v) and/or 10% sunflower oil (v/v) [16].Higher concentrations (nearly 9 g/l of glycolipids) have been observed after cultivation of Ustilago maydis for 12 days on medium with 50 g/l of technical glycerol [17].The authors upped the MS concentration to 32 g/l by adding some amino acids, B vitamins, ammonium citrate, mannose and erythritol (20 g/l) into the medium containing 50 g/l of technical glycerol [17].In [18] it has been established that strain P. aeruginosa WAE synthesizes 2.6 g/l of MS if grown on biodiesel production waste.
Note that in most papers, concentration of technical glycerol in the culture medium for MS producers has been 5% (v/v) or 50 g/l [14][15][16][17].In [17] the authors investigated the influence of higher concentrations of substrate on synthesis of glycolipids.However, increasing the concentration of technical glycerol to 80 g/l resulted in amount of synthetized glycolipids reduced twice.
Thus, the rate of MS synthesis by A. calcoa ceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMB B-7405 on biodiesel production waste is not only comparable to that described in the literature, but also surpasses those for many well-known producers.
Fried sunflower oil as a substrate for surfactant synthesis.Table 3 shows the results of MS synthesis by A. calcoaceticus ІМV В-7241 cultivation on various sunflower oilcontaining substrates.
Experiments showed that use of the inoculum grown on molasses resulted in 1.7-2.7 times less amounts of MS if strain was cultivated on fried and unrefined sunflower oil compared with those on purified (refined) substrate (Table 3).At the same time, emulsification index of 50-fold diluted culture liquid changed but slightly.However, if in the inoculum preparing medium molasses was replaced with refined sunflower oil, increased synthesis of microbial surfactants has been observed for fried and unrefined oil compared with the values for refined substrate.Note that using of inoculum grown on molasses and sunflower oil was not accompanied by significant changes in the emulsification index (Table 3).
In literature sources there is enough information on the use of oil-containing substrates for synthesis of microbial surfactants [19][20][21][22][23].However, in most cases, producers of MS are cultivated mainly on refined plant oils or oil production wastes (sludge).
Much less researches are dedicated to synthesis of MS on fried oil.For example, Pseudomonas fluorescence MFS03 cultured on 2% fried plant oil synthesized 4.2 g/l of MS [20].Cultivation of P. aeruginosa PB3A on the medium containing fried oil (1%) was accompanied by synthesis of 0.3-0.6 g/l of MS [21].P. aeruginosa ATCC 9027 grown on overfried sunflower oil (initial concentration of 15 ml/l, followed by introduction of 20 ml/l at 72 nd hour of growth) produced rhamnolipids in concentration of 8.5 g/l [23].
Transformation of aromatic compounds in surfactants.Data on the synthesis of surfactants by R. erythropolis IMV Ac-5017 grown in medium with different concentrations (0.5-1.5%) of aromatic substrates is shown in Table 4.
The results showed that the strain IMV Ac-5017 is able to use phenol and toluene at concentration of 0.5% as sources of carbon and energy for biosynthesis of surfactants (conditional surfactant concentration 3.3 and 1.3, respectively).Higher concentrations of phenol and toluene appeared to be toxic to R. erythropolis ІМV Ас-5017.Benzene and naphthalene even in low concentrations inhibited the biosynthesis of MS (MS* did not exceed 0.6).Given that the aromatic compounds in concentrations above 0.5% inhibited the synthesis of surfactants by strain IMB As-5017, in further studies A. calcoaceticus IMV B-7241 and N. vaccinii IMB B-7405 were grown in medium with lower concentrations of substrates (0.3-0.5%).Utilization of aromatics by N. vaccinii IMV B-7405 was accompanied by the formation of extracellular metabolites with surface-active and emulsifying properties.The maximum rates of MS synthesis (MS* 2.3-2.6% and Е 24 70-75%) were observed when strain IMV B-7405 had been cultivated in medium containing 0.5% naphthalene, N-phenylantranilic acid and phenol.
The strain A. calcoaceticus ІМV В-7241 is able to synthesize MS when grown on wider variety of aromatic substrates than the strains R. erythropolis ІМV Ас-5017 and N. vaccinii ІМV В-7405 (Table 5).The highest conditional concentration of MS and Е 24 (up to 75%) were observed after cultivation of strain ІМV В-7241 on medium with 0.5% phenol and benzoic acid.
Analysis of literature [24][25][26] has showed that some microorganisms under the cultivation conditions on aromatic substances are able to synthesize metabolites with surface-active and emulsifying properties.For example, Brevibacillus sp.PDM-3 [24] and Pseudomonas sp.USTB-RU [25] produced surfactants on phenantrene, and a member of Acinetobacter genus (strain USTB-X) Note.* -Р  0.05 compared to control (conditional concentration of MS produced by strain ІМV Ас-5017 on hexadecane).** -Р  005 compared to control (emulsification index for strain ІМV Ас-5017 on hexadecane).The measurement error for emulsification index did not exceed 5%.synthesized MS on pyrene [26].In [27] it has been shown that cultivation the strain P. aeru gino sa NY3 on medium with mixture of polycyclic aromatic compounds (5 ml/l fluorene, anthracene, phenanthrene, pyrene and fluoranthene) was accompanied by their decomposition by 10-20% within 24 h.The strain NY3 was shown to be able to synthesize rhamnolipids, although the authors studied the chemical composition of MS growing P. aeruginosa NY3 on glucose and glycerol [27].
The authors of [24][25][26][27] indicate that the ability to synthesize such extracellular metabolites greatly facilitates the assimilation of aromatic substrates by microorganisms.
Effect of surfactants on degradation of complex oil and heavy metal pollutions in water and soil.For now purifying of water and soil from oil is mostly done with biological products that are lyophilizated biomass (or paste) of oil-oxidizing bacteria [11].However, microorganisms introduced into oil contaminated ecosystems need some time to adapt to new conditions.Thus it would be more effective (compared to bio-augmentation) to implement another purification method, biostimulation (involving introduction of various substances, nutrients etc., which stimulate autochthonous, natural microbiota).Effective stimulants of natural oil-oxidizing microbiota are microbial surfactants [4,10,11,28,29], and the best removal of hydrocarbons is achieved by the use of microorganisms able to assimilate the oil and simultaneously synthesize surfactants.Hence, as preparations for treatment of the oil pollution we used culture liquid containing the cells of oiloxidizing bacteria and the MS they produced.
Table 6 shows data on destruction of complex pollution of oil and heavy metals in the presence of MS of A. calcoaceticus IMV B-7241.After culture liquid was added to water containing 3 g/l of oil and a mixture of cations of three heavy metals (Cu 2+ , Cd 2+ , and Pb 2+ ), the level of oil degradation was 90-92%, and in the case of increased concentration of oil in water (up to 6 g/l) the level of its degradation decreased slightly (to 85-88%).Intensification of oil degradation in the presence of surfactants is caused by the activation of natural oil-oxidizing microbiota that is evidenced by its increase in 100-1000 times by the end of the experiment.
Further experiments showed the possibility of using R. erythropolis IMB Ac-5017 the culture liquid for the destruction of complex with heavy metals oil pollutions in soil, at that copper cations stimulated the degradation of oil in the presence of surfactants (Table 7).Similar patterns were found during the study of the effect of A. calcoaceticus IMV B-7241 surfactants on the purification of oil and heavy metals contaminated water (Table 6).
Results given in Tables 6 and 7 support our previous findings about the role of MS of N. vaccinii ІМV В-7405 in degradation of complex oil and heavy metal pollutions in water and soil [30].Here it is also established that Note.* -Р  0.05 compared to control (degradation of 3 g/l oil in water in the presence of MS without metal cations).** -Р  0.05 compared to control (degradation of 6 g/l oil in water in the presence of MS without metal cations).Duration of experiment was 20 days.copper cations show a stimulating effect on oil degradation.
We suppose that one of the mechanisms causing increased oil degradation in the presence of low concentrations of copper cations may be Cu 2+ stimulating the activity of alkane hydroxylases (the first enzymes of hydrocarbon catabolism) of both MS-producing strains and natural oil-oxidizing microbiota.

