JERZY SO B IE SZ C Z A Ń SK I, A N N A RODZIEW ICZ, R EG IN A ST EM PN IEW IC Z
MICROBIOLOGICAL TRANSFORMATION OF HERBICIDES IN CATABOLIC PROCESSES
PART II. THE EFFECT OF DIURON ON THE RESPIRATION ACTIVITY OF SELECTED BACTERIAL STRAINS 1
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
Diuron (herbatox) 3-/3, 4-dichlorophenylurea/l, 1-dim ethy- lurea [13] belongs to the group of urea herbicides (abbrev. : DMU, DCMU, 3, 4 DDU).
This is a crystaline substance, slightly soluble in w ater (at 25° — 42 ppm), of long-term residue action of 4-6 m onths, and even up to 3 years. It acts through the root system of plants. In a m ixture w ith other herbicides it is applied to fight weeds in beets, maize, sunflower [5].
Urea herbicides are sufficiently dorable and resistan t to hydrolytic degradation under soil conditions, and also to oxidation through chemical agents. They are degraded by m icroorganisms (Pseudomonas, Bacillus, Sarcina, Xantomonas, Pénicillium and Aspergillus) [11] utilizing e.g. m onuron as a carbon source. Using 14C the authors have shown th a t diuron persisted in the soil for 70 days. A fter 90-130 days almost the total 14C was recovered in form of 14COz [113, w hich would indicate th a t urea herbicides can be an energy source for microorganisms. In study we exam ined diuron in th at respect.
M A TE R IA L A N D M ETH ODS
— D egradation of diuron by the population of soil microorganisms. The Wrocław black earth from the plot of IUNG (Institute of Soil Science and Cultivation of Plants) was chosen.
106 J. S ob ieszczań sk i at al.
— Exam ination of bacterial strains. The source of exam ined m icro organisms was black earth soil samples :
preincubated w ith diuron (1.5 ppm) in the percolator for 32 days, treated in a pot experim ent 13 days before the experim ent w ith diuron at 30 ppm,
the control — the soil w ithout a herbicide.
Respiration activity of the population was determ ined by the direct
W arburg method [4, 6]. Into the m ain cham ber of the W arburg respiro-
m etric vessel 3 ml of the sample were introduced. One gram of soil was suspended in a m ixture : 1 ml of a phosphate buffer of pH 7.2 and 1 m l of the K earney synthetic culture medium (for 1000 ml of w ater : K0HPO4 —0 . 8 g, KH2P 04 — 0 . 2 g., M gS04 • 7 H20 — 0 . 2 g, N aN 03 — 5 g,
C aS04 • 7 H20 — 0.05 g, NH4Mo7024 • 4 H 20 — 0 . 0 0 1 g.
Into the side arm of the W arburg vessel, as respiration substrates
0 . 1 ml of :
— diuron solution — 2 0 ppm,
— glucose so lu tio n — l°/o,
— a m ixture of solutions of both these substances in the proportion
I : 1 was introduced.
The chimney of the vessel was filled w ith 20% KOH, into which strips of filter paper had been put (uptake of evolving C 0 2).
3 3 bacterial strains were isolated from the chosen soil enriched
w ith diuron. For fu rth e r exam ination we selected 7 bacterial strains most often occurring in the exam ined soil, characterized by growing
in the presence of 1 0 0 and 1 0 0 0 ppm of diuron.
The genus affiliation of these strains, the origin and conditions of isolation are given in Table 1.
