arsenic contaminated soils

7/28/2019 Arsenic Contaminated Soils

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Research Art icles Aresenic-Co ntaminated Soils

Arsenic-Contaminated SoilsGenetically Mod ified Pseudomonas spp. and Their Arsenic-Phytoremediation PotentialOlg a I. Sizova 1, Vlad imir V. Koch etko v 1, Shamil Z. Validov 1, Alexa nder M. Bor onin 1, Paul V. Kosterin 2. an dYelena V. Ly ubu n 21 nstitute o f Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 5 Pros pekt Na uki,Pushchino 142290, Moscow Oblast, Russia2Institute of Biochemis try and Physiolo gy of Plants and Mic roorga nism s, Russian A cadem y of Sciences, 13 Prospe kt Entuziastov,Saratov 410015, Russia* Corresponding author: Dr. Paul V. Kosterin ([email protected])

IntroductionThe Conv ention on the P rohibit ion of the Development, Pro-duction, Stockpiling and Use of Chemical Weapons and onTheir Destruction (Paris 1993) stipulates that the vesicantchemical-warfare agents mustard and Lewisite be destroyedfirst of all. Lewisite [dichloro(2-chlo rovinyl)arsine; CI-CH =CH -AsCI:] is an arsenic (As)-conta ining agent. Its industrial detoxi-fication gives rise to sodium arsenite, an alkaline-hydrolysisproduct of Lewisite. Thus, alongside the mining and metal-

lurgical industr ies and the wide, uncontrolled, heavy appli-cation of As-containing pesticides up to the mid-20th cen-tury, the industrial destruction of Lewisite may soon b ecomea major potential source of As on the planet.Soils often contain high concentrations of various naturaland man-made compo unds of As. As is regarded to be mod-erately phytotoxic because, l ike Se, Cd, Zn, Mn, and Crions, As ions are inhibitory at solution concentrations of 1to 100 mg/L. (In contras t , the highly phyto toxic Hg, Be, Sn,Ag, and Pb adversely affect test organisms when present atsolution concentrations of up to 1 mg/L.) [1] . Arsenites[As(III)] and arsenates [As(V)] are most important for theinteraction with soil biota. Arsenites are powerful inhibi-tors of sulfhydryl groups. Th ey inactivate microbial enzymesand attack plant-cell membranes, thus suppressing root func-tion on contact with roots or causing a rapid necrosis oncontac t wi th leaves . Arsenates do no t damage membranes ,because they do not react with su lfhydryl groups; however,a rsenates do af fec t phosph ory la t ion in m i tochondr ia [2] .Mo st As present in soil is sorbed by soil colloids, mainly bythe clay compon ents Fe oxide and A1 oxide. Sorp tion of Asby clay depends on the type and pH of soils, the presenceand state of Fe and AI, the am ou nt and type of clays in soils,and the p resence of o rgan ic compounds. The amount o fadso rbed As increases with an increase in clay content. Theam ount o f so luble As takes about four m onths ( f rom one tosix) to decrease to a cons tant value, and the rate of decreaseis different for different soils. Init ially , the a dsorp tion of ar-senicals proceeds fast; then, a long-term transformation ofAs into low-soluble form s begins [1] .Sorption of As reduces the toxicity of the element. Data avail-able indicate that arsenicals are more toxic in st iff , sandysoils than they are in fine-structured, clay soils. This fact isunderstandable when it is considered that clay soils have ahigh sorption capacity toward As [2J.Bacteria take an active part in global As-cycling in the bio-sphere and detox ify toxic agents in soil by either degradingthem or t ransformin g them in to less tox ic compou nds. Mi-croorganisms resistant to As(III) /As(V) occur in varioustaxonomic groups. The mechanisms of As(III) /As(V) resis-

JSS-JSo ils&Sed iment s2(1) 19 23(2002){('~ecamedpublishers D-86899Landsberg. Germanv and F{ Worth/TX USAoTokvo Japan~ lndien-Seoul, Korea

