ROCZNIKI GLEBOZNAW CZE, t. X X X II, NR 3, W ARSZA W A 19Ô1
JER ZY SO B IE SZ C Z A Ń SK I, A N N A RODZIEW ICZ, R EG IN A STEM PN IEW IC Z
MICROBIOLOGICAL TRANSFORMATION OF HERBICIDES IN CATABOLIC PROCESSES
PART I. THE EFFECT OF VENZAR ON THE RESPIRATION ACTIVITY OF SELECTED GENERA OF SOIL BACTERIA1
In stitu te of P reserv a tio n and Food T ech n ology, A g ricu ltu ra l U n iv e r sity of W rocław
herbicides slightly soluble in w ater (at 25° 6 ppm). To weeds it is a photosynthesis inhibitor. Soil bacteria are little susceptible to this herbicide.
In the presence of Bacillus sp. venzar m arkedly increased the respiration and decreased the photosynthesis of leaves of Sinapis alba and Spirodella polyrrhiza as compared w ith the action of venzar itself and control [8]. Our aim was to test w hether and to w hat extent venzar can influence the metabolism of bacteria resistant to high doses of this herbicide, and particu larly their respiration activity.
The tested bacterial strains were isolated from previously percolated soils : sandy-loam y soil, the W rocław black earth, sandy soil Yugoslavian black-earth treated w ith low doses of venzar (1.5 ppm). The origin of individual bacterial strains, their genus affiliation as well as the con ditions of isolation are given *n Table 1. To isolate the strains of bacteria under exam ination the following media were chosen : the K a u f m a n synthetis medium [1 2], the K e a r n e y synthetic medium [1 1], the Capek medium [7] for Pseudomonas, S a n d s and R o v i r a [15] and a soil ex tract (flood w ith 1.5 1 of tap w ater 1 kg of soil, sterilize after
/V; i
Venzar (3-cyclohexyl-5, 6-trim ethyleneuracil) belongs to
CO
M A TER IA L ’A N D M ETH ODS
1 Supported fin a cia lly by the U SA D epartm en t of A gricu lture. Res. S ervice G ran t Fg. Po — 335. P resen ted at In tern a tio n a l S y m p o siu m “T estin g of ch em ica l su b sta n ces for e x o to x ic o lo g ic a l e v a lu a tio n ,” M ay 1979 G SP , M unich— N eu h erb erg.
9 4 3. Sobieszczański at al. T a b l e 1 L i s t and o r i g i n o f b a c t e r i a l s t r a i n s t e s t e d B a c t e r i a l s t r a i n P e r c o l a t e d c o i l + v e n z a r ppm C u ltu re medium f o r i s o l a t i o n + v e n z a r ppm Symbol Gijn u s V-217 B a c i l l u s sp lo am y -san d y 0 K earney 1000 V-218 B a c i l l u s sp loam y-sandy+2^ c e l l u l o s e 0 K earney 0 V- 92 B a c i l l u s op b la c k d e g ra d e d 1.5 K earney 100 V- 25 B a c i l l u s sp lo am y -san d y 0 s o i l e x t r a c t 100 V- 76 B a c i l l u s sp lo am y -san d y f e r t i l i z e d 3 Саре к 100 V- 20 B a c i l l u s зр loam y -san d y 9 oynthe t i c , Kaufman 100 V- 16 B a c i l l u s m y co ld es lo am y -san d y f e r t i l i z e d 3 Kaufman s y n t h e t i c 100 V- 80 Pseudom onas f Хиогезсепз Y ougoolav b l a c k - e a r t h 1.5 f o r Pseudomonao 1000 V-209 Paeudonona3 f l u o r é s c e n s Y ougoolav black-earth-i-2%
c e l l u l o s e 1 .5 f o r Ploudomonas 100 V-208 Pseudom onas f l u e re n c e n a Y ougoslav b la c k -e a rth + 2 %
c e l l u l o s e 1 .5 f o r Pseudom onas 1000 V-105 Pseudom onas f l u o r é n ceno b la c k d e g ra d ed 1 .5 K earney 100 V- 65 Pseudomonas f lu o r e c c e n s loam y -san d y f e r t i l i z e d 9*0 f o r Pseudom onas 100 V-212 Pseudom onas a e r u g in o s a Y ougoslav b la c k -e arth + 2 ? £