T a b l e 1 C h a r a c t e r i z a c i o n o f th e exam ined b a c t e r i a l S t r a i n s B r c r o r i a l s t r a i n s I s o l a t e d f r o n s o i l w ith d iu ro n ppm I s o l a t i n g c u l t u r e mcdiimi d iu ro n ppm Sym bols S p e c ie s D-58 B a c i l l u s s u b t i l i s b la c k s o i l , p o t s e x p e rim e n t 3 Kaufman e y n t h o t i c 10C0 A-11 Pseudomonas op. blaclc 3 o i l p e r c o l a t i n g sy stem 1 .5 s o i l e x t r a c t 100 D-5 9 Pseudom onas sp. san d y -lo aru s o i l , роЬз e x p e rim e n t 0 10C0 D-39 Pseudom onas gd. san d y -lo ara 3 o i l , p o ts e x p e rim e n t 0 s o i l e x t r a c t 1000 D-48 C o ry n o b a cteriu m sp. san d y -lo am n o il, p e r c o l a t i n g sy stem 1.3 Kaufmr.n s y n t h e t i c 100 D-31 B ac te riu m op. b la c k s o i l ( . o l a t i n c sy stem 1 .5 Kaufman s y n t h e t i c 100 D-14 B a c te riu m sp. san d y -lo am s o i l , p o t e e x p e rim e n t 0 s o i l e x t r a c t 1000
To isolate the bacterial strains the following media w ere employ ed : th e K aufm an synthetic m edium '[10] and a soil ex tract [16]. Weighed portions of diuron w ere added to sterile hot media under sterile conditions in quantities given in Table 1.
The K earney liquid cultures of bacteria (composition as above) cultivated for 24 hours on a medium were centrifuged at 10 000 r.p.m. The separated bacterial cells were washed w ith physiologic salt solution and suspended in a phosphate buffer on pH 7.2. One m illilitre of the cell suspension containing 150 fig of protein (estimated after L o w r y [12]) was introduced into the m ain chambers of the W arburg vessels. Side arm s of the vessels, each, contained 0.1 ml of solution of :
— diuron 20 ppm, — glucose 50 ppm,
— a m ixture of both these substances,
— the control w ithout medium (endogenic respiration).
A fterw ards in the same apparatus the cell respiration at 30° was examined. Oxygen uptake was determ ined in m icrolitres (ц,1), reading out the results every 60 m inutes over a period from several to a dozen or so hours. A fter completion of the experim ent the diuron residue was extracted from the solution previously introduced into the W arburg vessel, and determ ined by thin -lay er chrom atography on starch gel G-60. Diuron was extracted w ith chloroform in proportion 1 : 10. The development system in chrom atography was piridine-chloroform (1 : 100), or chlcroform -nitrom ethane 1: 1. Plates were chlorinated and detected w ith starch, estim ating them according to stand ard spots [14].
R E SU L T S A N D D IS C U S S IO N
R e s p i r a t i o n a c t i v i t y o f t h e s o i l . The results of the exam ination (Fig. 1) showed th a t in the W rocław black earth
preincu-F ig. 1. O x y g en u p ta k e b y th e b lack , earth , p ercolated for 32 days
108 J. S ob ieszczań sk i at al.
bated w ith diuron (1.5 ppm) in the percolator a m icrobial population was formed, capable to utilize diuron as a sole carbon source, and at th e same tim e to m aintain the unchanged endogenic respiration. Howe ver, glucose added as l°/o respiration substrate was not oxidated (Fig. 1A). This interesting fact is being studied separately, the m ore so because the m icrobial population from the control soil (percolated w ithout a herbicide) did not oxidate diuron, bu t it very well utilized glucose (Fig. IB). Likewise a m icrobial population, form ed in the soil enriched w ith diuron at 30 ppm during 13 days of incubation, utilized also very intensely diuron as a carbon source, b ut not before a 24-hour adapta tion in the W arburg vessel.
In this experim ent the endogenic respiration of microorganisms was stopped, and glucose was not oxidated either (Fig. 2A).
Fig. 2. O x y g en u p tak e by the b lack earth, in cu b a ted for 13 days in a pot Л — w ith diu ron at 30 p pm , В — w ith o u t a h erb icid e
In the control soil w ithout a herbicide the m icrobial population, sim ilarly not before a 24-hour adaptation in the W arburg vessel, increa sed endogenic respiration, but it utilized diuron more poorly than a m ic robial population previously preincubated w ith diuron, whereas it oxi dated glucose very well (Fig. 2B).
