Identification of spore-forming strains involved in biodegradation of acifluorfen

Res. Microbiol.

© INsri: t~r PASTEUR/ELSEVIER

Paris 1996

1996. 147, 193-199

Identification of spore-forming strains involved in biodegradafion of acifluorfen M.G. Fortina o)(*), A. Acquati (t) and R. Ambrosoli (2) (t) Dipartimento di Scienze e Tecnoiogie Alimentari e Microbiologiche, Sezione di Microbiologia Industriale, Universitd degli Studi di Milano, 20133 Milano (Italy), and ¢2)Dipartimento di Valorizzazione e Protezione delle Risorse Agroalimentarl, Uniwrsitia degli Studi di Torino, 10126 Torino (Italy)

SUMMARY

We isolated and identified four spore-forming bacteria from activated sludges and soil, three of which wer~ able to degrade acifluorlen. Biochemicalcharacteristics,DNA base composition and DNA-DNA homology indicated that the degcading strains belonged to the species Bacillus thuringiensis, Ciostridium perfringens and Closlridium sphMIok~s, The fourth strain, identWmdas C. sphenoides and showing the same charof the corresponding degrading strain, was unable to metabolize ncifluorfen, Thus, the plasmkl content of these strains was analysed to study the possible correlation between the presence of extrachromosomal elements and the ability to degrade this herbicide.

Key-words: Acifluorfen, Biodegradation, Bacillus thuringiensis, Clostridium perfringens, Clostridium sphenoides; Identification, Plasmids, Spores, Herbicide.

INTRODUCTION The widespread use of pesticides and herbicides has raised considerable concern about their persistence in the environment. Indeed, these compounds can be found in almost any natural habitat. Microorganisms can play an important role in the degradation and removal of toxic chemicals with consequent decontamination of polluted environments. The possibilities for biodegradation are dependent upon the metabolic potential of indigenous microorganisms and the capacity to adapt to phy-

Submitted March 13, 1995, accepted August 17, 1995. (*) Correspondingauthor.

sico-chemical conditions in the natural environment. For this reason, many attempts are being made to evaluate the possibility of applying pure cultures and enriched or specifically engineered strains to enhance biodegradation of specific compounds (Rosenberg, 1993). Acifluorfen, 5-(2-chloro-4-(trifluoromethyl) phenoxy)2-nitrobenzoic acid, is a diphenylether herbicide widely used in pre-emergence control of broad-leaved weeds in soybean fields. The action of microorganisms in the degradation of nitrodiphenylether herbicides in soil was pointed out by several authors (Niki and Kuwatsuka,

194

M.G. FORTINA ET AL.

1976; Schmidt and Braune, 1987; Oyamada and Kuwatsuka, I989), but biodcgradation of acifluorfen has been described in very few works (Gennari and N~gre, 1990; Perucci and Scarponi, 1993; Andreoni et aL, 1994). In a previous paper (Gennari et aL, 1994), we isolated, from activated sludges of a wastewater treatment plant and from soil with a long history of acifluorfen applications, mixed cultures that were able to reduce acifluorfen to aminoacifluorfen; almost quantitative conversion was observed when the medium contained the herbicide as sole carbon source. Considering the importance and the scarcity of existing reports on the microflora responsible for this degradation, we isolated from mixed cultures four spore-forming bacteria that constituted the majority of the microflora present, three of which were able to degrade herbicide faster than the parent mixed cultures. This paper deals with the identification and characterization of these spore-forming bacteria and their potential application as pure cultures to enhance the above-mentioned degradation. Additional studies on the possible correlation between the presence o f p l a s m i d s and the ability to degrade acifluorfen were also undertaken.

MATERIALS AND METHODS

Bacterial strains and growth media Four spore-forming bacteria, designated C l, C3, C4 and C5, were isolated in our laboratories from mixed cultures taken from activated sludges of a wastewater treatment plant and from soil treated with acifluorfen. Three reference strains, Bacillus thuringiensis DSM 2046T, Clostridium perfringens DSM 756"r and Clostridium sphenoides DSM 632~" were used for comparative studies. Clostridium indolis DSM 755 T was used as a control strain in spectrophotometric hybridization experiments.

DMSO DSM EDTA LB PY

= = = = =

dimethyisulphoxide. DeutscheSammlungyon Mikroorganismen. ¢thylenediaminetetraacetieacid. Luria-Bertani(medium). peptone/yeastextract(medium).

