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Pure & A p p l . Chern., Vol. 69, No. 11, p p . 2357-2369, 1997.P rinted in Great Britain.Q 1997 IUPAC

Perspect ives in b io degradat ion of alkanes an d PCBsJ. Kiga,J. Burkharda, K. DemnerovBa,J. KoSt'6la, T. Macekb, M. MackovP, and J.PazlarovPa Prague Institute of Chemical Technology, Technickd 5, 166 28 Prague 6Prague 6, Czech RepublicInstitute of Organic Chemistry and B iochemistry CAS, Fleming. n. 2, 166 10

Abstract: Mixtures of indigenous soil bacteria were applied to remediate local groundwaters and soil polluted with petroleum derived substances. Implementation of threemo nth s remediation protocols resulted in a decline of the amount of petroleum derivedcontaminants from an initial concentration of 1-10 g.kg-' soil dry weight to an average of0.2 5 g.kg-' soil dry weight. We also studied genetic and biochemical properties of thebacterial strain Pseudomonas C12B. It was originally isolated for its ability to utilisealkylsulfates and alkylbenzensulfonates as the sole source of carbon and energy. PCBbiodegradation was studied using two biological models, bacterial co-cultures and plantcells cultivated in vitro. An industrial mixture of polychlorinated biphenyls (Delor 103)containing about 60 congeners of different degrees of chlorination (an average of threechlo rines per biphenyl molecule) was used. Bacterial co-cultures acquired fromenrichment protocols were tested in laborato ry and semi-pilot experiments. Pilotexperiments were performed in a two step process in a ground water decon tamination un it(working volume 5m3) which w as operated semi-continuously. After 45 days of operationth e initial PCB concen tration had decreased to 20% . In laboratory experiments PCBdegradation using plant cells cultivated in vitro was also performed. Different cultures ofvarious species differing in their growth parameters and morphology (amorphous,differentiated shoot forming or "hairy root"), transformed or nontransformed byAgrobacterium, were used. Differentiated or hairy root cultures exhibited betterdegradative abilities than undifferentiated amorphous cultures.Key Words: biodegradation, alkanes, PCBs, bacteria, plant cells.INTRODUCTION

In recent years a wide spectrum of microorganismshas been found t o possess the ability to efficiently degradenumerous xenobiotics. Oil-derived substances, mainly n-alkanes belong to the most easily biodegradablecom poun ds, whereas polychlorinated biphenyls (PCBs) are considered to be the most persistent pollutantsope n to degradation by a limited number of species. In the Czech Republic aside from other environmentalcon tam inan ts these above-mentioned compounds represent extremely widespread soil and water pollution.There aremany reasons for pollution in the C zech Republic: industrial accidents, traffic accidents, irresponsiblebehaviour, former bases of Soviet Army primarily polluted by oil and kerosene and imperfect legislation. Oneexample of an industrial accident in 1984 is the leakage of PCBs t o the river Skalice from Technological Unitlocated in RoimitAl pod RadhoSt6m. Periodical monitoring of the river sediments during the following eigh tyears proved t he extreme persistence of PCBs in an untreated system. In Table 1 there are listed someprevious Soviet Army bases heavily polluted mainly by oil derivatives.Aliphatic hydrocarbons are assimilated by a wide variety of microorganisms, but not all species are capableof utilising aliphatic hydrocarbons as growth substra tes. Whilst many details of a microbial hydrocarbonmetabolism have already been elucidated (ref l) , including the enzymology (ref 2), regulation and genetics (ref3), less attention has been paid to the primary interaction of microorganisms and the hydrocarbon involved.The substrate specificity and biochemical propertie s of bacterial strain Pseudomonas C12B , which is used inour laboratory, were described by KoSt'Al et al. (ref 4),whilst the genetic characteristics were summarized onlyrecently. Coculture ASANOL, (ref 5) was used for practical bioremediation cases.

2357

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2358 J . KAS e t a / .

I

TABLE 1 : The Main Centres of Soviet Army Bases in the CzechRepublic

I

I IPlace Area Location[b21

Mlada BoleslavRalskoMiloviceBohosudovOlomoucVysokC Mj%oPlzebLibava

1002911636501036

Northern BohemiaNorthern BohemiaNorthern BohemiaNorthern MoraviaNorthern MoraviaEastern BohemiaWestern BohemiaNorthern Bohemia