c e l l u l o o e 0 .0 f o r Pseudom onas 1000 V-204 Pseudom onas a e r u g in o s a loam y -san d y 1 .5 f o r Poeudomona3 100 V-211 Pseudom onas a e r u g in o s a sandy 1 .5 f o r P3eudomonao 1000 V-1CO Pseudomonas a e r u g in o s a b la c k degoaded 1 .5 f o r Pseudom onas 1000 V I-5 Pseudom onas a e r u g in o s a lo am y -san d y 0 .0 f o r Pseudom onas 100 V-206 Pseudomona3 a e r u g in o s a Y ougoolav b l a c k - e a r t h 1 .5 f o r Pseudom onas 1000 V-223 Pseudomonao a e r u g in o s a lo am y-sandy 1 .5 f o r Pceudomona3 1000 V-23a Pseudam onas a e r u g in o s a loam y -san d y f e r t i l i z e d 1.5 K earney 1000 V-205 Pseudom onas a e r u g in o s a loam y -san d y 1.5 f o r Pseudom onas 0 v-11 Pseudomonao a I c a l i g e n e s lo am y -san d y 9 .0 Kaufman s y n t h e t i c 1000 V-1 P3eudomonas a l c a l i g e n e o lo am y -san d y 9 .0 Kaufman s y n t h e t i c 0 VIV-3 Pseudomonas s p e c i e s b la c k d e g ra d e d f e r t i l i z e d 0 .0 K earney 1000 V-4a C o ry n e b a c te riu m s p . loam y -san d y 9 .0 Kaufman s y n t h e t i c 1000 V-84 C o ry n e b ac te riu m s p . sandy 1 .5 K earney 100 V-36 C o ry n e b a c te riu m o p . lo a m y -запйу 0 s o i l e x t r a c t 100 V-2 1 6 A r th r o b a c te r s p . loam y -san d y 0 K earney 1000 VVI-3 A r th r o b a c te r o p . b la c k d e g ra d e d f e r t i l i z e d 0 K earney 100 V-5 M icro co ccu s s p . lo am y -san d y 9 .0 Kaufman s y n t h e t i c 100 VDC-1 S e r r a t i a sp, b la c k d e g ra d e d f e r t i l i z e d 0 K earney 1000 V-10 N o c a rd ia sp, lo am y -san d y f e r t i l i z e d 0 f o r Pseudom onas 100
24 hours and filter the liquid through filter paper and enrich w ith KH2PO4 — 0.5 g/1, glucose — 10 g and agar 16 g, and then sterilize again). A p art of it was enriched w ith 100 and 1000 ppm of venzar. A corres ponding herbicide dose was suspended in a small volume ethanol and added to previously sterilized media. 32 bacterial strain coming from four successive microbiological analyses were examined, th a t is : 17 strains of genus Pseudomonas, 7 strains of genus Bacillus, 5 strains of genus Corynebacterium, one Nocardia, sp., one Serratia sp. and one Micrococcus sp.
Ëffect ö£ venzar on respiration activity 95 m ethod in the W arburg apparatus at 27°C in vessels of 3 m l volume [5, 6]. One-day liquid b roth cultures of individual bacterial strains were centrifuged (10 000 r.p.), and washed w ith psysiological salt solu tion, as well as suspended in a phosphate buffer of pH 7.2. One m illilitre of this suspension was introduced into the m ain cham ber of the vessel. The same am ount of suspension was dried at 105°C up to a constant w eight in order to determ ine the dry mass of cells which was between 10 and 12 mg/ml. Into the side arm s of the W arburg vessels the respiration substrates w ere introduced : a solution of w atersoluble dose of venzar (6 ppm) and 0.1°/o glucose solution, or m ixtures of both these compounds. For each bacterial strain the oxygen uptake and carbon dioxide evolution in the presence of the above-m entioned respiration substrates were examined, and the respiration factors RQ were calcu lated. The m easurem ents were usually carried out over 8 hours, read ing out the level of m anom eters every half an hour [17]. A part from the exogenic respiration, endogenic respiration was also examined. The results of the gas exchange were expressed in m icrolitres (jil) of the taken up or evolved gas for one mg of dry mass of bacteria. On the basis of the obtained results the oxygen uptake curves and the carbon dioxide evolution curves were plotted.
By the m ethod of th in - layer chrom atography the undegraded h er bicide and its m etabolites in the liquid from the W arburg vessels was determ ined after completion of gas exchange investigation. Venzar was extracted w ith ethyl acetate, and the chrom atographic plates covered w ith silica gel G-60 were developed in the system benzene : acetone (6 : 1) and then sprayed w ith a starch reagen t [14].