Strain C I and B. thuringiensis DSM 2046 were grown in aerobic conditions in Luria-Bertani (LB) medium (Maniatis et aL, 1989) at 37°C. Strains C3, C4 and C5, the corresponding type strains C. perfringens DSM 756 and C. sphenoides DSM 632 and C. indolis DSM 755 were cultured in reinforced elostridial medium (RCM) (Atlas and Parks, 1993) at 30°C in an anaerobic chamber under 85 % nitrogen, 10% hydrogen and 5% carbon dioxide atmosphere.

Identification Morphological characteristics

After macroscopic inspection of the colonies, morphological properties, Gram reaction, motility, sporulation and the presence of parasporal crystals were examined with the phase contrast microscope. The Gram reaction was observed on very young cultures (6-12-h old). For anaerobic strains, spomlation tests were performed after 10-20-30 days of incubation in chopped meat agar (Holdeman et aL, 1977). Biochemical characteristics

For aerobic cultures of strain CI and the corresponding reference strain, conventional tests including catalase, lecithinase and gelatinase reaction, citrate and propionate utilization, nitrate reduction, anaerobic production of gas from nitrate, formation of indole, Voges-Proskauer reaction and growth in the presence of lysozyme were performed according to Claus and Berkeley (1986). Carbon source utilization was performed using the "API-50-CI-IB" system (APl system, Bio-Merieux) according to the instructions of the suppliers. Tests were conducted at 37*(:: in aerobic conditions and readings were performed at 18-24 and 48 h. For anaerobic cultures of strains C3, C4 and C5 and the corresponding reference strains, conventional tests included production of catalase, gelatinase, lecithinase, lipase, indole, H2S and nitrate reduction. Tests were carried out according to Cato et al. (1986). The acid production from carbohydrates was determined in culture tubes using PY broth (Atlas and Parks, 1993) as basal medium. The carbon sources, sterilized separately by filtration,

PYG RCM SDS SSC

= =

peptone/yeast extract/glucose (medium). reinforced closzridial medium.

= sodiumdodccy|sulphate. = salinestandardcitrate.

SPORE-FORMING STRAINS IN BIODEGRADATION OF A CIFLUORFEN

were added to the sterile basal medium to give a final concentration of 0.2 %. Alternatively, the "APi20-A" system was employed. Tests were incubated at 30°C under anae'obic conditions and readings were performed at 18-24 and 48 h. Preparation o f DNA

The aerobic and anaerobic cultures were grown respectively in nutrient broth and RCM broth. Bacterial cells were harvested by centrifugation at the end of the log phase of growth, washed using a 0.5 % SDS/50 mM EDTA (pH 8) solution, subsequently washed with distilled water and twice with 0.15 M NaCI/0.1 M EDTA pH 8. DNA was isolated by the method of Marmur (1961). DNA base composition

The guanine-plus-cytosine (G +C) content of the DNA preparations was determined by the thermal melting point (Tin) method of Marmur and Dory (1962) using a "Gilford Response" spectrophotometer (Ciba Coming Diagnostics Corp., OH) fitted with a circulating temperature bath. The DNA of Escherichia coil strain B (Sigma, St. Louis, MO), containing 50,9 tool% G+C, was used as internal standard (Manachini et aL, 1985).

DNA-DNA hybridization

The extent of D N A reassociation was determined spectrophotometdcally according to the procedure of Seidler and Mandel (1971), Seidler et al., (1975) and Kurtzman et al. (1979). The reaction was carried out under optimal conditions (25°C below the melting temperature) in 5xSSC buffer (IxSSC is 0.15 M NaCI plus 0.015 M sodium citrate) containhag 20% dimethylsulphoxide (DMSO). Plasmid extraction

For detection of plasmid DNA, the alkaline extraction procedure described by Maniatis et al. 0980) was followed. Agarose gel electrophoresis, staining and evaluation of gels were performed as described by Maniatis et al. (|989), Molecular weight of the plasmids obtained was determined from standard curves with a supercoiled DNA ladder (Gibco-BRL, UK) containing 16.21, 14.17, 12.14, 10.10, 8.06, 7.04, 6.03, 5.01, 3.90 and 2.97kb plasmids. For further analysis of CCC and OC forms of plasmids, second dimensiolt electrophoresis of the plasrnid DNA was performed according to Hintermann et al, (1981).