Table 1 . Synthesis of extracellular MS by IMВ B-7405, ІМВ Ас-5017 і ІМВ В-7241 grown on medium with varying glycerol types Strain MS (g/l) if grown on glycerol
* -Р  0.05 compared to control (concentration of MS produced by strains grown on purified glycerol).

Table 2 . The influence of the nitrogen source concentration in the medium with technical glycerol on MS synthesis by IМV B-7241, IМV Ac-5017, and ІМV B-7405
Note. * -Р  0.05 compared to control (concentration of MS produced by strains ІМV Ас-5017, ІМV B-7241, and ІМV B-7405 on basal medium containing 1.3, 0.35 and 0.5 g/l of nitrogen source respectively).

Table 3 . Synthesis of MS by ІМV В-7241 cultured on medium with sunflower oil (4%)
Note. * -Р  0.05 compared to control (concentration of MS synthesized by strain ІМV В-7241 on refined sunflower oil).** -Р  0.05 compared to control (emulsification index for cultivation of strain IMV B-7241 on refined sunflower oil).The measurement error for emulsification index did not exceed 5%.

Table 7 . Degradation of complex oil and heavy metal pollutions in soil (20 g/kg) in the presence of R. erythropolis ІМV Ac-5017 culture liquid (5%, v/v) Concentration of cations in soil, mmol Concentration of residual oil, g/kg Oil degradation,%
Note. * -Р  0.05 compared to control (concentration of residual oil in soil in the presence of MS without metal cations); ** -Р  0.05 compared to control (oil degradation in soil in the presence of MS without metal cations).