;\thi';of's par:;oral FAppl Biochem Biotechno1 (2013) 170:880 894Doll O. 1 007/$ 1 201 0-0 13-0246-7

Biosurfactants of R&odococczzs eiy/&ropo/A IMV Ac-5017Synthesis Intensification and Practical Application

Tetyana Pirog ' Anna Sofilkanych . Teq'ana ShevchukMariya Shulyakova

Received: !3 June 2Ql2 /Accepted: !S April 20{3 /Ptlblished online: 25 April 2013€) Springer Science+Business Media New York 20 13

Abstract hltensiHication of the surfactant synthesis by R/zodococcus eryr/z/'opo//s IMV Ac-5017 on different substrates, including industrial waste, as well as the use of surfact&Htpreparations for oi deglada€io:} wen studied. }{ wa$ estabiisfted bat i\e addition of $ulB&i ate(0.2 %) and citrate (0. I %) into the medium with ethanol, /z-hexadecane, or glycerol (1--2 %)was accompanied by an increase of conditional surfactant concentration by 1.5-1.7 timescanlpared to the indexes in the !neditlm without organic acids. Tile intensification ofsurfactant synthesis in the presence of fumarate and citrate is caused by the increasedactivity of isocitrate lyase (by 1 .2-1 5-fold) and enzymes of the surfactant biosynthesis (by2--4.8-fold) compared to their activity in the medium without precursors. The possibility ofsurfactant synthesis intensification (by 3-4-fold) while cultivating of R. eryfh/opo/fs IMVAc-5017 in the medium with oil containing substrates(2 %) and glucose(0. 1%) was shown.The introduction of 0.01 mM Cu2* in the exponential growth phase of strain IMV Ac-5017in the medium with ethanol accompanied by the increasing conditional surfactant concen-tration by 1 .9 times. The highly efHcient remediation (92-95 %) of oil (2-2.6 g/L) and Cu2'polluted water acer treatment with surfactant preparations (native cultural liquid) at lowconcentrations <5 %) was determined

Keywords Rhodococc s ery//zropo/is IMVAc-501 7 Surfactant . Biosynthesisinto si$:cation , Precursors . Oii degradation

Introduction

Microbial surfactants (biosurfactants) are widely used in different fields of industrythat is why the demand on synthetic surfactants is increasing constantly. At the same

T. Pirog ' A. Sofilkanych ' M. ShulyakovaNational University of Food Technologies, 0 1 60 1 Volodymyrska stt 68 Kyiv, Ukraine

T. Pirog (BI) ' T. ShevchukInstitute of Microbiology and Virology of National Academy of Science of Ukraine, D03680, 1)SP,Zabolotnogo str, 1 54 Kyiv, Ukrainee-mail: tapirogGBnuR.ed\l.ua

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time, the pace of biotechnology development and increased environmental concernshas been of great interest to scientists using microbial surfactants as an alternative tochemical ones rJ],

Biosurfactants have a number of advantages aver synthetic substances: biodegrad-ability (when they appear in the ecosystem, they degrade easily in simple sub-stances; at the same nine, the use of chemicals leads ta great harm), synthesizedby microorganisms from cheap raw materials (for example, the wastes after difTeientproduction), the stability of properties in a wide range of pH and temperatures, notoxicity, and the complex chemical structure that is hardly produced by chemicalsynthesis.

Due to the amphyphylic structure of molecules, biosurfactants have differentproperties, which is why they are used in oil industry and mining, in chemical andfood industry, agriculture, and also in environmentally safe technologies For bioreme-diation [1].

Despite the outstanding properties of biosurfactants and the advantages over thesynthetic analogues, their industrial production is much lower than chemical ones. Thehigh-cost of biosynthesis and the process of biosurfactants extraction from the culturalliquid, as well as the low concentration of biosurfactants in Ihe cultural liquid, haverestrained global industrial production of biosurfactants [2]. That is why tile investi-gations devoted to the solution of these prob]ems became topical and main inbiotechnology of microbial surfactants

In the previous work, the oil-oxidizing bacteria identiHled as Rbodococczfs e/'/hropo/isIMV Ac-5017 were isolated from the oil-polluted samples of soil. The ability of the strain tosynthesize the metabolites with surface-active and emulsifying activity during the cultivationon different hydrophobic (pz-hexadecane, liquid paraffin) and hydrophilic (glucose, ethanol)substrates was detenninedt3].