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t a n c e , d e t e r m i n e d b y p l a s m i d a s w e ll a s c h r o m o s o m a l g e n e s,h a v e b e e n d e s c r i b e d f o r E s c h e r i c h i a c o l i , A l c a l i g e n e sfaecalis , S taphylococcus xylo sus , S . aureus , Ac idiph i l iumm u l t i v o r u m , D e s u l fo v i b r io sp . , D e s u l f o m i c r o b i u m s p . a n dP s e u d o m o n a s [ 3 - 8 ] ;P h y t o r e m e d i a t i o n i s a p r o m i s i n g e n v i r o n m e n t a l t e c h n o l o g yfo r so il r es t o ra t i on [9,10 ]. So rgh um an d sun f l ow er can be goodc a n d i d a t e s t o c l e a n u p a r s e n i c - c o n t a m i n a t e d s o i l s [ 1 1 - 1 3 ] .P l an t s c an be i nvo l ved i n so i l r ehab i l i t a t i on i nd i r ec tl y , v i a thes u p p o r t o f s y m b i o t i c a n d p l a n t - r o o t - a s s o c i a t e d b a c t e r ia . T h erh i zosphere o f p l an t s i s r i ch i n roo t exudat es , se rv i ng as car -b o n a n d n i t r o g e n s o u r c e s f o r r h i z o s p h e r e m i c r o o r g a n i s m s .F l u o r e s c e n t p s e u d o m o n a d s s u c h a s P . putida, P. f luorescens,P. aureofaciens, P. chlororaph is, a n d P . corrugata are t he p re-d o m i n a n t g r a m - n e g a t i v e b a c t e ri a i n t h e r h iz o s p h e r e . C e r t a i ns t r a i n s o f t h e s e p s e u d o m o n a d s c o n t r i b u t e m u c h t o t h e i m -p r o v e m e n t o f p l a n t g r o w t h a n d d e v e l o p m e n t [ 14 ].T h e a i m s o f th i s w o r k w e r e :* T o o b t a i n g e n e t i c a l ly m o d i f i e d P s e u d o m o n a s s t r a i n s r e -s i s t an t t o As( I I I ) / As(V) .| T o a s se s s t h e e f f e c t s c a u s e d b y th e s t ra i n s o n t h e g r o w t h

o f s o r g h u m o n s o d i u m - a r s e n i t e -c o n t a i n i n g s o i ls .1 Exper imentalB a c t e r i a l s t ra ins and growth condi t ions . T h e P s e u d o m o n a ss t r a i n s u s e d w e r e t h e r h i z o s p h e r e s t r a i n s P. lquorescens 3 8 a ,P. aureofaciens B S 1 3 9 3 ( s t r a i n n o . V - 2 1 8 8 D , A l l - R u s s i aC o l l e c t io n o f M i c r o o r g a n i s m s ) , a n d P . putida 5 3 a ( s t r a i nn o . V - 1 7 4 3 D , A l l - R u s s ia C o l l e c t i o n o f M i c r o o r g a n i s m s )[15 ,16 ] . E. col i K 8 0 2 , h a r b o r i n g t h e c o n j u g a t i v e A s ( I I I ) /A s ( V ) - r e s i s t a n c e p l a s m i d p B S 3 0 3 1 [ 1 7 ] , w a s u s e d a s t h ed o n o r t o o b t a i n p l a s m i d - c o n t a i n i n g d e r i v a t iv e s o f t h er h i z o b a c t e r i a . P . f luorescens 3 8 a , P . put ida 5 3 a , a n d P .aureofaciens B S 1 3 9 3 w e r e c h o s e n b e c a u s e t h e y a r e h i g h l ya n t a g o n i s t i c t o a w i d e r a n g e o f p h y t o p a t h o g e n i c f u n gi a n db a c t e r i a , a n d t h e y c a n s t i m u l a t e p l a n t g r o w t h .B a c t e r i a w e r e g r o w n i n L u r i a - B e r to n i ( L B ) b r o t h , o n L B a g a r ,o r i n M 9 s y n t h e t i c m e d i u m [ 1 8 ] , w i t h g l y c e r o l a s a c a r b o na n d e n e r g y s o u r c e . G r o w t h t o o k p l a c e a t 3 0 ~ e i t h e r i n p e t r id i s h e s o r i n E r l e n m e y e r f la s k s c o n t a i n i n g 1 0 0 m L o f li q u i dm e d i u m a n d s h a k e n a t 1 5 0 r p m .Conjuga l t rans fer o f the As-res i s tance p l a s m i d p B S 3 0 3 1 .T w e l v e - h o u r d o n o r a n d r e c ip i e n t c u lt u re s ( 1: 2) w e r e s p r e a do n t o t h e s u r fa c e o f a g a r - s u p p l e m e n t e d L B m e d i u m b y u s in ga s t e r i l e g l a s s s p a t t l e . A f t e r b e i n g i n c u b a t e d f o r 1 2 h , t h ec e ll s w e r e w a s h e d o f f w i t h 0 . 8 5 % N a C l a n d w e r e a p p r o p r i -a t e l y d i l u t e d . T h e m a t i n g - m i x t u r e d i l u t i o n s w e r e p l a t e d o ns e l e c t iv e m e d i a c o n t a i n i n g 5 0 0 l a g/ m L A s . T h e f r e q u e n c y o ft h e c o n j u g a l p l a s m i d - t r a n s f e r w a s c a l c u l a t e d a s a r a t i o o ft r a n s c o n j u g a n t n u m b e r t o d o n o r - c e l l n u m b e r .T ran sform at ion o f the rec ip ient s t rains w i t h p la s m i d D N A .P l a s m i d D N A w a s i s o l a t e d a s d e s c r i b e d i n [ 1 8 ] . F o r t r a n s -f o r m a t i o n , a 1 00 -1 aL p o r t i o n o f o v e r n i g h t c u l tu r e w a s g r o w ni n 2 m L o f L B m e d i u m f o r 2 h. T h e c u l t u r e w a s t h e n t r a n s -f e r r e d a s e p t i c a l l y i n t o t e s t t u b e s a n d w a s s e d i m e n t e d b y c e n -t r i f u g a t i o n ( 3 0 0 0 r p m fo r 1 5 r a in ) a t 0 ~ T h e s u p e r n a t a n t