R E SU L T S A N D D IS C U S S IO N
The effect of venzar on the respiration activity of various genera of bacteria was different (Figs 1-7). Most bacteria of genera Bacillus (Fig. 2), Pseudomonas and Arthrobacter (Figs 1, 4) show ability to oxidation of venzar to equal degree as glucose and in some cases venzar was even b etter utilized (Figs 1, 2). The bacteries of genus Pseudo
monas aeruginosa and one strain Arthrobacter sp. very intensely oxidated
the m ixtures of glucose and venzar (Fig. 4).
The respiration factors RQ for bacteria oxidating venzar were higher than this would result from the stechiom etrie reaction (Table 2)
9 6 j. Sobieszcżański at ai.
Fig. 1. T h e resp iration of Pseudomosci strain s u tilizin g va rio u s carbon sources
The genus Pseudomonas prevailed quantitatively in the population of isolated bacteria resistan t to high doses of venzar in the substrate (100 and 1000 ppm) and most intensely utilized this herbicide as a carbon source (Fig. 1). A fter completion of the investigation of gas exchange in the liquid from the W arburg vessels, in most cases no residues of undegraded herbicide were detected, while there appeared new m e tabolites (Table 2). For the genus Bacillus venzar was also a carbon source. A decrem ent of about 75°/o of this herbicide was noted chro- m atographically bu t no m etabolites w ere detected. Factors RQ were higher for venzar, which could indicate the predom inance of
decarbo-Ëffect of venżar on respiration activity 97
F ig. 2. T h e resp ira tio n o f B a c illu s sp. and M icrococcus sp. strain s — v a rio u s carbon sou rces
xylation processes, incomplete oxidation of the herbicide and of the evolution of yet other gases besides C 0 2, or of oxidation typical of organic acids w hich could be form ed in th e degradation process of venzar. It is not known w hich of the possibilités m entioned was the cause of increased values of the respiration factors RQ.
J. Sobieszczański at al.
F ig. 3. T he e ffe c t of g lu co se and v en za r on th e resp ira tio n of v a rio u s b a cteria l strain s
The other genera of bacteria : Corynebacterium, Serratia, Nocardia,
Micrococcus, Arthrobacter and Pseudomonas alcaligenes oxidated venzar
m ore poorly (Figs. 2, 5, 6, 7) or they could not adapt them selves to the utilization of venzar as a respiration substrate. Thus they have shown a very small respiration activity. A t the same tim e a disturbance in respiration processes was observed, m anifesting itself by breakdow n and stoppage of respiration, the respiration factors RQ reaching in these experim ents irratio nal values (Table 2). The above-m entioned results indicate th a t venzar changed the m etabolism of these bacterial strain s
A w ater-soluble dose of venzar (6 ppm) acted stim ulating on the respiration of some of bacteria of genera Pseudomonas, Bacillus and
Arthrobacter (Figs. 1, 2, 4), the stim ulation being proportional to both
Effect of venzar on respiration activity 99
Fig. 4. T h e e ffe c t of g lu co se and v en za r m ix tu r e on th e resp ira tio n on P se u d o m o n a s
a eru g in o sa and A r th r o b a c te r sp. stra in s
by K a r p i a k, I w a n o w s k i {10], can stim ulate or inhibite dispro portionately oxygen and oxygen-free processes.
G enerally, one can say th a t 50®/oi of th e bacteria exam ined were susceptible to the presence of venzar, which m anifested itself by a disturbance in respiration processes. In this group w ere present forms of Corynebacterium sp., Serratia sp., eight strains of genus Pseudomonas sp. and two strains of genus Bacillus sp. The other bacterial strains of genus : Bacillus, Pseudomonas and Arthrobacter utilized venzar as
j[Q(j j. Śobieszczanski at al.
F ig. 5. T h e e ffe c t of g lu co se and a m ix tu r e of g lu co se and v en za r on th e resp ira tio n o f v a rio u s b a cteria l stra in s
a substrate in processes of oxygen respiration, thus contributing to its rem oval from the medium.
The obtained results stress the necessity of fu rth e r investigations on m icrobial degradation of herbicides, w hich we w ill carry out in our successive experim ents whose justification is also found in the studies b y : A l e x a n d e r [1, 2], M i a y a m o t o a n d O n t a v o i [13], B a l l a g [3, 4], G o ł ą b , A l t h a n s {9], Ś o b i e s z c z a n s k i [16].
