A NEW FACULTATIVE ANAEROBE CAPABLE OF GROWTH ON CHLOROBIPHENYLS MICHEL SYLVESTRE AND JOHANNE FAUTEUX

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1 J. Gen. App!. Microbial., 28, (1982) A NEW FACULTATIVE ANAEROBE CAPABLE OF GROWTH ON CHLOROBIPHENYLS MICHEL SYLVESTRE AND JOHANNE FAUTEUX Centre de recherche en bacteriologie, Institut Armand-Frappier, Universite du Quebec, C.P.100, Laval, Quebec H7N 4Z3 (Received September 25, 1981) Strain B-206 is a new facultative anaerobe, non flagellated, gram negative rod, able to grow on 4-chlorobiphenyl (4CB) and to degrade it. When this strain was grown on 4CB as sole growth substrate, two types of cultures were obtained; either the culture became pink or yellow with the formation of a black pigment. The cultures turning yellow were the more active ones and they were obtained with a large inoculum size or low oxygen tension. 4-Chlorobenzoic acid (4CBA) was detected in both pink and yellow cultures but in much higher concentration in yellow cultures. This compound appeared to be the ultimate metabolite of 4CB in both types of growth since strain B-206 could not grow or degrade 4CBA and that the concentration of 4CBA did not decrease even after the appearance of the yellow compounds. The presence of 4CBA and yellow compounds that differs spectrophotometrically at acid and basic phs suggest that 4CB is degraded via a meta cleavage between the 1 and 2 position. The absence of any other metabolites than a pink pigment and 4CBA in the pink cultures provides no evidence that in this type of culture there would be some other mechanism of cleavage of the biphenyl molecule. In the yellow culture type, there was also formation of a black pigment. This pigment, like the pink compound, possessed all the characteristics of the phenolic type pigments. Strain B-206 could also grow on biphenyl, 3-chlorobiphenyl, 2-chlorobiphenyl, benzoic acid but it was unable to grow on any of the monochlorinated benzoic acids. Although this strain could not grow on Aroclor 1254, we have evidence that it is partially transformed by this strain. Polychlorinated biphenyls (PCB's) are a matter of concern for our ecosystem. (1-3). In this respect, any information on their biodegradation is of outmost importance. Several strict aerobic bacterial strains have already been described as being able to oxidize low chlorine containing PCB isomers. These bacteria include members of the genus Pseudomonas (4, 5), Achrornobacter (6), Acinetobacter (7), Alcaligenes (7, 8) and Nocardia (4). 61

2 62 SYLVESTRE and FAUTEUX VOL. 28 Using the technique of SYLVESTRE (9) we isolated from different environments several bacterial strains capable of growth on 4-chlorobiphenyl (4CB). Most were strictly aerobic gram negative rods. Strain B-206, isolated from an activated sludge sample was a facultative anaerobic bacterium which grew on 4CB with the production of yellow intermediates and black pigments. This research was carried to characterize this strain. Microorganisms. MATERIAL AND METHODS Strain B-206 was isolated from an activated sludge sample (9). The strain was kept lyophilized in bovine serum until required. It was propagated on nutrient agar slants or in liquid medium No. 30 (9) to which was added trypticase 0.4 %, yeast extract 0.01 % and glucose 1 %. As it will be seen in the results, 4CB could not be used as growth substrate in the propagation medium. Chemicals. The 4CB was purchased from Aldrich Chemical Co. (Milwaukee, Wisc.), the ortho chlorobiphenyl (2CB) from Pfaltz and Bower (Stanford, Conn.), the meta chlorobiphenyl (3CB) from ICN Pharmaceuticals Inc. (Cleveland, Ohio). Samples of purified 2CB, 3CB and 4CB from Analabs (North Haven, Conn.) were also used. Aroclor 1254 was graciously provided by Mosanto Chemical (St. Louis, Mo.). All other compounds including biphenyl (BP), benzoic acid (BA), 2-chlorobenzoic acid (2CBA), 3-chlorobenzoic acid (3CBA) and 4-chlorobenzoic acid (4CBA) were from Aldrich Chemical Co. Taxonomical characterization of strain B-206. Detection of flagella was done by electron microscopy from a culture on SIM medium (Difco, Detroit, Mich.) incubated 48 hr at 18. This same medium was used to test for indole and H2S production. Tests for oxidation and fermentation of sugars were performed using Difco O.F. basal medium. The test for denitrification was as described by Stanier et al. (10). The oxidase test was described by HUGH (11). Catalase was demonstrated by the addition of peroxide to a culture of B-206 grown on mannose. The patch method was used for the determination of extracellular enzyme production. Starch hydrolysis was tested by flooding trypticase soy agar plates supplemented with 1 % (w/v) of starch with Lugol iodine after 48 hr of growth. Production of lipase was tested following the technique of SIERRA (12). Proteolytic activity was determined by liquefaction of gelatin in nutrient gelatin and digestion of casein using TSA plus 10 % (w/v) skim milk powder. Detection of cellulase was done by the technique described by DAIGNEAULT-SYLVESTRE and KLUEPFEL (13). Cultivation of strain B-206 on 4CB and other substrates. The basal liquid minimal medium No. 30 (MM No. 30) was used for all these assays. The desired amounts of growth substrate were weighed directly into a sterile 125 ml Erlenmeyer flask, then 25 ml of medium No. 30 containing 0.01 % yeast extract were added. In most of the experiments the inocula were prepared from 18 hr old cells