l iq u id w a s d e c a n t e d , a n d 5 m L o f 0 . 1 M M g C I 2 w a s a d d e dt o t h e s e d im e n t . T h e s e d i m e n t w a s r e s u s p e n d e d a n d c e n t ri -f u g e d a t 3 0 0 0 r p m f o r 1 0 m i n . T h e s u p e r n a t a n t w a s d e -c a n t e d , a n d 5 m L o f 0 .1 5 M M g C I 2 w a s a d d e d t o t h e s e d i-m e n t . T h e s e d i m e n t w a s r e s u s p e n d e d a n d k e p t i n ic e f o r 25m i n . N e x t , t h e s e d i m e n t w a s c e n t r i f u g e d f o r 1 5 m i n , t h es u p e r n a t a n t l iq u i d w a s d e c a n t e d , a n d 1 m L o f 0 . 1 5 M M g C 12w a s a d d e d . T h e s e d i m e n t w a s r e s u s p e n d e d a n d k e p t i n i c eu n t il u s e d . F o r t r a n s f o r m a t i o n , 2 0 1JL o f p l a s m i d D N A w a sa d d e d t o 2 0 0 la L o f c o m p e t e n t c e ll s . T h e m i x t u r e w a s l e f t t os t a n d i n i c e f o r 1 h a n d w a s t h e n h e a t e d f o r 2 m i n a t 4 2 ~T h e n , 2 m L o f w a r m L B m e d i u m w a s a d d e d a n d t he m i x -t u r e w a s l e f t t o s t a n d f o r 5 m i n . T h e c e ll s w e r e f u r t h e r c u l -t u r e d f o r 2 h a n d w e r e p l a t e d o n s e l e c t iv e m e d i u m c o n t a i n -i ng 500 l ag /mL As .Screening f o r p l a s m i d s . C u l t u r e s w e r e t e s t e d f o r t h e p r e s -e n c e o f p l a s m i d D N A b y t he c o n v e n t i o n a l m e t h o d o fE c k h a r d t [ 19 ], b a s e d o n t h e i d e n ti f ic a t i o n o f p l a s m i d D N Ab y a g a r o s e g e l e le c t r o p h o re s i s w i t h e t h i d i u m b r o m i d e s t a in -i n g . T h e e l e c t r o p h o r e t i c c o n d i t i o n s w e r e 3 0 V f o r 3 0 m i na n d 1 0 0 V f o r 4 h. T h e 0 . 8 % a g a r o s e g e l s w e r e s t a i n e d w i t he t h i d i u m b r o m i d e f o r 1 5 r a i n a n d w e r e p h o t o g r a p h e d u n -d e r u l t r a v i o l e t i ll u m i n a t i o n b y u s i n g a r e d f il te r . T h e s t a b i l -i t y o f p l a s m i d p B S 3 0 3 1 w a s t e s t e d a s r e p o r t e d i n [ 2 0 ].D e t e r m i n a t i o n o f t h e m i n i m u m i n h i b i t o r y c o n c e n t r a t i o n s .T h e m i n i m u m i n h i b i t o ry c o n c e n t r a t i o n s ( M I C s ) ( b a c t e r ia l -r e s i s t a n c e le v e ls ) o f s o d i u m a r s e n i t e a n d s o d i u m a r s e n a t ew e r e d e t e r m i n e d a s d e s c r i b e d b y C a r l i n e t a l . [ 4 ] .P l a n t i n o c u l a t i o n , s o i l p r e p a r a t i o n , a n d c e ll e n u m e r a t i o n .S o r g