Also polychlorinated biphenyls(PCBs) are a family of compoundswith a wide range of industrialapplications in heat transfer fluids,dielectric fluids, hydraulic fluids,flame retardants, plasticizers,solvent extenders and organicdiluents. Although the manufactureof PCBs has been forbidden (in theCzech Republic in 1984), they arestill an environmental problembecause of their presence inelectrical transformers and landfills.In the United States and the UnitedKingdom, complex PCB mixtureswere manufactured under the tradename Aroclor. In the CzechRepublic the commercial name ofthe similar mixture was Delor (see Table 2.). All these mixtures consist of a number of cong eners which differin the number and distribution of chlorines on the biphenyl nuclei. Possible congeners (20 9) were describedaccording to IUPAC nomenclature. About 150 congeners have been reported in the environment. PCBs haveentered into soil and sediment environments as a result of improper disposal of industrial PCB w astes andleakage of PCBs from electric transformers. Their thermal and chemical stability, resistance to chemicalcorrosion, and general inertness have contributed to their persistance in the environment. Chemically stableTABLE 2: Assignment of Congeners to Peaks of Delor 103 Analyzedon Gas C hromatograph-electron Capture Detector

Peak No. IUPAC No. Substitution

123456789101 11213141516171819202122

5 + 815 +181716 +3226312820 +33 +5345524947 +75484437 +42 +594 1 +6 496747066 +88 +9510177 +110

2 34,42,2,42,2,32,3,52,4,52,4,42,3,32,2,3,62,2,5,52,2,4,52,2,4,42,2,4,52,2,3,53,4,42,2,3,42,2,3,6,62,4,4,52,3*,4,52,3,4,42, 2,4,5,53,3 ,4,4

+2,4+2,2,5+2,4,6

+2,3,4 +2,2;5,6

+2,4,4,6+2,2:3,4 +2,3,3,6+2,3,4,6

+2,23,4.6 +2,2,3,5,6+2,3,3,4,6

lipophilic PCBs are easilytransported through the foodchain. The concentrationsometimes reaches thousands of ngof PCB 1 of water, which is alsooften contaminated by petroleumproducts or chlorohydrocarbons.Furukawa et al. (ref 6) andBedard et al. (ref 7) were amongthe first who reported thebiodegradation of thesecompounds.Indigenous microorganismscapable of effective PCBsbiodegradation were isolated fromthe various sites of the CzechRepublic and described byBokvajova et al. (ref 8) .KaStfinek et al. (ref 9) publishedthe results from combinedmicrobial treatment and sorption ofPCBs from polluted ground water.Current research efforts focus onthe use of plants forbiodegradation of variousxenobiotic compounds. Thistechnology - phytoremediation, isstill in development. Different

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Biodegradat ion of alkanes an d PCBs 2359

projects that target organic contaminants in the water phase (e .g. trinitrotoluene or trichloroethylene) lookpromising, however, more research on less mobile contaminants, e.g. PCBs, is needed before undergoing pilotand large-scale field testing (ref 10). Metabolic transformation and the biochemical pathways leading to thedegrada tion, solubilization and for example in the case of chlorinated compounds also to dechlorination arenot totaly understood, but preliminary results are quite promising. Qualitative differences between plant andbacterial bioremediation showed that this action could be of great environmental importance due to thetendency of plant cultures to transform xenobiotic compounds (PCB etc.) into water soluble products (ref 11).The scope of this paper is to present the practical approaches to bioremediation used in the C zech Republic,and to show some of the generally accepted conclusions found in the course of experiments.

MATERIALS AND METHODSSubstratesAlkanesFor screening of the ability of isolated strains to degrade n-alkanes an industrial mixture of alkanes C,, - C,,was used. Pure alkanes used as g rowth substrates were purchased from Fluka, Switzerland.Polvchlo rinated b iqhenvlsCommercial mixture of polychlorinated congeners produced as Delor 103 (see Table 2) was used throughout.PCBs producer in the f o m r Czechoslovak Republic was Chemko-Str62nC. In field experiments deg radationof two types of Delor was followed: Delor 103 (D 103)with maximum of five chlorine atom s per biphenylmolecule, and Delor 106 (D 106), that was highly chlorinated. Usage of PCBswas forbidden in the Czech Republic in the beginning of the 1980s, but prevailing environmental contaminationis still caused by these products.

Used

Alkane degrad ing bacteria: coculture ASANOL which was developed in our department using an enrichmenttechnique, comprised the following genera: Acinetobacter, Klebsiella, Pseudom onas. Strain PseudomonasC12B (NCIMB 11753) was used throughout for physiologic and genetic studies under lab oratory conditions.PCBs degrading bacteria: several cocultures were developed in our department using soil from the pollutedsites of the Czech Republic by an enrichment technique (319A, 319B, Z 1 ,2 2 ,2 3 ) and the individual strainswere isolated from these mixed cultures. According to preliminary identificationsal isolated strains belongto genus Pseudomonas.