3 1982 A New Facultative Anaerobe Capable of Growth on Chlorobiphenyls 63 grown in the same medium No. 30 with added yeast extract (0.01 %), glucose (1 %) and trypticase (0.4%). The grown cells were washed and the suspension adjusted spectrophotometrically to the desired concentration. The cultures were incubated at 29 with agitation (250 rpm) under normal atmosphere or in a controlled environment incubator shaker (New Brunswick) adjusted between 5 to 12 % C02 in air. For the anaerobic growth tests, the media were transferred under a gas mixture of 10 % C02-10 % H2-80 % N2 immediately after autoclaving. Inoculations and incubations were carried out under the same gas mixture. Bacterial platings, when required, were made on nutrient agar. Estimation of cell activities and characterization of metabolic intermediates from 4CB. Cultures grown on 4CB were filtered through a Whatman paper No. 40 to remove residual growth substrate. The cultures were then acidified to ph 1.2 with nitric acid and immediately extracted with ethyl acetate. The extract was then dehydrated on ammonium sulfate, concentrated by evaporation in vacuo and chromatographed on silica gel by TLC using one of the following development systems: Benzene-dioxane-acetic acid ( ) (BDA); benzene-acetic acid (1001) (BA). The disappearance of the growth substrate or the appearance of metabolic intermediates were quantified by gas chromatographic analysis. The residual 4CB from the culture retained on the filter paper was injected in the chromatographic column as an ethyl ether solution. The intermediates were extracted from the TLC plates with methanol, then evaporated and solubilized in a known amount of ethyl ether for injection on the GLC column. The column used was a 2 m by 2 mm I.D. glass column packed with 3 % SE 30 on 80/100 Supelcoport from Supelco Inc. (Bellafonte, PA). The chromatograph was a Tracor model 220 gas chromatograph and the oven was programmed between 50 and 230. The disappearance of growth substrates and the appearance of intermediates were quantified by integrating the area under the corresponding peaks. RESULTS Identification of strain B-206 Strain B-206 is a non sporulating, non flagellated gram negative rod (0.5x 1.5 to 3 µm). It occurs singly or in short chain of two or three cells. In nutrient broth, it grew at 15, and 37 but not at 4 and 42. The optimum growth temperature was 29 but good growth was obtained at 37 in nutrient broth. On SIM medium, H2S is produced, but no indole. The organism is oxidase and catalase positive and gave a strong urease reaction. None of the following extracellular enzymes as detected by the tests referred to in MATERIAL AND METHODS, including RNase, DNase, cellulase, caseinase, gelatinase and lipase were produced. Gas was produced from nitrate. Finally, the test for oxidation or fermentation of sugars on OF medium were negative when the tubes were read after less than 10 days. Except for the motility, these characteristics are those of the bacterial

4 64 SYLVESTRE and FAUTEUX VOL. 28 group IVe isolated from urine specimens (14). However, when the glucose containing OF media were incubated for period exceeding 10 days, the media turned slightly yellow after approximately 14 days of incubation either aerobically or anaerobically. When these cultures were transferred in identical media and incubated under identical conditions the medium turned yellow in a few days suggesting a faster transformation of the glucose (either oxidative or fermentative depending on the growth conditions). Therefore, it appeared that strain B-206 could oxidize or ferment glucose although very slowly. The strain grew well on medium No. 30 containing glucose 1 %, trypticase 0.4 and yeast extract 0.01 %, whether aerobically, anaerobically or under 5 % CO2 in air. When strain B-206 was inoculated from nutrient broth to medium No. 30 containing glucose alone (without trypticase or yeast extract) a lag period exceeding two to three weeks was required before the log phase of growth started. However, by successive transfers on this same medium the lag period was reduced to a few hours. Thus, although yeast extract and trypticase stimulated growth on glucose or 4CB, there was no need for a growth factor. Experiments for oxidation-fermentation of sugars on OF medium were then made with such an adapted strain on glucose and this strain was able to oxidize and ferment trehalose, xylose, sucrose, dextrose and maltose but not rhamnose, raffinose, arabinose or inositol. Sugar fermentation is not aerogenic. There is no corresponding description of a similar facultative anaerobe in the Bergey's Manual of Determinative Bacteriology (8th edition). It differs from Klebsiella and Shigella in being oxidase positive. In several respects it resembles the genus Plesiomonas except that it is non motile, it is slightly positive for H2S, it is resistant to the vibriostatic agent 2, 4-diamino-6, 7-diisopropyl pteridine (0/129) and it does not produce indole. Growth of B-206 on 4CB Strain B-206 could grow on 4CB and degrade this substrate. It was also noticed that colored intermediates and pigments were released in the medium during growth. The growth pattern on 4CB was however very erratic. Sometimes the culture would turn yellow in the first 24 hr of growth (Fig. 1, curve b), in this case the growth would proceed without a lag period and the cell yield was high; there was also production of a black pigment after 72 hr to 120 hr of growth. Other times, the culture would turn pink after 24 to 48 hr of growth. These pink cultures grew much slower (Fig. 1, curve a). The influence of various other factors on the biodegradability of 4CB by strain B-206 was also evaluated. We did not notice any effect of the growth substrate concentration up to 1 % (w/v) or of the addition of co-substrates (lactate, succinate, acetate) on the biodegradability of 4CB. The addition of yeast extract (0.01 %) stimulated the growth of B-206 on 4CB although at this concentration, yeast extract alone could not support the growth of the organism. When the culture was started with less than 1 x 108 cells/ml, and it was incubated in shake flask under normal atmosphere, the growth