h u m (Sorghum saccharatum c v . P i s h c h e v o y e - 6 9 , T h eS o r g h u m R e s e a r c h I n s t it u t e , S a r a t o v , R u s s i a ) s e e d s w e r e in -o c u l a t e d w i t h s u s p e n s i o n s o f th e P s e u d o m o n a s s t r a i n s ( d e n -s it y, 1 08 c e l ls / m L ) , g e r m i n a t e d f o r 2 4 h a t 2 4 ~ a n d s o w ni n t o p r e p a r e d s o il . P l a n ts w e r e g r o w n i n a g r o w t h c h a m b e rw i t h a 1 2 - h l i g h t p e r i o d a t 2 5 ~ a n d a 1 2 - h d a r k p e r i o d a t2 0 ~ T h e r e w e r e t h r e e e x p e r i m e n t s , w i t h fi v e r e p l i c a t e s p e re x p e r i m e n t . U n i n o c u l a t e d p l a n t s s e r v e d a s c o n t r o l s .F o r s o i l p r e p a r a t i o n , g r a y f o r e s t so i l w a s p a s s e d t h r o u g h as ieVe (1 ram ) and pac ked i n po t s (50 g o f so i l per po t ) . Tos t u d y A s t o x ic i t y , w e u s e d a n a q u e o u s s o l u t i o n o f s o d i u ma r s e n i t e , a p p l i e d t o s o il to f in a l A s - c o n t e n t s o f 7 5 a n d 1 0 0m g / k g o f so i l. S o il w a s m o i s t e n e d w i t h t h e s o l u t i o n , a n d t h ep r e p a r e d s o r g h u m s e e d s w e r e s o w n i n p o t s , o n e p e r p o t .S u b s e q u e n tl y , th e p l a n t s w e r e w a t e r e d w i t h t a p w a t e r a t r e g u -l a r i n t e rva l s .F o r e n u m e r a t i o n o f b a c t e r i a l c e l l s o n t h e r h i z o p l a n e , t h er o o t s o f t h e p l a n ts w e r e e x t e n s iv e l y w a s h e d i n t a p w a t e r,r i n s e d i n s t e r i l e t a p w a t e r , p l a c e d i n 5 m L o f s t e r i l e p h y s i -o l o g ic a l s a li n e, a n d v i g o r o u s l y s h a k e n . T h e r o o t s w e r e t h e nr e m o v e d f r o m t h e s a l i n e a n d w e r e w e i g h e d . T h e r e s u l t a n ts u s p e n s i o n w a s s u c c e s s iv e l y d i l u te d 1 : 1 0, a n d 0 . 1 m L o f e a c hd i l u t i o n w a s p l a t e d o n L B a g a r . A f t e r t h e p l a t e s w e r e i n c u -b a t e d f o r 2 4 h a t 3 0 ~ w e e s t i m a t e d th e c o l o n y - f o r m i n gu n i t s ( C F U s ) f o r e a c h s t r a i n . T h e C F U s w e r e t h e n e s t i m a t e dp e r g r a m o f r o o t . T h e c o l o n ie s w e r e r e p l i c a t e d o n t o a g a r -c o n t a i n i n g s e le c ti v e m e d i u m s u p p l e m e n t e d w i t h s o d i u m a r -s e n i te ( 5 0 0 l a u r e L ) , a n d t h e n u m b e r o f c el ls c o n t a i n i n g p l a s -m i d p B S 3 0 3 1 w a s c a l c u l a t e d .