195

Table I, Carbon source utilization by aerobic strains, Utilization Amyg "daline Arabinose Arbutine Cellobiose Esculin Fructose Gentiobiose Giuconate Glucose Glycerol Glycogene lnositol Maltose Mannitol Mannose N-acetyl-glucosam. Ribose Salicin Sorbitol Sucrose Tagatose Trehalose

Reaction given by B. thuringiensis Strain C 1 DSM 2046 -/+ + + + + + + + + -/+ + -/+ +/+ + + -/+ + +

+ + + + + + + + + +/+ + + + + + + + + + + +

The strains did not utilize adonitol, arabitol, chcto-gluco~ nate, erythritol, fucose, galactose, inuline, lactose, lyxose. melezitose, melibiosc, raffinose, rhamnose, s0rbosc, turanose. xylitol or. xylose.

RESULTS AND DISCUSSION

Identification of the aerobic strain CI Strain C 1 was an aerobic, Gram-positive, motile microorganism. The cells were rod-shaped and 1.0 to 1.8 by 2.5 to 5.0 pan, and occurred singly and in short chains. Strain C I was sporeforming: the endospores were oval with sporangia not d e f i n i t e l y s w o l l e n in a s u b t e r m i n a l position, Parasporal crystals, in parallel with spore formation, were easily visible by phase contrast microscope. Table I shows the sugar utilization profiles of strain C1 and reference strain B. thuringiensis DSM 2046. Other characteristics of strain C 1 were as follows: catalase positive, anaerobic growth in glucose broth, citrate utilized, gelatin

M.G. FORTINA ET AL.

196

Table II. Fermentation profiles of anaerobic strains.

Fermentation Arabinose Ceilobiose Esculin Fructose Glucose Inositol Lactose Maltose Mannitol Mannose Melezitose Raff'mose Rhanmose Ribose Salic'~n Sorbitol Sucrose Trehalose

Xylose

Reaction given by Strain C3

C. perfringens

(7. sphenoides

DSM 756

DSM 632

+/+ + + +/+ + + + + +/+ + -

-/+ + + + + ND + + + + -/+ + + ND + -/+ + +

+ + + + + + + + + + + + +/+ + + + +

+I-

Strain C4

Swain C5

+ + + + + ND + + + + + + ND + + +

+ + + + + ND + + + + + + ND + + +

ND=not determined.

hydrolysed, Voges-Proskauer positive, arginine dihydrolase-produced, growth in presence o f lysozyme, indole not formed, nitrate reduced to nitrite, lecithinase and lysine decarboxylase not produced. A DNA G + C m o l % of 35.2 was found for this strain. Strain C1 showed 92% DNA relatedheSS with the reference strain of B. thuringiensis and 0 % relatedness with control D N A f r o m C. indolis DSM 755 T, chosen as the unrelated organism.

meat agar slants incubated at 30 and 37*(2 for 2-3 weeks, but the strain resisted heating at 80°C for 10 rain and after 45-min treatment with an equal volume of 95 % ethanol. These characteristics suggested the presence of spores, responsible for resistance to heat and to alcohol (Cato et al., 1986). C u l t u r e s in P Y G w e r e t u r b i d w i t h a smooth sediment; abundant gas w~': produced in d e e p agar cultures. O p t i m a l temperature for growth was between 30 and 45°C. There was n o growth at 25*{2 and little or none at 50°C.

Identification of the anaerobic strain C3

Table II shows the sugar fermentation profiles of strain C3 and reference strain C. perfringens DSM 756. Other characteristics were as follows: indole not reduced, nitrate reduced, lecithinase produced, gelatin hydrolysed, lipase not produced, resazurin poorly reduced.

Ceils of anaerobic strain C3 in PYG broth culture were Gram-positive, non-motile rods that occurred singly and rarely in short chains, and were 0.9 to 2.0 by 2.0 to 5.0 llm. Spomlation did not occur either in PYG broth or on chopped

A DNA G + C m o l % o f 27.8 was found for this strain, and 98% DNA relatedness with the type strain of C. perfringens. The control DNA f r o m (7. indolis DSM 755 T s h o w e d only 6 % relatedness with strain C3. On the basis of these data, strain C3 was identified as C perfringens.

The morphological, biochemical characteristics and DNA hybridization identified the swain with B. thuringiensis.