Effect of venzar on respiration activity 101
F ig. 6. T he d istu rb an ce in resp ira tio n of P s e u d o m o n a s and A r t h r o b a c t e r sp.
102 J. S ob ieszczań sk i at al.
T a b l e 2
The e f f e c t o f v e n z a r and g lu c o s e on g a s exchange o f th e t e s t e d g e n e ra o f b a c t e r i a and th e d e crem en t o f v e n z a r from th e medium
RQ
B a c t e r i a l s t r a i n s Respiration substrates V enzar r e s id u e № Genus v e n z a r 6 ppm glucose 0.1% % v e n z a r + + g lu c o se % Rf o f metabo - lite3 V-217 V-218 B a c i l l u j s p . B a c i l l u s s p . 1 .1 7 0 .9 1 5 .3 0 1 .4 0 2 .9 0 1 .0 9 0 70 0 .3 9 0.92 V-92 B a c i l l u s s p . 0 .9 8 1 .2 7 1 .0 5 20 V-25 B a c illu 3 s p . 1.12 1 .2 7 0.42 25 V-76 B a c i l l u s s p . 1.20 1.12 1.40 20 V-20 B a c i l l u s sp. 1 .2 7 0 .8 1 _ 0 V-16 B a c i l l u s m vcoidc3 1 .0 6 0 .2 9 0 .2 0 ^8 V-80 V-209 V-208 V-105 Pseudomonas Pseudomonas Pseudomonas Pseudomonas f l u o r e s c e n s f l u o r e s c e n s f l u o r e s c e n s f l u o r e s c e n s 1.0 8 0 .7 0 1 0 .6 4 1 .3 0 0 .6 8 i r r a t i o n a l 0 .4 7 0 .6 7 0 .8 2 2.10 0 .7 5 0 0 20 100 0 .4 7 0 .2 5 0 .2 9 0 .6 6 0 .4 0 0 .9 4 V-65 Pseudomonas f l u o r e s c e n s 0 .6 8 0 .8 4 _ 95 V-212 V-204 Pseudomonas Pseudomona3 a e r u g in o s a a e ru g in o c a 1 .0 0 0 .4 4 0 .3 5 0 .3 3 0 .7 0 0 .3 8 0 0 0 .4 7 0 .8 8 0 .9 5 V-211 V-100 Pseudom onas Pseudomonas a e r u g in o s a a e r u g in o s a 1 .3 0 1 .6 0 1.00 0 .8 2 - 0 20 0 .1 7 0 .3 7 ---V I-5 V-206 V-223 Pseudomonas Pseudom onas Pseudomonas a e r u g in o s a a e r u g in o s a a e r u g in o s a 1.12 0 .9 1 1 .2 0 7 .4 0 0.52 2 .5 0 -50 50 100 0 .3 2 0 .3 2 --- ---V-82a Pseudomonas a e r u g in o s a 0 .3 8 0 .2 8 0 .4 7 90 V-205 Pseudomonas a e r u g in o s a 1 « 40 4 .4 0 _ 0 V-11 Pseudomonas a l c a l i g e n e s 1.0 5 0 .4 5 1.18 0 V-1 Pseudomona3 a l c a l i g e n e s 1 .1 7 1,00 _ 0 VIV-3 Pseudomonas s p e c i e s _ _ _ 0
V-4a Corynebactei■ium s p . 11 .8 0 0 .2 5 25
V-34 C orynobacterium s p . 0 .1 2 0*26 _ 95 V-36 C o ry n o b a c te r iu n 0 .1 9 0 ,2 3 7 .3 0 100 V-216 A r th r o b a c te i' я р . 0 .3 6 2 1 .7 20 W T-8 A rth ro b acfcei■sp. _ _ 0 V-5 M icro co ccu s 1.08 25 VIT-1 S e r r a t i a 4 .4 1.6 4 .3 0 50 V-10 N o c ard ia 2 .0 7 1.12 20 CONCLUSIONS
1. Bacteria of genera : Bacillus, Pseudomonas, Nocardia, Corynebac- terium, Serfßtia, Microçoccus and Arthrobacter are susceptible to high doses of venzar (100 nad 1000 ppm) in the medium.
2. Most bacteria of genera : Psedomonas B tillus and Arthrobacter can utilize this herbicide as a carbon source in energetistic processes, thus contributing to the removal of venzar from the medium.