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Research Ar t ic les Aresen ic -Con tam ina ted So ils

2 R e s u l t s a n d D i s c u s s i o nW h e n t h e b a c t e r i a w e re g ro w n i n LB b ro t h a n d M 9 g l y c -e ro l - c o n t a i n i n g m e d i u m u n d e r n o n s e l e c t i v e c o n d i t i o n s fo r144 h , the s tab i l i ty o f p lasmid pBS 3031 in P . fluorescens38a , P . putida 5 3 a , a n d P . aureofaciens B S 1 3 9 3 w a s 1 0 0 % .Ex t e n s i o n o f t h e g ro w t h t i m e t o 1 6 8 h r e s u l te d i n th e l o ss o fp B S 3 0 31 f ro m t h e p s e u d o m o n a d s . I n P. aureofaciens B S 1 3 9 3 ,p l a s m i d s w e re l o s t b y 5 % ( r ic h m e d i u m ) a n d 1 % (d e f i c ie n tme d ium ) o f the cel ls ; in P . fluorescens 3 8 a , b y 1 3 % o f t h ecells; and in P. putida 53a , by 5% of the cel ls . Af te r 264 h o fc e ll c u lt i v a t i o n , p l a s m i d s w e re l o s t b y 1 3 % ( r ic h m e d i u m )a n d 3 % (d e f ic i e n t m e d i u m ) o f t h e P. aureofaciens B S 1 3 9 3cel ls , by 33% of the P . f Iuorescens 3 8 a c e l ls , a n d b y 1 8 % o fthe P. putida 53a cel ls .As(III)/As(V)-resis tance s tudies show ed tha t E fluorescens 38aand P . putida 53a w ere As-sens it ive , wi th M ICs o f 0 .23 m Ms o d i u m a r s e n it e a n d 1 0 m M s o d i u m a r s e n a te . P . aureofaciensBS1393 p rove d to be the mos t As-res i s tan t , wi th M ICs o f 2 .5m M s o d i u m a r s e n i t e a n d 1 5 0 m M s o d i u m a r s e n a t e . To i n -crease the As res i s tance in these s tra ins , we p er fo rm ed con ju -ga l t rans fer o f p lasmid pBS3031 which was p rev ious ly foun din the cl in ical isolate P. aeruginosa BS3031 [17].P lasmid pB S3031 i s 45 k i lobases in s ize , i s con jug a t ive , andi s c a p a b l e o f f u n c t i o n a l e x p re s s i o n i n E. col i a n d Pseudomo-has c e ll s, p ro v i d i n g fo r t h e i r r e s i s ta n c e t o s o d i u m a r s e n i t e a t5 0 0 - 2 0 0 0 p g / m L . P C R a n a l y s i s f o u n d t h a t p B S 3 0 3 1 c o n -ta ined a gene , arsC, c o d i n g fo r a r s e n a t e r e d u c t a s e . Th u s ,the mec han ism o f res i s tance to As(I II )/As(V) was d e te rm inedt o i n c l u d e t h e r e d u c t i o n o f AsO4 3- to as O 2- and the ac t iverem oval o f As(II I) f rom the ce l l.We p erfo rme d con juga l t rans f er ( f requency , 10 - s) o f pBS3031f ro m t h e d o n o r s t r a i n, E . col i K 8 0 2 , t o t h e r e c i p i e n t s t r a i n sP. fluorescen s 3 8 a a n d P . aureofaciens B S 1 3 9 3 . N o p l a s m i dt r a n s f e r o c c u r r e d d u r in g m a t i n g o f s t r a in K 8 0 2 w i t h P . putida5 3 a ; t h e r e fo re , w e p ro d u c e d a p l a s m i d -c o n t a i n i n g d e r i v a -t iv e o f t h is s t r a in b y t r a n s f o rm a t i o n o f p l a s m i d D N A ( f re -quency , 10 -4 ) . Th e p resence o f pBS3031 in the t rans fo rm an tsa n d t r a n s c o n ju g a t e s w a s c o n f i r m e d b y E c k h a r d t 's m e t h o d .Th e M IC s o f s o d i u m a r s e n i t e a n d s o d i u m a r s e n a t e fo r th et r a n s co n j u g a n ts a n d t r a n s f o r m a n t s w e r e ( m M ) : 1 7 a n d 1 5 0fo r P . fluorescens 3 8 a (p B S 3 0 3 1 ) , 1 7 a n d 4 0 0 fo r P. putida5 3 a (p B S 3 0 3 1 ) , a n d 1 7 a n d 3 0 0 fo r P. aureofaciens B S 1 3 9 3(pBS30 31) , respec t ive ly .Th e o r i g i n a l a n d p l a s m i d -c o n t a i n i n g rh i z o s p h e r i c p s e u d o -m o n a d s w e re u s e d fo r i n o c u l a t in g s e e d s o f s o rg h u m , t o t e s tfo r b a c t e r i a l e f f e c ts o n p l a n t s g ro w n o n s o i l c o n t a m i n a t e dw i t h s o d i u m a r s e n it e .Th e l e a f t ip s o f u n i n o c u l a t e d c o n t ro l p l a n t s b e g a n t o y e l l o wa n d t o w i l t o n d a y 7 ; t h e p l a n ts i n o c u l a t e d w i t h t h e o r i g i n a lp s e u d o m o n a d s , o n d a y 1 4 ; a n d t h e p l a n t s i n o c u l a t e d w i t hthe p lasmid-con ta in ing pseudo mon ads , on da y 2 l . The p lan t st h a t s u rv i v e d th e n d e v e l o p e d n e c ro t i c s p o t s o n t h e i r le a v e s .A t a b o u t 3 0 d a y s a f t e r th e s t a r t o f t h e e x p e r i m e n t s , t h e p l a n t ss t o p p e d g ro w i n g . Th e n u m b e r s o f v i a b l e p l a n t s w e re e s t i -