To study the ability of plants to degrade PCBs various plant cell, tissue and o rgan cultures from the C ollectionof the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sc iences, have been used. Fromsome species both amorphousundifferentiated callus cultures and shoo t forming terratoma cultures w ere usedas starting material, while in o ther species callus cultures were compared with strains obtained by geneticaltransformation using Agrobacterium tum efaciens and A. rhizogenes (see Table 3). These cultures includedundifferentiated ones, shooty terratomas and hairy root clones. Some of the stock c ultures were already keptin liquid media as suspensions of small aggregates, some as submerged root cultures, othe rs were transferredfrom agar to liquid media just before the incubation with PCB s.

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2360 J. dS e t a / .MediaMicroorPanismsAll types of microbial cultures were cultivated on simple mineral media described earlier (Refs 4,8) andsupplemented with suitable carbon source. Alkane degrading bacteria were cultivated with a mixture of n-alkanes or with individual aliphatic hydrocarbons (c6 - q 6 ) . PCBs degrading bacteria were cultivated onbiphenyl, which was also present in all Delor 103 degrading experiments at the laboratory stage.Plant Ce lLPlant cell cultures were cultivated using media prepared according to Linsmeier and Sko og (ref 12), 2% ofthe sucrose w as used as carbon source (ref 13). The biodegradation experiments were performed in liquidmedium of the same composition, but without any grow th regulators added.

. . . .ultivation conditions - microorganismsAlkanes. .i v m n on a g a r t e s n gaseous vaDours of a :The cell suspension (0.1 ml)was inoculated on agarplates with basal salts liquid medium and the cells were cultivated in alkane vapours a t 28 C.Cultivation in liquid med ia: The organisms were grown in batch cul ture made up of a basal salts liquid medium.For growth experiments 500mlErlenmayer flasks containing 100 mlof medium were inoculated with testedstrains and shaken at 28C. Concentrations of alkanes used and details about different cultivation conditionsand modes are given by KoSt'91 et al. (ref 4).Microo rgan sms for the decontaminat ion process were grow n in a three s tep procedure: flask-bioreactor oftotal volume 7 1- bioreactor of total volume 70 1 (ref 5) .

TA BL E 3: Plant Cell Cultures Used for PCB Degradation and'heir Morphological CharacteristicsPlant cell cultures Agrobacterium Type of culturetransformation

Solanum aviculareKKl NAVI-3AVR- 1Armoracia rusticanaK62KK50K54Solanum nigrumSNC90SNT-2SNC-9LAtropa bella-donn aR l B CAngelica sylvestrisCucurbita meloMe1

ATR-1ANG-X

nonoYesnononoYesYesYesnoYesnono

callus, nondifferentiatedcallus, nondifferentiateddifferentiatedcallus, nondifferentiatedcallus, nondifferentiateddifferentiatedhairy rootdifferentiatedcallus, nondifferentiatedcallus, nondifferentiatedcallus, nondifferentiatedcallus, nondifferentiatedcallus, nondifferentiated

Polychlorinated biphenyls. .Cultivation on agar -1ates in gaseousw o u r s of PCBs: Cell suspension (0.1d) as inoculated o n agar plates withbasal salts liquid medium and the cellswere cultivated in PCB vapours at28C.

Cultivation in liauid media: Themicroorganisms were grown in batchculture made up of a basal salts liquidmedium (ref 8). For growthexperiments 500ml Erlenmayer flaskscontaining 100 ml of medium wereinoculated with tested strains andshaken at 28C under conditionsdescribed by Bokvajova et al. (ref 8).For biodegradation experiments theDelorl03 concentration was 50mg.1.'.M i c r o o r g a n i s m S f o r t h e&contaminatio n process were grown ina three step procedure: flask -bioreactor total volume 7 1 - bioreactortotal volume 70 1 (ref 9) .

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Biodegradation of alkanes and PCBs 2361. . * .ultivation conditions - ulant cell cu ll u m

4g of inoculum cultivated on the solid medium were aseptically transferred to 100 mlof the liquid medium in300ml Erlenmayer flasks. 10 mg per 100 ml of Delor 103 (methanol solution) was added to each flask.Cultures were incubated with PCBs 14 days on a ro tary shaker at 26 C . The process was finished by heatingin a water bath (8 0-1 00C ) for 15 minutes. Further treatment was the same as with microbial cells.Procedures

Residual alkanes were estimated by gas chromatography on GS Labio GC-94 equipped with a fused silicacapillary column coated with polyethyleneglycol. The flame onization detec tor (FID) was operated at 230C.