The criteria of biosurfactant synthesis in medium with /z-hexadecane, which were shownfor R. err/zropo/fs IMV Ac-5017, can be compared to the ones of the otller Rhodococcusstrains that were described in the publications [4, 5]

However, in comparison with the other representatives of genus R#odococcui, thestrain we have selected has the following advantages: (1) synthesizes biosurfactant inthe medium with total salt content of 3.15 g/L (fol the other Rhodococcus--up to10 g/L); (2) does not need the trace elements and yeast extract in the medium; (3)produces biosurfactant with a higher yield from substrate. It was detenllined that thesurfactant synthesized by R. e/7ffropo/is IMV Ac-5017 consists of glyco-, phospho-,and neutral lipids, which torn the complexes with the substances of polysaccharideand peptide nature [3]

The aim of the present work--development of approaches allowing to increase theef'nciency of biosurfactant synthesis by R. er/#/opo/is IMV Ac-50 1 7 on different carbohy-drate substrates, and also to investigate the possibility of practical use of these products.

Materials and Methods

C)bject of Research

Object of research--Rhodococcus ezy/hlopo/is EK- I strain, registered in MicroorganismsDepositary of Institute of Microbiology and Virology, the National Academy of Sciences ofUkraine under the number IMV Ac-5017

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Medium composition and conditions of R. efyfh/lipo/is IMV Ac-501 7 cultivation

Bacteria were grown in the liquid mineral medium (g/L distilled water): NaNO3--1.3,NaCl1--1.0, Na2HPO4' 12HzO--0.6, KH2PO4---O. 14, MgSO47H2O..- O. 1 , FeSO4 7HzO.0.00}, PH 6.8-7.0.

rz-Hexadecane, ethanol, glycerol, and oil-containing substrates (waste of oil and fats

manufacturing, fried sunflower oil) were used as the carbon and energy sources in concen-tration of 1--2 % v/v.

In one of the variants the organic acids (sodium citrate and sodium fumarate in concen-tration of 0. 1 % and 0.2 %, respectively) or glucose (1-3 g/L) were added in the medium atthe beginning of the stationary growth phase of strain IMV Ac-5017. Citric acid was alsoused in concentration of 0.08 % instead of sodium citrate. The concentrations of sodiumcitrate (0. I %) and citric acid (0.08 %) were equimolar by carbon. The sodium fumarate,sodium citrate and citric acid were added into the medium as 10 % solutions

Taking into account thai fulnarate and citrate (citric acid) were additional carbon sourcesand their presence in the medium led to the changing ofcarbon concentration, and also of theC/N ratio, which was canceled in the control flasks by adding higher concentrations ofcarbon sources.

In one of the variants only the fumarate was added into the medium with ethanol andglycerol at the beginning of the stationary growth phase of IMV Ac-5017 strain, and theneutralization ofcultural liquid with the citric acid solution was conducted while the pH wastncreasmg.

The Cu2+, Cd2+, and Pb2+ were added into the nutritive medium in the concentrations of0.0 I and 0.05 mmol/L at the beginning of the cultivation, at the exponential and stationarygrowth phases to test the influence of metals on the growth of R. ery/;z/'opo/fs IMV Ac-5017and the surfactant synthesis. These metals were added as 1 % solutions of salts5HzO, CdSO4' 8HzO, and Pb(CH3C00H)4.

The culture in the exponential phase was used as the inoculum and added in concentrationof 5 % of nutritive medium volume. The concentration of the corresponding carbon sourcein the medium for the inoculum obtainment was 0.5 % v/v. The cultivation was carried out in750 ml flasks, containing 100 ml of medium, on a shaker (220 rpm) at 30 'C during 24-

The biomass was determined by a gravimetrical method. The double treatment of culturalliquid with hexane or petrol ether was carried out to remove the residual n-hexadecane or oil,respectively, before the cell sedimentation (5,000xg, 30 mln). The necessity of this operationwas caused by the precipitation of n-hexadecane or oil with cells that overestimate thebiomass level.