m a t e d o n c e a w e e k . U n i n o c u l a t e d c o n t ro l p l a n t s b e g a n t od ie on day 14 (F ig . l ) . W i th 100 m g/kg o f As, the l ast p lan t sd i e d o n d a y 4 0 ; w i t h 7 5 m g / k g o f A s, o n d a y 6 0 . W h e ng ro w n w i t h 7 5 m g / k g o f A s , t h e p l a n t s i n o c u l a t e d w i t h t h erh i z o s p h e re p s e u d o m o n a d s s u rv i v e d b e t t e r . Th e p l a n t s i n -o c u l a t e d w i t h P . putida 5 3 a a n d i t s p l a s m i d -c o n t a i n i n g d e -r iva t ive g rew a nd deve loped no rm al ly (F ig . 2) . A s imi la r p ic -t u r e e m e rg e d w h e n p l a n t s w e re t r e a t e d w i t h t h e o t h e r s t r a in s( n o t s h o w n ) . T h e b e s t r e s u l t s w e r e o b t a i n e d w i t h P .aureofaciens B S 1 3 9 3 . W i t h 7 5 m g / k g o f A s , n o d i f f e r e n c e sw e re o b s e rv e d b e t w e e n t h e o r i g i n a l r h iz o s p h e r i c s t r a in s a n dt h e p l a s m i d -c o n t a i n i n g d e r i v a t iv e s .

6 84

i I421 I1i l l I I

14 21 28 35 49 56Days

B75 rng/kg As ]9 100 mg/kg As]

F ig . 1 : T ime co u r se o f th e d e a th o f co n t r o l ( b a c te r i a - u n t r e a te d ) p l a n ts

6 45

g 4L-s 3~ [3 5 3 a

9 5 3 a ( p B S 3 0 3 1 ) J

Z 1

O ,7 14 21 28 35 49 70 98

DaysF i g . 2 : T i m e c o u r s e o f t h e d e a t h o f R putida-treated p la n ts g r o wn o n so i lc o n t a i n in g 7 5 m g / k g o f A sH o w e v e r , t h e r e w e re d i f f e r e n c e s w i t h 1 0 0 m g / k g o f A s. O nd a y 3 5 , f o r e x a m p l e , t h e p l a n t s t r e a t e d w i t h t h e o r i g in a l P.fluorescens s t r a i n 3 8 a s u rv i v e d b e t t e r t h a n d i d p l a n ts t r e a t e dwi th i t s p lasm id-con ta in ing der iva t ive (F ig. 3 ) . A s imi la r p ic -t u r e w a s o b s e rv e d fo r p l a n t s t r e a t e d w i t h t h e o t h e r s t r a i n s(Figs . 4 and 5).In each exper imen t , we m easured average p lan t -he igh t s . Wi th75 moffkgof As , the g rea tes t he igh ts w ere ach ieved by the p lan t streated w ith the plasmidless P. putida 53a; wi th 100 mg/kg o fAs, by the plants t reated with the plasmidless P. aureofaciensBS 1393 (Table 1). Th e plasm id-conta ining derivat ives of theses t rains were no t so s t imula t ing to p lan t g rowth .