The m entioned results of exam ination show th a t diuron disturbed oxygen uptake processes in soil and itself became a better carbon source for the adapted m icrobial population than glucose. It is possible th a t diuron was a repressor of enzymes oxidating glucose [15]. Diuron inhi bited also the endogenic respiration or caused the disturbance in respi ration processes, th eir intensity depending on the introduced dose of diuron and on the duration of its action (Figs 1, 2).
diuron and came from a control soil w ithout a herbicide, utilized first of all glucose as a carbon source, and no diuron at all or only a small p art of it.
T h e u t i l i z a t i o n o f d i u r o n . The utilization of diuron аз
a source of carbon and energy in the respiration processes in predo m inating bacterial strains resistan t to high doses of diuron in the
substrate ( 1 0 0 and 1 0 0 0 ppm) varied and depended on both the strain
and composition of the substrate and time (Figs 3 and 4), nam ely :
F ig. 3. O x y g en u p tak e b y b a cteria l stra in s iso la ted from a soil in cu b ated w ith a h igh dose of d iuron (100 ppm)
— Bacteria Pseudomonas sp. (D-59) and Bacterium sp. (D-14) in ten sely utilized diuron as a sole energy source, and did not utilize glucose, or utilized it only in a m ixture w ith diuron (Bacterium sp. D-14) — Fig. 4,
— Bacterium sp. (D-31) and Bacillus subtilis (D-58 utilized first glucose and then diuron b u t did not utilized m ix tu re diuron + glucose (Fig. 4),
— Pseudomonas sp. (D-59) oxidated diuron m ore intensively th an glucose (Pseudomonas sp. D-39) oxidated both substrates applied (Fig. 4),
— the strain Bacterium sp. (D-14) showed a stim ulating effect of glucose on the utilization of diuron although glucose itself was not separately utilized (Fig. 4),
— Corynebacterium sp. (D-48) — Fig. 3, and Pseudomonas sp. D-39) — Fig. 4, poorly utilized glucose in the presence of diuron (in a m ixture). Corynebacterium sp. D-48 did not utilize diuron (Fig. 3).
Basing oneself on the bacterial strain Pseudomonas sp. (A -ll), it was additionally established th a t optimal respiration substrates for
n o J. Sobiesżczański at al.
respirom etric exam ination w ere : diuron at 20 ppm and glucose at 50 ppm (Fig. 5).
F ig 4. O x y g en u p tak e by b a cteria l stra in s of g en era : B a cillu s sp., P s e u d o m o n a s sp.,
B a c t e r i u m sp.
The results of our investigations show th a t diuron selectively af fected the m icroflora of the soil, w hich is confirm ed by other w orkers [2, 3, 7].
They say th a t urea, triazine and carbam ate herbicides select m icro bial population in the soil, and at the same tim e m ajor disturbances
Fig. 5. O x y g en u p tak e by b acteria P s e u d o m o n a s sp. (A-11) in th e p resen ce of v a rio u s co n cen tra tio n s of rasp iration su b stra tes
in respiration processes in sandy soil and non-fertilized soil [7] were noted.
The resutls of our study show th a t individual adapted bacterial strains are capable of degrading diuron. We observed an intense oxi dation of this herbicide, e.g. by Pseudomonas sp. and Bacterium sp.
A fter completion of m anom etrie m easurem ents ńo herbicide residues were found in the reaction liquid from the W arburg vessels. K e a r n e y and K aufm an [9, 11] as well as M a i e r - B a d e [13] say w hat react ions can take place during the microbial degradation process of aniline herbicides. A t the first stage it is a hydrolytic degradation of this compound to dichloranilines, C 0 2 and amines. A fu rth e r degradation of the arom atic ring involves hydroxylation [17], deam ination and deha- logenation.