168 h

Detemlination of Indexes of Growth and Biosurfactant Synthesis

The surfactant synthesizing capacity was estimated by adopting the following criteria

(1) Surface tension (a;) of the cell-free cultural liquid, which was measured with a semi-automatic tensiometer (LAUDA TDIC, Gemlany). Before the indicator as was defined,the supematant of the cultural liquid was preliminarily washed with hexane from rz-hexadecane residues or with petrol ether from oil residues, which have surface-activeproperties and significantly decrease the real value of surface tension.For a rapid test of the quantitative content of surfactant in the cultural liquid, we usedthe index of conditional surfactant concentration (CSC), which was defined as the

(2)

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The concentration of ethanol in nutritive medium was 2.0 vol.%. The emulsification index was determined Forthe native cultural liquid; duration of cultivation was 1 20 h

cluster of small dry flaked, some of which stayed on the water surface and the others settleddown to the bottom of reservoirs after the mixing. The data on determination of concentra-tion of residual oil acer 30 days of the experiment is shown in Table 5.

At the next stage complex water pollution was modeled and the possibility of itstreatment in the presence of surfactants was studied. Oil (2 g/L) and Cu2" (0.01 and0.05 mM) were added into the water (2 L), and then it was treated with surfactant preparationof R. eU/hrapo/fs IMV Ac-5017 based on post fennentation cultural liquid (5 %, Table 6).

The data presented in Table 6 show that in the presence of Cu2+ the degree of olldestruction increased. Control of water microflora, conducted during the experiment,showed 1--2-fold increase in the total number of microorganisms in all samples treated withsurfactant (Table 7).

Discussion

The Hlrst articles that described the representatives of the genus Rhodococcus as producers ofsubstances with surface-active properties appeared in 1970--1 980s [20--22]. Later, thein6omiation about surfactants synthesis by Rhodococcus was summarized in reviews [4,5], and has been extended [23--25]. Taking into account that there are many bacteria of

Table 5 Efficiency of oil polluted water treatment with surfactants' preparations of R. eryr/lropo/ff TMV Ac501 7

Surfactant

preparationConGentmtion ofsurfactant preparation, %

Number oftt'eatments

One

'l'wo

One

I'woOne

'T'wo

T-wo

Concentration ofresidual oil. g/L

Oil degradationdegree, %

CHIN!at !squid 5

10

15

30

5

15

30

Without treatment (control)

0.20£0.0 !0.32 J:0.020.36t0.020.2810.01

0.44£0.020.32£0.020.38£0.020 .32J: 0 .02

2 .6 :E 0 . 1 3

92.3£4.687.7J:4.488.2£4.489.2J:4.583 . I :t4.285.714.385.4£4.387.7£4.40

Supematant

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Table 4 Influence of CuZ* on growth af R. er]/f/i/opo/fs IMV Ac-501 7 and surfactant synthesis  MomentofCuZ+addition CuZ+concentration. mM Biomass,g/L CSC E24, %

Beginningof process 0.01 0.18:E0.009 1.9£0.10 40£2

0.05 0. 1310,007 1 .8:E0.09 4 1 t2Exponential phase 0.01 0.25£0.013 5.6:L0.28 47£2

0.05 0.55£0.028 2.8i:0. 14 39£2

Stationalyphase 0,0} 0.67:to.033 2.8i:0.14 4S£2

0.05 0.90j:e.845 3.} £O.}6 4 1 £2

Control 0 0.?5£0.038 3.0£0.15 43£2

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Table 6 Remediation of oil polluted (2 g/L) water by surfactant preparations of R. eryfAropo/fs IMV Ac-50 17in the pfese ce of copper cano! s

Type of pollution Concentration of residual oil, g/L Oil degradation degree, %

oil0ii+Cu2+ 0.01 mMOil+Cuzt 0.0S mM

Control (without surfactant)

! .0£0.0500. I to,0050.5£0.0251 .82£0.09 1

S0£2.59514.875 1: 3 . 8

9£0.5

The content of residual oi} w© determined at 20th day of the experiment

R/zodococcus genus known today as producers of surfactants, each new strain must havesome advantages compared to the existing ones.