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Fig. 3: T ime course of the death of R fluorescens-treated plants grown onsoil containing 100 mg/kg o f AsFig. 4: T ime course of the death of R aureo fac iens- t rea ted plants grownon soil containing 100 mg/kg of As

Fig. 5: T ime course of the death of R pu t ida - t rea ted plants grown on soilcontaining 100 mg/kg of As Fig. 6: Average heights (cm) of plants treated with the P s e u d o m o n a sstrains used

Table 1 Average plant-heights (cm) measured af ter t reatment with the P s e u d o m o n a s strains (day 98)S t r a in He ig h t w i t h S t a n d a r d S t a n d a r d He ig h t w i t h S t a n d a r d S t a n d a r d75 mg/kg deviation error 100 mg/kg deviation error

of As of AsP. aureofaciens BS139 3 19.6 2.3 1.02 20.0 2.3 1.15P. aureofaciens BS 1393 (pBS3031 ) 18.0 3.08 1.37 15.4 4.87 2.18P. put ida 53a 22.2 3.11 1.39 18.4 1.84 0.87P. put ida 53a (pBS3031) 17.4 2.5 1.12 17.0 4.96 2.48P. fluorescens 38a 19.4 1.14 0.5 17.6 6.3 2.82P. fluorescens 38a (pBS30 31) 18.2 1.3 0.58 13.0 5.56 2148

Table 2: Microbial-cell numbers p er gram of root (est imated on day 98)

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The p resence o f p lasmid pBS303I in the rh izosphere s t ra inss l igh t ly inh ibi t ed p lan t g ro wth as comp ared wi th the e f fec tpro duce d by the plasmidless s t rains (Fig. 6) . The s l ight inhi-bi t ion was caused by al l the plasmid-containing derivat ives .

Analys i s o f the so rghum-rh izosphere popu la t ions o f theP s e u d o m o n a s s t rains used in this s tudy sh owed that the plas-mid-con taining derivatives were characterized by increased cell-num bers present on plant roots , in comp arison with the origi-nal, plasmidless strains (Table 2). It is l ikely that plasmidpBS3031 ensures the selective advan tage tha t the s t rains pos-sessing this plasmid have in sodium-arsen i te-containing soi l .

3 C onc l us i ons9 I t i s possible to increase the survivab i l i ty of sor ghu m

growing in sod ium-arsen i t e -con ta in ing so i l by us ingrh izosphere pseudomonads .9 The p resence o f the As( I I I ) - /As(V)- res i s tance p lasm idpBS3031 offers the s t rains a certain select ive advantagein arseni te-contaminated soi l .

9 The p resence o f pBS3031 impai rs p lan t g rowth due tothe As-resis tance me chanis m determ ined by this plasmid:the t ransform at ion o f the less toxic arsenate into the mo retox ic bu t water -so lub le a rsen i t e by arsenate reductaseand the act ive removal of arseni te from bacterial cel ls .

9 Such a mecha n i sm makes i t poss ib le to develop a micro -bial -ass isted phytor eme diat ion technology for the cleanupof As-con tam inated so il s and i s the on ly poss ib le way o fremo ving the soi l -sorbed arsenates fro m the environment .

R eferences[1] Alexeyev YuV (1987): He avy metals in soil and in plants.Agropromizdat Publishers, Leningrad [in Russian][2] Ga ma yur ova VS (1993): Arsenic in ecology and in biology.Na uka Publishers, M oscow [in Russian][3] Kau r P, Rosen BP (1992): Plasmid-encoded resistance to ar-senic and antimony. Plasmid 27, 29-40[4] Carlin A, Shi W, Dey S, Rosen BP (1995): Th e ars operon ofE. coli confers arsenical and an timo nial resistance. J Bacteriol177, 981-986[5] Phung LT, Silver S (1996): Ba cterial heavy metal resistance:New surprise. Annu Rev Microbiol 50, 753-7 89[6] Rosenstein P, Peschel A, Wieland B, Gotz F (1992): Expres-sion and regulation of the antimony, arsenite, and arsenateresistance opero n of Staphylococcus xylosus plasmid pSX267.J Bacteriol 174, 3676-36 83