K a u f m a n [9] stresses th a t oxidating processes are an im portant stage of microbiological degradation of aniline derivatives. К a r p i a к [8] dem onstrated th a t urea herbicides can stim ulate the respiration activity of bacteria. Also Pajewska, cited after [3], corroborates the stim ulation of respiration processes of bacteria under the influence of herbicides.
Investigations by A l e x a n d e r [1], S o b i e s z c z a ń s k i et al. [16] indicate th a t herbicides m ay be degraded by soil microorganisms in the catabolic way. A l e x a n d e r [1] thinks th a t it can take place thanks to enzym atic changes in the cells of microorganisms w ithout a change of genotype. Sim ilarly, Bishop and Jagendorf, cited after [2], say th a t m onuron and diuron inhibit the phosphorylation, w hereas linuron in bacteriostatic doses acted m utagenically on Rhizobium. It
112 J. S ob ieszczań sk i at al.
seems probable th a t in our investigations diuron induced the required enzymes in the exam ined bacterial strains resistant to high doses ol this herbicide, which enabled them to utilize this herbicide as a sole carbon and energy source, but at the same tim e it was observed th a t some of the strains (Fig. 4) lost their ability to utilize glucose in the respiration processes, which indicates of m ajor disturbances in th eir physiology. The obtained results perm it to sum up the study w ith the following conclusions.
C O N C L U SIO N S
1. D iuron disturbs respiration processes in the soil by way of a selective action on the tnicroflora, changes the intensity of these processes, causes th eir breakdown.
2. Even field doses of diuron (1.5 ppm) cause a disturbance in the oxygen uptake processes in a soil population.
3. Predom inating bacterial strains isolated from the soil preincubated w ith diuron acquire the ability to utilize this herbicide as a carbon source. However, some of them lose the ability to utilize glucose.
4. A m icrobial population in the soil, by utilizing diuron as a carbon source in respiration processe, prevents its accum ulation in soil.
5. The results point out the need for studies on the mechanism of herbicide degradation in catabolic microbial processes, which will be the subject of our successive investigations.
REFEREN CES
f l] A l e x a n d e r M. : B iod égrad ation : P ro b lem of m o lecu la r reca lcitra tio n and m icrob ial fa lla b ility . A d v a n ces in A ppl. M icrobiol. 1965, 7, 35.
[2] B a l i c k a N., K a s z u b i a к H. : E ffect of h erb icid es on soil m icroorgan ism s. P ost, m ikrob. 5, 1967, 1, 15-26.
[3] B a l i c k a N. , G o ł ę b i o w s k a J. : E ffect of h erb icid es on soil m icroorga n ism s in th e lig h t of in v e stig a tio n s carried out in P olan d . P ost, m ikrob. 9, 1972, 1, 117-136.
[4] B i e s z k i e w i c z E., C z e r w i ń s k a К .’: T rain in g on m icrob iology. PW N W arszaw a, 1971, 205-215.
[5] В ł a s z c z a k W. : S cien ce on d ise a se s and p ests of p lan ts, PW R iL 1971, 514. [6] B r z e s k i W. : P ractical training on b ioch em istry. PW RiL W arszaw , 1972,
289-327.
[7] H a u k e - P a c e w i c z o w a T. : H erb icid e e ffe c t on th e m icroflora a c tiv ity in soil. P am . puł. 1971, 46.
[8] K a r p i a k S., I w a n o w s k i H. : T he effect of herbicid es on soil m icroflora. V II. R esp ira tio n of b acteria iso la ted from m aize rh izosp h ere. A cta M icrob. P olon. 18, В 1969, 47-52.
[9] K a u f m a n D. : M icrobial m eta b o lites of a n ilin e -b a se d herb icid es. S o il S ei. A n. 26, 1975, 2, 5-15.
[10] K a u f m a n D., K e a r n e y P., S h e e t s T. : M icrobial d egrad ation of sim a - zine. A g ricu lt, and Food C hem . 13, 1965, 3, 238.