PCB concentration in material used (water media treated by m icrobial or plant tissue cultu res, slurry, sedimentand sorbent) was analysed by gas chromatography. Remaining PCBs content was extracted with hexane atambient tempera ture and according to the procedure described earlier (ref 4). A Hewlett-Packard 5890 gaschromatograph with an electron capture detector and a fused silica capillary column coated with animmobilized nonpolar phase was employed.

* .ination of m u n d water c o n t a m t e d w ith alkanesThe bioreactor was an open vessel with a capacity of 2m3 equipped by built-in polyamide netting with 5mmmeshes form ing a ve rtical surface in d istance of 100mm. The suspension of bac terial cells was immobilizedon the netting by adsorp tion. The content of bioreactor was aerated continuously by a compressor usingperforated distribution tubes. Contaminated water was pumped into the bioreactor from drill holes througha tempered vessel, into which solid phase nutrients (ammonium phosphate) were periodically added. Afterpassing through the bioreactor water returned to the infdtration drill holes. The water remained in the reactorfor 20-30 hours. During this process the content of nutrients, dissolved oxygen and the input and outlet valuesof the hydrocarbons were monitored. The temperature in the bioreactor was dependent on the weatherconditions and was kept lower than the optimum used for bacteria.Two step tech-for removing -from ground waterDecontamination for semi-pilot plants was arranged as shown in Fig. 1. Water was pumped from the well tothe reservoir (1) with a capacity of 3000 1 and further to the top of the stripping column (2) wherechloroethylenes were removed. Water was stored in the tank (3) . Sorp tion of PCBs on bentonite activatedby ferric sulphate took place in the sorption tank (4). The sorbent sedimented in the tw o sedim entation basins(5) . The retention time of water during sedimentation was 130-170 min. After several sorption cycles, thesufficient amount of the slurry with entrapped PCBs in the parallel couple of basins (5) was used in a furtherstep as a bioreactor with following parameters: volume: 1700 1, content of mud with so rbed PC Bs: 5 %, usedbacteria: co-cu lture of indigenous strains, inoculum: 2% ,nutrients: 0.7% of comm ercial fertilizer, aeration:periodical, time of decontamination:40 days.

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2362

7.4

J. KAs e t a / .

activatedcarbon 4-filter

'4 ContaminatedWUtBtreservoir

with active

r-filter4-

4.

5.b

+,orptionSettler-

bloreactor

settler-ioreactorBoth branchesoperate alter-natively assemers orSettler bioreactors

bloreactorI 6 . a

settler-ig, 1 Schem e of Semi-pilot Unit for Clean-up Process of Ground Water

The method of Rheinwald et al. (ref 14) was followed. lo3 o lo4cells from an overnight culture ofPseudomonas C12B w ere inoculated into 2 m l of nutrient broth containing mitomycin C at concen-trationsfrom 5 to 25 p g d ' and shaken at 28C until growth occured (48 hours). Aliquot of the cell suspension

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Biodegradation of alkanes and PCBs 2363with sublethal concentration of mitomycin C (5 pg.mY') was 5x diluted and 0.1 ml portions were spread onnutrient agar plates. Colonies were then tested for growth on n-decanoic acid, n-decane and laurylsulfate.

We used the m ethod of Wheatcroft et al. (ref 15) which is a modification of that of Eckha rdt (ref 16). Themethod is based on the cell lysis directly in the agarose gel prior to electrophoresis. To suit particularproperties of Pseudomonas C12B, we had to further modify the method as follows: because the cells ofPseudomonas C12B lysed readily when exposed to cold, the entire procedure was performed at roomtemperatu re. To furthe r slow down cell lysis, the concentration of n-lauroylsarcosine solution used forwashing the cells had to be decreased to 0.06% (w h) . The electrophoresis (distance of electrodes 150mm)was run at 100 V backwards for 15minutes and 20 V forw ards for 1 hour followed by 100 V for an other hour.

Resistance to m ercury was determined on nutrient agar plates with 100 k g HgCl, .d l.o test resistanceagainst various antibiotics, the antibio tic disks supplied by Lachema-Chem apol (Ne ratovice, Czech Republic)were placed o n freshly inoculated Mueller Hinton Agar plates (Diagnostics Pasteur, France). Grow th wasexamined after 1day incuba tion at 28C.RESULTS

AlkanesOur work was focused on resolving two mainproblems; (a) - environmental-technologicalapplicationand (b) - physiological and genetic characteristics of microorganisms involved.a) environmental-technological application .The principle of the method w as the use of microorganisms coming directly from the contamina ted locality,after their selection in the laboratory and their production to the necessary quantity. From the samples of soilwhich were taken from several localities, around 300 strains were isolated after cultivation on the mineralmedium with kerosene as the only carbon source (ref 16). Their ability to degrade oil pollution was tested .The isolated strains were identifed and species selected were not pathogenic or conditionally pathogenicmicroo rganisms. For the locality VysokB M p o , a coc ulture consisting of three strains was suggested:Acinetobacter DBM 155, Acinetobacter DBM 163 and Micrococcus DB M 153.On the basis of de tailed monitoring of the con tents of the oil substances in the so il and ground water in thislocality, the following biodegradation technologies w ere used; (i) - surface decontamination of the soil in situ,