[7] Ma sy JM , Santini JM , Pauling BV, O'N eill AH, Sly LI (2000):Tw o new arsenate/sulfate-reducing bacteria: Mechanisms o farsenate reduction. Arch Microbiol 173, 49-57[8] Cai J, Salmon K, DuBow MS (1998): A chrom osom al ar soperon homologue of Pseudomonas aeruginosa confers in-creased resistance to arsenic and antim ony in Escherich ia colLMicrobiology 144, 2705-2713[9] Mace k T, Mackov~i M, K ~ J (2000): Exploitation of plantsfor the removal of organics in environmental remediation.Biotechnol Adv 18, 23-34[10] Trapp S, Karlson U (2001): Aspects of phytorem ediation oforganic pollutants. JSS - J. Soils & Sediments 1, 37-43[11] Shcherbakov AA, Kochetkov VV, Za kha rov a EA, Lyubun YeV,Sherbakova LF, Tka ch VI, Brudnik W, Mandych VG, KosterinPV (2000): The extraction-phytoremediation tandem as analternative method for reclam ation of soils contam inated byarsenic compounds. In: Abstracts of the Presentation-Work-shop fo r the Scientific and Technical Inn ovat ion Project 'Bio-technology-200 0', 26-2 8 September 2000, Pushchino, Rus-

sia, pp 154-155[12] Lyubun YeV, Za kh aro va EA, Shcherbakov AA, Kosterin PV,Koche tkov VV, Sizova OI (2001): Indirect p hytoreme diationof arsenic-containing c om pou nds from soils. In: Abstracts o fthe ISEB 2001 Phytoremediation Meeting, 15- 17 Ma y 2001,Leipzig, Germany, p 93[13] Zak haro va EA, Lyubun YeV, Kosterin PV, Shcherbak ov AA,Kochetkov VV, Sizova OI (2001): Direct phytoremediationof arsenic-containing com pou nds fro m soils. In: Proceedingsof the First European Bioremediation Conference, 2-5 July2001, Chania, Greece, pp 489- 491[14] Lugtenberg B. (ed) (1986) Recognition in mic robe -pla nt sym-biotic and pathogenic interactions. NA TO A SI Series, Vol H4. Springer Verlag, Berlin[15] Mo rdukh ova EA, Kochetkov VV, Loban ova EV, Slepen'kinAV, Boronin AM (2000): Effect of sodium salicylate on thepopulation of the rhizobacterium Pseudomonas aureofaciensin the wheat rhizoplane and adjacent soil. Mikrobiologiya69, 844-849 [In Russian].[16] Kochetkov VV, Skvortsova NP, Dubeikovsky AN, BoroninAM (1993): Pseudomonas putida strain VKM V-1743D forthe production of a prepa ration used for stimulating plantgrowth and for protecting plants against fungi of the genus

Fusarium and bacteria of the genus Erwinia. Russian Federa-tion patent no 1805849. Byull Izobr 12, 138 [In Russian][171 Kozlova EV, Suzina NE, Karpov AV, Duda VI, Boronin AM(1999): Effect of arsenite o n the physiological and cytological

properties of Pseudomonas putida strains capable of degrad-ing polycyclic aromatic hydrocarbons. Mikrobiologiya 68,179-1 86 [In Russian[.[18] Sa mb roo k J, Fritsch EF, IVlaniatis T (1989): M olecu lar clon-ing: A laborator y manual, 2nd ed. Cold Spring Ha rbo r Labo-ratory, Cold Spring Harbor, NY[19] Eck hard t T (1978): A rapid me thod for the identification o fplasmid desoxyribonucleic acid. Plasmid 1,5 84 -5 88[201 K ochetkov VV, Boronin AM (1984): A compar ative studyof plasmids control l ing naphthalene biodegradat ion byPseudomonas bacteria. Mikrobiologiya 53, 639-645 [InRussian]

Received: Novem ber 13th, 2001Accepted: March 19th, 2002OnlineFirst: March 28th, 2002

JSS - J S oils & Sedim ents 2 (1) 2002 23

arsenic contaminated soils

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