A significant advantage of the isolated strain IMV Ac-5017 is the ability to synthesizesurfactant on a vast variety of hydrophilic (glucose, ethanol, glycerol) and hydrophobic(liquid paraffin, n-hexadecane, sunflower oil) substrates, while the majority of R#odococcusstrains foml a surfactant mainly while growing on hydrocarbons. Strain IMVAc-50 1 7 growsand synthesizes surfactant in a simple mineral medium containing no trace elements andgrowth factors, and synthesizes mainly exocellular surfactant [19], while the otherrhodococci--both associated with cells and exocellular surfactant [4, 5]. ]t should be notedthat our world [3] was one of the first that reported on the synthesis of surfactant byrhodococci on ethanol.

One of the global environmental problems of today is the search 6or methods of disposalor the reuse of industrial wastes. Moreover, hazardous wastes are not only wastes containingtoxic substances (such as phenol and its derivatives), but the wastes entering the environ-ment in uncontrolled amounts, for example, oil-containing wastes (wastes after oil and fatproduction, fried oil after use in food establishments and etc.). Biotechnological methodsallow industry to recycle wastes and to obtain practically valuable biologically activesubstances or biomass.

Despite the large number of publications on the synthesis of microbial surfactants onindustrial waste [2G-29], information about rodococci growing on such substrates is very[imited. ]t is known that R. ery/hlppo/fs ] 6 LM.USTHB synthesized surfactants in a mediumcontaining 3 % of fried sunflower oil [30], and Rhodococczls sp. BS32--in a mediumcontaining 20 g/L of rapeseed oil [3 1]. However, in these studies the authors did not showthe quantitative determination of surfactant (in g/L), and therefore it is not possible tocompare their results with data reported in this article. Our studies have shown that the rate

Table 7 Control of microorganisms' quantity irt wafer during the oil degradation process

'type of pollution Quantity of microorganisms(CFU/ml) on the day

7th 21 st 28th

oilOil+Cu2+ 0.01 mMOil+Cu2+ 0.05 mM

Contra! (without surfactan!)

S.3 . 104

1 .6 . 1 0s

2.0. 10s

3.6. 1 0'i

7.2. 104

2.6. 10s

3.0. 10s

! .7. tQs

8.0 . 1 04

2.8 . 10s

3.9 . 10s

3.1 . 10s

8.3 . 104

3.3 . 1 0s

4.7 . 10s

3.8 . 10s

Quantity of microorganisms in the water before the contamination was 3.2. 1 0a CFU/ml

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presence of copper canons may occur in the activation of alkane hydroxylases--Horstcatabalic enzymes of n-alkanes as in the strain IMV Ac-501 7, and the indigenous oxidizingmicroorganisms. Thus, it is known [45, 46] that Cu2+ p]ays a key ro]e in the physiology andactivity of methanotrophs. Methane oxidation by inethanotrophs is canned out by membranebound and/or soluble methane oxygenase belonging to the class of alkane hydroxylases, aswell as enzymes of catabolism of lz-alkanes. The dependence of growth criteria (growth rate,biomass concentration, the economic factor) on the content of copper canons in the culturemedium was detemlined for many methanotrophs [45]. Our future research wi]] focus onstudying of the influence of copper on the activity of all(ane hydroxylase of strain IMV Ac-

We established the possibility of increasing the emciency of surfactant biosynthesis by R.eryrhropo/zs IMV Ac-5017 as a result.of: (1) use of oil containing substrates as the carbonsources, including wastes of oil and fat industry and catering establishments; (2) introductionof biosynthesis precursors (glucose, C4-dicarboxylic acid) and regulators of lipogenesis(citrate) into the medium with non-carbohydrate substrates; (3) introduction of copperGallons (0.01 mM) into ethanol containing medium in the exponential growth phase. Itwas established that surfactants ofR. ery//zropo/is IMV Ac-50 1 7 intensiHled processes of oildegradation in polluted water. The possibility of using surfactant preparations (nativecultural liquid) in low concentrations (5 %) for the effective (83-95 % degradation of oilat the concentration of 2.0 g/L) treatment of water, containing heavy metals, was detennined.

5017

Acknowledgments The authors thank Darien Perrett (Cambridge Exams Manager, Teacher of English inInternational House Language Center, Kyiv) for the help in the preparation of the manuscript.

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