... tiJ ' g0v )

f ' * 9Time a7 0IFig. 2 Decontamination Course in Ground Water

0 1997 IUPAC, Pure and Appl ied Chemist r y69,2357-2369

(ii) - decontamination of the soil atthe decontamination area and (iii)- sanitary pumping ofcontaminated water and the use ofthe bioreactor.In all cases the coculture of theabove named microorganims wasused for biodegradation of oilsubstances ( details see KaStfineket al. 1992) (ref 16).The decontamination course ofground water in bioreactor isshown on Fig.2. It can be seenthat water was cleansed practicallyto the purity prescribed by

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2364 J. KAS e t a / .

Pseudomonas C12B OC T plasmid alk. system

.n-alkanes C9 - C12' no rubredoxin detected.plasmid encoded.NOT cP 2 group.very specific inductors

* n-alkanes C6 - C10.contains rubredoxin.plasmid encoded. ncP2 incomp. group. ess specific inductors

Czechoslovak standard for drinking water, i.e. less than 0.05 m g l ' oil. For decontamination of soil specialdecontamintion area w as used.

suggest that there is not too much diversityin alkane oxidation system among thegram negative bacteria, we have foundimportant new features (see Table 4) aftercloser examination in Pseudomonas C12Bof a similar system. The system is plasmidencoded (the only one plasmid known tocode the alkane biodegradation was theOCT plasmid, belonging to the IncP2incompatibility group and encodingresistance to mercury and the ability to

cfu =colony forming units (vital count) per m culture

Using radiotracer methods, we determined the pathway for n-decane utilisation by this strain. Theseexperiments are not completed yet, but all results so far obtained suggest that n-decane is oxidised viacorresponding alcohol and aldehyde to n-decanoic acid, which is further metabolised by either a- or p-oxidation.

PCBs level in the cultivation

Microbial degradation of PCBs was developed on the laboratory scale for aerobic bac teria. In the beginning,we were focused on the study of physiological characteristics of the involved microorganisms.TABLE 5 : Influence of Delor 103Concentration on Viability of Strain A number Of bacteria found afterNo.2 the biodegradation experiments,using the vital count technique,

was in agreement with PCBsreduction (data not shown). Inenvironmental conditions(polluted soil and water)Delor 103 cfu after 24hrs cfu after 1week(pV50ml medium)

0440400

4 . 0 ~ 1 0 ~ 9 . 8 ~ 1 0 ~1.2x10' 2.8~10~8 . 5 ~O8 7 . 2 ~ 1 0 ~9.2~10' 2.0~107

enormous differences exist inPCBs con centration because thePCB congeners are waterinsoluble. The effect of increased

The com parison of growth abilities of the pure culture (strain No. 2) and coculture 319 is presented in Fig.3. The growth kinetics of these two microbial populations were different. The growth curve of isolate No.

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Biodegradation of alkanes and PCBs 2365

2 was steeper and followed classical growth kinetic patterns belonging to the coculture.

, strains involved in this study has been100 planned.E 80 The decomposition of PCBs on thee6 602 40 The concen tration of PCBs in the0* 20

semi-pilot scale was developed for thecleaning of ground water in the area ofmE5 a factory in the E lbe (Labe) river basin.g!- water reached thousand s of ng.T', bothtypes of Delor (Delor 103 and Delor106) and also som e chloroethyleneswere present. The substantial part ofI the PCB s was adsorbed on particles of1 2 3 4 5 6 7 8 9 1011121314151617181920211 1 3 1 9 0 2 1 peak No.

'0 ' 2 ' 4' 6 8 ' 1 0 ' 1 2 ' 1 4 ' 1 8 ' 1 ~ ' 2 0 ' 2 2 ' 2 4 ' 2 6 ' 2time (hours)Fig. 3 Grow th Curves of Coculture 319 and Isolate No.:

on Biphenyl.Squares =coculture 319, double triangles=isolateNo.2

In the first sedimentation basin (see Fig.1) suspension contained 5% of solids and in the second sedimentationbasin suspension contained 3% of solids. The deposit was composed of natural sed iment, bentonite, ferric andcalcium sulphates and flocculant. The deposit in the second sedimentation basin contained a larger amountof very fine particles. The different composition of deposits also correlated with differences in the ratio ofhigher and lower chlorinated PCBs congeners. While in the first sedimentation basin the ratio of D103:D106in the slurry was 7 and in the solids 17 , the same ratio in the second sedimentation basin was 10 in both theslurry and solids.