E ffect of ven zar on resp iration a c tiv ity 103
3. Venzar at 6 ppm stim ulated respiration processes in bacteria of genera : Bacillus, Pseudomonas, Arthrobacter.
4. In some of genera of bacteria (Corynebactrium sp., Serratia sp.,
Nocardia sp., Micrococcus sp.) venzar disturbed respiration processes.
R EFEREN CES
[1] A l e x a n d e r M. : B iod égrad ation : P rob lem of m o lecu la r reca lcitra te and m icrob ial fa lla b ility . A d v a n ces in A ppl. M icrobiology 7, 1965, 35-80.
[2] A l e x a n d e r M. : M icrobial foorm ation of environm ental pollutants. A d van ces in A ppl. M icroobiology 18, 1974, 1-64.
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[6] B r z e s k i W. : P ractical training on bioochem istry. PW RiL, 1972, 317. [7] B u r b i a n k a M. , P l i s z k a A. : Food m icrob iology. Ed. IV , 1977, 487. [8] C z e r w i ń s k i W., W ę g r z y n T. : The effect of in teraction of ven zar and
b acteria on p h o to sy n th esis of p lan ts. S o il Sei. A nn. 26, 1975, 2, 73-77. [9] G o ł ą b T., A l t h a n s A. , W o o l e n H. : D ég ra d a tio n of tr iflu r o lin in soil.
IV th In tern a tio n a l Congr. of P est. Z ürich, 1978, 5, 505.
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[14] P i s s J. : In v e stig a tio n s on th e p h y sic o -c h e m ic a l m ethod of th e d esa ctiv a tio n rate of 2 -c h o r o -4 -e th y la m in o -6 -iz o p r o p y la m in o -S -r ia z in e in so il (D octor’s th esis). W SR W rocław , 1970.
[15] S a n d s D., R o v i r a A . : Iso la tio n of flu o r e sc e n t p seu d om on as w ith a s e le c t iv e m ed iu m . A ppl. M icrobiol. 20, 1970, 3, 513.
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[17] Z e c e r o v E., K h r i p u n o v a J.: E lek tro m etrich esk ii m etod izu ch en ia tk a n e - vo g o d yk h an ia. B io k h im 26, 1961, 1, 86-92,
104 J. S ob ieszczań sk i at al.
J. SO BIESZCZAŃSK I, A. RODZIEWICZ, R. STEMPNIEW ICZ
M IK RO BIO LO G IC ZN A T R A N SF O R M A C JA H E R BICY DÓ W W PRO CESA CH K A TA B O L IC Z N Y C H
CZĘŚĆ I. W PŁY W V EN Z A R U N A A K TY W N O ŚĆ ODDECHO W Ą W Y BR A N Y C H RO D ZA JÓW BA K TE R II G LEBOW YCH In sty tu t P r z e c h o w a ln ictw a i T ech n o lo g ii Ż y w n o ści AR
w e W rocław iu
S t r e s z c z e n i e
B adano 32 szczepy b a k terii w y o d ręb n io n e z różn ych g leb uprzednio p erk o lo - w a n y ch w od ą za w iera ją cą n isk ą zaw artość v en za ru [tab. 1 ]. Szczepy te n ależały do rodzajów : P se u d o m o n a s, B a c illu s, C o r y n e b a c te r iu m , S e r r a tia , N o c a rd ia , M icro -
coccu s i b y ły oporne na d a w k ę 100 i 1000 ppm v en za ru w p odłożu. P o słu g u ją c się
bezp ośred n ią m etod ą W arburga b adano p roces od d ych an ia szczep ów b a k terii ozn a czając ilość p ob ieran ego tlen u i w y d z ie la n e g o d w u tlen k u w ę g la w ob ecn ości v e n zaru, g lu k ozy i m iesza n in y obu su b sta n cji jako su b stra tó w o d d ech ow ych . V en zar
ok azał się dobrym su b stra tem o d d ech ow ym dla w ię k sz o śc i b a k terii z rodzajów :
P seu d o m o n a s, B a cillu s, A r th r o b a c te r i sty m u lo w a ł od d ych an ie ty ch szczep ów b a k
terii. P o zo sta łe szczep y b ak terii w y k a z a ły w ob ecn ości v en za ru n isk ą a k ty w n o ść odd ech ow ą i zab u rzen ia p rocesów w y m ia n y g azow ej.
Prof . d r J e r z y S o b i e sz c za ń s k i
I n s t y t u t P r z e c h o w a l n i c t w i T e c h n o lo g ii Ż y w n o ś c i AR W r o c la w , ul. N o r w i d a 25