[11] K e a r n e y P., K a u f m a n D. : D eg ra d a tio n of h erb icid es. M. D ek k er Inc., N e w Y ork 1969, 79-111.
[12] L o w r y O., R o s e n b r o u g h N. : P ro tein m ea su rem en t w ith th e fo lin p h en ol reagen t. J. B iol. C hem . 193, 1951, 265.
[13] M a i e r - B a d e H. : H erb icid e und ih re R ü ck stän d e. V erl. E u gen U lm er, S tu ttg a rt 1971, 176.
[14] P i s s J. : In v e s tig 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 lo r o -4 -e th y la m in o -6 -iso p r o p y la m in o -S -tr ia z in e in so il (D octor’s th esis, typ escrip t), W SR W rocław , 1970.
[15] R o m a n o A., K a r n b e r g H. : R eg u la tio n of sugar u tiliz a tio n by A s p e r
g illu s n id u la n s. B iochem . B iop h ys. A cta 158, 1968, 3, 491-493.
[16] S o b i e s z c z a ń s k i J., R o d z i e w i c z A., S t e m p n i e w i c z R. : M icro b io lo g ica l tra n sfo rm a tio n s of h erb icid es in ca ta b o lic processes. P a rt I. V enzar e ffe c t on th e resp iration a c tiv ity of soil. Rocz. glebozn. 1981.
[17] T o c z k o W. : H y d r o x ila tio n rea ctio n s in bacteria. B io lo g ica l o x id a tio n . P ol. T ow . B ioch em iczn e, W arsaw 1971, 129.
J. SO BIESZCZAŃSK I, A. RODZIEWICZ, R. STEMPNIEW ICZ
M IK RO BIOLO G IC ZN A T R A N SF O R M A C JA H E R BICY DÓ W W PR O C ESA C H K A T A B O L IC Z N Y C H
CZESC II. W PŁY W D IU R O N U N A A K TY W N O ŚĆ O D DECHO W Ą G L EBY I W Y BR A N Y C H SZCZEPÓW BA K TE R II
In s ty tu1 P rzech ow aln ictw a i T echnologii Ż yw ności AR w e W rocław iu
S t r e s z c z e n i e
P o słu g u ją c się b ezp ośred n ią m etodą W arburga badano zab u rzen ia w p o b iera n iu tlen u przez g leb ę u p rzednio trak tow an ą diuronem .
C zarna ziem ia w r o cła w sk a w d o św ia d czen ia ch w a zo n o w y ch b y ła p erk o lo w a n a w o d ą z a w iera ją cą 1,5 ppm diu ron u i in k u b o w a n a p rzez 13 dni w tem p era tu rze 25°C z d aw k ą 30 ppm diuronu. S u b stra ta m i od d ech o w y m i były: d iu ron — 20 ppm i glu k ooza — 1 %.
D iu ron za k łó ca ł proces p ob ieran ia tlen u przez p o p u la cję g leb y , co zależało od w y so k o śc i d a w k i i czasu d ziałan ia. S am b y ł źród łem en erg ii. B ad an o ta k że szczep y b a k terii w y o d ręb n io n e z tej g leb y i z id en ty fik o w a n o je jako rodzaje: P s e u
d o m o n a s sp., B a cillu s sp., C o r y n e b a c te r iu m sp. i B a c te riu m sp. B y ły one oporne
n a w y so k ie d a w k i h erb icyd u (rzędu 100 i 1000 ppm) i w yk a za ły zdolność do k o rzy sta n ia z diuronu jako źródła w ęg la . Z arów no u p op u lacji, jak i w y o d ręb n io n y ch szczepó w za n o to w a n o u tratę zd oln ości do u tlen ia n ia g lu k o zy w m ia rę u zd oln ień do w y k o r z y sty w a n ia diuronu.
Prof. d r J e r z y S o b i e sz c z a 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 a i T e c h n o lo g ii Ż y w n o ś c i A R W r o c l a w , ul. N o r w i d a 25