Finally Fig. 4. represents thebiodegradation patte rn of cocu lture 3 19and strain No. 2, which was derivativedfrom the parent coculture. Data fromseveral experiments were collected andthe average values are shown on thisgraph.Using GC analysis, we measured thedecrease of the original content of Delor103 individual cong eners. Results on Fig.4 are expressed in percents of remainingcongeners after the biodegradation. Theindustrial PCBs mixtures were verycomplex and the estimation of theindividual congeners concentrationspresented a difficult analytical task.Therefore, EPA has recommended

Both basins were periodically aerated. The air was injected from inlets located at the bottom and in regularintervals parallel branches (F ig.l,5a,5b) were supplied. The initial pH was 7 .4 in the first sedimentation basinand it was 5 .3 after biodegradation. In the second sedimentation basin the pH changed from pH 7.6 to 7.4 .

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2366 J. KAS e t a / .

First sediment basin Second sediment basinD 103 n gn D I06 ng/l D 103 ng/l D I06 ng/l

26 36911860

3237 341 5 5636 563 601 2 073

The biodegradation cou rse in the sedimentation basins suspension is shown in Table 6. After 5 weeks ofincubation, when PCB concentration did not exhibit further decrease, in the first sedimentation basin @a), seeFig.1 the level of D 103 was found to be 6 500 ng.T'. It represented 25% of the initial concentration. In thesecond basin (5b) after the sam e elapsed time, the D 103 concentration decreased to 2 OOO ng.l'l, which wasalso 25% of the original content. The concentration of D 106 decreased from 3 700 to 600 ng.1-l in the firstbasin. In the second basin the content of D 106 changed from 900 to 300 ng.l". Content of D 106decreasedin the first sedimentation basin to 15% of the initial value and in the second basin it decreased to 30% of tha t.

decontamination basins wasfollowed separately. In the firsttank the content of D 103decreased from 8 OOO to 2 000ng.T', which was 25% of thevalue at the beginning ofbiodegradation. In the solidsfrom the second basin theconcentration of D 103

O K K l N I K 6 2 Kl R l B C m S N C 9 0

For comparison, in the slurry treated under laboratory conditions (in shake flasks, temperatu re 28C) the D103 concentration decreased to 6% of its initial value which was 8040 ng.T' in the solids , and the con tent ofD 106 decreased from 479 ng.1.' to 30% of this value after 22 days of biodegradation. Under laboratoryconditions, lower-chlorinated congeners were subjected to more substantial degradation than higher-chlorinated congene rs. In the first sedimentation basin, higher-chlorinated congeners were degraded in asimilarmanner as lower-chlorinatedcongeners. The different results can be attributed to the difference at the

peak N o .

biodegradation time, the temperature and aeration conditions in the laboratory andsynergistic effect of natural microorganisms in the basins was also possible.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 IS 16 17 18 1 9 2 0 21 22

Fig. 5 Efficiency of Delor 103 Degradation by Some Plant Cell Cultures

in the basins. The

To establish a reliable plant model system, in the first step the analytical approach was optimized for the useof plant material (ref 18) by changing, inoculum age and size and also biodegradation time period.Furthermore cultures of more species with various levels of differentiation were compared.

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Biodegradation of alkanes and PCBs 2367The overall PCB metabolising capability and also degradation of individual congeners greatly differed fromstrain to strain. The results obtained for some of the tested cultures are summarized in Tab le 7. Figure 5shows an example of the typical degradation course of individual congeners by some plant cultures. Thehighes t ability to metabolize PCB revealed some Agrobacterium transformed cultures of S. nigrum and S .aviculare. These cultu res degraded PCBs under the given conditions up to 40 50%of the starting contentof PCBs (lOmg/lOOml).From the results obtained until now we can conclude that in contrast to microorganisms (induction bybiphenyl) plant cells are able to biodegrade chlorinated compounds without previous induction and in thepresence of other carbon source (sucrose). Among the tested cultures transformed cells were more effectivethan non-transformed ones, and differentiated cultures were more effective than amorphous non-differentiatedmes. Cultures of Solanun nigrum degraded PCBs with higher efficiency than othe r species.