SPORE.FORMING STRAINS IN BIODEGRADAT:ON OF ACIFLUORFEN

A

Identification of the anaerobic strains C4 and C5 The two anaerobic strains designated C4 and C5 showed the same phenotypie characteristics. Cells in PYG broth culture were motile rods, 0.3 to 0.9 by 1.5 to 7.5 IJan, and occurred singly or in short chains. They were Gram-negative within 8-10 h. Sporulation occurred readily on chopped meat agar slants incubated at 30°C for 3--4 days. Spores were oval, subterminal or terminal, with sporangia not definitely swollen. Cultures in PYG broth were turbid and gas was detected in deep agar cultures. Optimal temperature for growth was 30--37°C. There was little growth at 25 and 450C, Table II shows the sugar fermentation profiles of strains C4 and C5 and reference strain C sphenoides DSM 632. Other characteristics were as follows: indole reduced, leeithinase and lipase not produced, nitrate not reduced, gelatin not hydrolysed, resazurin poorly reduced. A DNA G + C tool% of 41.5 and 42.1 was found for strains C4 and C5, respectively. Strains (24 and C5 showed 96 and 97 % DNA relatedness. w..slx~tively, with the type strain of C. sphenoides. C. indolis DSM 755 T, which phenotypically most closely resembles C. sphenoides, was shown genetically to be only partially related, showing 30% DNA relatedness with the type strain of C. sphenoides and 28 and 32% relatedness with strains (24 and C5, respectively. Biochemical tests and DNA hybridizati(,n results identified the two strains (24 and C5 with C. sphenoides. Plasmid DNA content We detected plasmid bands in two degrading strains. Strain C 1 showed the presence of four plasmids in the CCC form, with molecular weights of 2.05, 2.2, 7.0 and 11.7 kb; strain C3 showed six CCC molecules of ] .5, 1,75, 8.4, 10.5, 12.0 and 14.5 kb. Figure 1 shows the electrophoretic pattern of the plasmid DNA isolated from strains C1 and C3, and figure 2 shows an example of second dimension eleetrophoresis of plasmid DNA from strain C3. The possible role of these plasmids in the degradation of aeifluor-

[3

C

: ::! %::11.

~

,,

-

197

~'!i/¸

~

.:.~

~,~.

:

Fig. 1, Agarose gel eleetrophoresis of purified plasmid DNAs. Lane A ffireference mobility plasmids; lane B ffiplasmid DNA from B. ~huringiensis strain C1 ; lane C = plasmid DNA from C. perfringens strain C3; chrfchromosomal DNA ; the numbers refer to CCC form of plasmid DNAs, as performed by two-dimensional eleetrophoresis.

fen is not yet clear. We are at present carrying out curing experiments to correlate the presence of plasmids with specific phenotypie traits. As for strains (24 and C5, which differed only in the ability of strain (24 to metabolize acifluorfen, we presumed that this ability was correlated with the presence of extrachromosomal dements. However, both strains were plasmid-free. Thus, in this case, the ability of strain C4 to degrade the herbicide was probably harboured at the chromosomal level.

Identification de souches ~porog~nes agents de la biod~gradation de I'acifluorfen Nous avons isol6 et identifi6 quatre bact6ries sporog~nes ~t partir de boues actives et de terrain, dont trois capables de d6grader l'aeifluorfen.

M.G. FORTINA ET AL.

198

FIRST DIMENSION SECOND

DIMENSION

p l 1. -c,.

Pl 2--I> p l 3 .--~> p14 -..l>

05 .-t> 0 6 --~

p15 -~> p16

Fig. 2. Banding pattern of plasmid DNA from C. perfringens strain C3 after two-dimensional agarose gel electrophoresis. Symbols on the left side refer to the six plasmids in the CCC form (from pl ! to pl6) and to the OC form (0~, 0 6) of the corresponding pl5 and pl 6 plasmids, as confirmed by second dimension eleetrophoresis. The symbol 0* refers to the OC forms newly originated from CCC forms (C) after UV irradiation of the correspor, Jing plasmid (pl) originally present in the non-irradiated plasmid DNA preparation.

Les caract6ristiques biochimiques, la composition en bases de I'ADN et l'homologie ADN-ADN indiquent que les souches biod6gradantes appartiennent aux espcSces Bacillus thuringiensis, Clostridium perfringens et Clostridium sphenoides. La quatfi~me souche identifi6e comme C. sphe. noides et ayznt les m6mes caract&istiques que celles de la souche correspondante mais active, n'6tait pas capable de mEtaboliser l'acifluorfen. On a par consequent analyse le contenu en plasmides des souches dans le but d'Etudier l'6ventuelle corr61ation entre la presence d'E1Ements extrachromosomiques et la capacitE de degrader cet herbicide.

Mots-cl~s : Acifluoffen, Biod6gration, Bacillus thuringienis, Clostridium perfringens, Clostridium

sphenoides; Identification, Plasmides, Spores, Herbicide.

References

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