TABLE 7: Ability of Various Plant Cell Cultures to Degrade PCBsCulture Morpholog y PCB biodegradation

Solanum aviculareK K l NAVI-3AVR-1 (T)*Armoracia rusticanaK62KK5 0K5 4Solanum nigrumSN C 90 (T)SNT-2 (T)SNC-9L (T)Atropa bella-donnaR lB CAngelica sylvestriCucurbita meloMel-2

ATR- 1(T)ANG-X

callus, nondifferentcallus, nondifferentdifferentiatedcallus, nondifferentcallus, nondifferentdifferentiated

hairy rootdifferentiatedcallus. nondifferentcallus, nondifferentcallus, nondifferentcallus, nondifferentcallus. nondifferent.

20 %0%56 %35 %35 %44%

40 %53 %20%20%42%

0%10%* (T)=cultures transformed by Anrobacterium

CONCLUSIONSrhe feasibility of bioremediation for the cleanup of contaminated sites starts with an assessment of thedegradation potential of the contaminants. This assessment includes evaluations of the ease or difticulty ofdegradation, the ability to achieve total mineralization, as well as the environmental conditions necessary formineralization. Regulatory officials and citizens demand information on the po tential degradation produc tsand their potential hazard.

Microbial transformations of organic compounds are frequently described using the terms detoxification,degradation, and mineralization. Detoxification is the transformation of the compound to some intermediateform that is less toxic. Degradation means that the parent compound no longer exists. Mineralization refersto the com plete conversion of the organic structure to inorganic forms e.g. H,O, O, and Cl-.When b ioremediating aliphatic hydrocarbons, the following should be considered. Firstly, tThe oxygen isrequired because biodegradativepathways are aerobic processes. Secondly, many microorganismsare capable

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2368 J . KAS e t a / .of aliphatic hydrocarbons degradation. Thirdly, soil normally contains an adequate inoculum of naturalorganisms for bioremediation, nevertheless the application of indigenous strains consortia as ASANOLstimulate and speed up the clean-up processes. Short hydrocarbons that have less than nine carbon atoms aretoxic for microorganisms and therefore bacterial biodegradation processes are not generally applied for theirbioremediation. How ever, bacterial strains evolved that are capable of dissimilating even these toxiccompounds (refs 19 and 20).There is more than one type of plasmid encoded enzyme system that gram negative bacteria use fordissimilation of short alkanes. We found a biodegradative plasmid that enables the strain Pseudomonas C12Bto gro w on n-alkanes from n-nonane through n-tridecane. Alkenes are oxidized by this system, too. T hismicroorganism uses the common biodegradative pathway that includes the oxidation of the primary carbonatom to form the corresponding primary alcohol, aldehyde and carboxylic acid.Both aerobic and anaerobic metabolism modes affect some biotransformation of PCBs. However, PCBschlorinated with four and more chlorine atoms per molecule are rather resistant under aerobic condition s. Fordegradation of multiple chlorinated PCBs the microbial consortia of selected organisms provide the optimalresults. The catabolic pathway of the entire PCB mineralization is encoded by two different sets of genes(chlorobiphenyl and chlorobenzoate degradation) that are normally not found in the same organism.Preparation of hybrid strains bearing the genes of both pathways represents the futu re of PCBs biodegradationtechnology.In addition, these days there is no approved technology in the Czech Republic which would efficiently removePCBs from the environment. The clean-up of PCB contaminated sites generally presents quite a difficultproblem and technologies used are costly and time consuming. Thus, bioremediation through the use ofspecific biodegrad ing bacteria seems to be an effective approach tow ards cleaning of such sites.Phytoremediation s defined as the use of green plants to remove, accumulate or render harm less environmentalcontaminants. Plants are the primary source of energy for all other above ground and for all below groundorganisms. They have evolved in an environment where they are continually assaulted with a wide array ofmicrobial, plant and, more recently, man-made toxins. In addition they have significant metabo lic activitiesin their roots and the shoo ts that may be exploited for phy toremediation. Phytorem ediation technology is arelatively new concept and although it is still in development it has been already shown to be an emergingtechnology that promises effective and inexpensive cleanup of certain hazardous waste sites. Also the role ofvegetation in the process of enriching in situ microbial populations for enhanced degradation of organics bythe provision of appropriate beneficial primary substra tes (plant exudates - sugars, amino acids, vitamins etc.)supplied by vegetation must also be taken into account.

ACKNOWLEDGEMENT:The work was sponsored by a grant PECO-CIPA-CT -3020 and the grants from the Grant Agency of theCzech Republic No. 104/94/1315 and No. 204/96/0499

REFERENCES1.2.3.

Ratledge, C. (1984).Mic robial conversion ofalkanes and fatty acids. JAOCS 61,447-453Providenti, M.A., Lee,H. &Trevors JC(1993) elected factors limiting the microbial degradation of recalcitran t compounds.J.lndustria1 Microbiol. 12,379-395Witholt, B.,de Smet, M.J.,Kingma, J.,van Beilen, J.B.,Kok, M., Lageveen, R.G. & Eggink, G. (1990).Bioconversion ofaliph atic compou nds by P seudomonas putida in multiphase bioreactors: Background and eco nomic potential. TrendsBiotechnol.8,46-52KoSt'6l J., Mackovfi M., Pazlarovfi J. &Demnerovfi K (1995). lkane assimilation ability of Pseudom onas C12B originallyisolated for degradation of alkvlsulfate surfactants. Biorechnol. Lett ers 17,765-7704.

5. Musil, P., J.,& ., DeA erovfi,K &Marek M (1990) SANOL-Biodegradative bacterial conso rtium. Czec h PatentPV 5161-906.7.

Furukawa, K., Tonomura, K. & Kamibayashi, A. (1978).Effect of chlorine substitution on the biodegradability ofpolychlorinated biphenyls. Appl. En viron. Micro biol. 35,223-227Bedard, D.L., nterman, R., Bo pp, L.H., Brennan, M.J., Haberl, M.L.& Johnson C. (1986).Rapid assay for screening andcharacterizingmicro organ isms or the ability to degrade polychlorinated biphenyls. Appl. E nviron . Microb iol. 51,761-768

0 1997 IUPAC, Pure and Applied Chemistry69,2357-2369

7/30/2019 6911x2357

13/13

Biodegradation of alkanes and PCBs 23698.9 .

10.1 1 .12 .13 .

14 .15 .16 .16 .17 .18 .

19 .20 .

Bokvajov& A., Burkhard, J., Demnerovh, K. &Pazlarovh, J.(1994). Screening and separation of microorganisms degradingPCBs. Environ. Healf hPerspecf.102,552-554KaStAnek F. , Kuncovh G., Demnerovh K., Pazlarovh J., Burkhard J. &MaMterovh Y. (1995) Laboratory and pilot-scalesorption and biodegradation of polychlorinated biphenyls ffom ground w ater. Infernat. Biodeferioration Biodegradaf ion 35 ,287-300Cunningham S.D., B erti W.R. and Huang J.W. (1995). Phytoremediation of contaminated soils. TZBTECH 13,393-397Groeger A.G. and Fletcher J.S. 1988). The influence of increasing chlorine content on the accumulation and metabolism ofpolychlorinated biphenyls by Pauls Scarlet Rose cells. Plant Cell Repor fs7 ,329-332Linsmeier E.M. &SkoogF (1965) Organic growth factor requirement of tobacco tissue cultures. Physiol. Plant. 18 , 100- 127Ma& M. (1989).Poroporo,Solanurn aviculare, Solanum laciniafum : in v ifr ocultivation and production of solasodine. In:Biotechnology - Agriculture and Forestry, Y.P.S. Bajaj (ed), Springer Verlag Berlin, Heidelberg, New York, P aris, Tokyo,Rheinw ald J. G., Chakrabarty A. M. and G unsalus I. C. (1973) A transmissible plasmid controlling camph or oxidation inPseudomonasputida. Proc. Naf .Acad. Sci. US A 70,885-9Wheatcroft R., McRa e D. G. and Miller R. W. (1990) Changes in the Rhizobium melilofi genome and the ability to detectsupercoiled plasmids during bacterioid development. molecular Plant - Microbe Interactions 3,9-17Eckhardt, T. (1978) A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid 1,584-588KaStAnek,F.,Demnerovh, K, HetflejS, J . , Kuberovh, I., KaSthnek, P., Bokvajovh , A. & Burkhard, J. (1993). Removing ofpolychlorinated comp ounds mainly PCBs from solid carriers. Czech Patent PV 1263-993Beilen J. B., Wubbolts M. G. and Witholt B. (1994) Genetics of alkane oxidation by Pseudomonas oleovorans.Biodegradation 5 , 161-174MacekT.,Mackovh M. OcenfiSkovh J., Demnerovh K., Pazlarovh J., K0en V. (1996) Peroxidase isoenzyme pattern and totalperoxidase activity changes in plant cells cultivated in vitro under abiotic stress. In: Plant Peroxid ases, Biochemistry andPhysiology. Obing er C., Burner U., Eb ennann R., PenelMusilP., Marek, M ., Demnerovh, K. &KaStAnek,F. (1992). Biodegradation of petroleum hydrocarbons on a location afterthe departure of the Soviet army. In: Soil Decontamination Using Biological Processes, Proc. InfSymposiumpp.231-250MusilP., Marek M., Demnerovh, K, Sajdok J.,&Wc J. &hk,. (1992). Degradation of kerosen e and other hydrocarbons.In Soil Decontamination Using Biological Processes Proc. Inf. Symposium, pp.726-732

443-467

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