5 1982 A New Facultative Anaerobe Capable of Growth on Chlorobiphenyls 65 Fig. 1. Growth of strain B-206 in conditions giving yellow or pink cultures. Two cultures were started from the same inoculum as described in material and methods. One culture was incubated with agitation under normal atmosphere to give a pink culture (curve a). The second culture was incubated with agitation under 7 % CO2 in air (curve b) resulting in a yellow culture type. The amount of yellow compounds (curve c) in the yellow culture was evaluated in arbitrary units from the absorption an ethyl acetate extract of the culture at 412 nm. curve was characterized by an initial drop of viable cell counts as seen in Fig. 1 (curve a), followed by an increase of approximately 1 to 2 log, and the medium would turn pink. Degradation of 4CB occurred in the first 24 hr of growth (Fig. 2) and almost no degradation was noticed afterward. In this case, production of 4CBA was very low. With a larger inoculum or in shake flask under 5 to 12 C02 enriched atmosphere, the cell numbers would immediately start to increase, and the culture became yellow within a few hours. There was a higher degradation rate of 4CB than in pink cultures, and the production of 4CBA was much higher (Fig. 2). The ph of the medium affected the growth response of strain B-206 to 4CB. At lower ph values, 6.6, 6.0, the strain grew poorly and a faint pink color appeared after a long period of incubation. At higher ph values 7.4, 7.8 and normal atmosphere and with an inoculum size higher than 1 x 108, the cultures turned pink within 24 hr. The optimum ph for growth on 4CB with production of yellow intermediates was between 6.9 and 7.2. Finally it was of

6 66 SYLVESTRE and FAUTEUX VOL. 28 INCUBATION PERIOD (HOURS) Fig. 2. Degradation of 4CB and production of 4CBA in yellow and pink cultures. Duplicate cultures containing mm 4CB were incubated in conditions giving the yellow (a) or pink (.) culture type and the disappearance of 4CB (-) 4CBA (---) were measured by GLC as described in the text. and appearance of noticed that, unexpectedly the cells lost their ability to grow on 4CB with repeated transfers on this substrate. For this reason, nutrient agar was chosen as propagation medium for this strain. Characterization of the yellow and pink cultures Since strain B-206 grew differently on 4CB depending on the growth conditions, we compared the intermediates in both types of cultures. Fig. 3 illustrates the TLC's on BDA system of ethyl acetate extracts from acidified culture medium. The rapid appearance of 4-chlorobenzoic acid (4CBA) in both types of culture was confirmed by GLC analysis (Fig. 2). The pink cultures did not appear to produce any yellow intermediates but contained a pink colored pigment which was very similar to the black pigment in the yellow cultures. The yellow cultures contained at least three different yellow compounds which were separated by TLC (Fig. 3). A yellow compound with an Rf of 0 and no characteristic absorption spectra appeared early in the cultures. Then, two other yellow compounds were detected in the growth medium of the 72 hr old culture. These last two compounds had a maximum peak of absorption between 400 and 450 nm in basic solutions and their rate of appearance is shown in Fig. 1(curve c).

7 1982 A New Facultative Anaerobe Capable of Growth on Chlorobiphenyls 67 Fig. 3. Comparison of TLC's from extracts of yellow and pink cultures of strain B Ethyl acetate extracts from acidified cultures were spotted on silica gel plates containing fluorescein and developped in the BDA system. (Y) yellow spot; (B) brown or black spot; (Y') spots carrying both 4CBA and a yellow compound. Characterization of the black pigment After 72 hr, the yellow cultures started to darken, first becoming brown then deep brown and black after 15 to 20 days of incubation. This pigment had characteristics similar to those described by HALLER and FINN (IS) which were produced by a Pseudomonas strain growing on 3CBA. The substance was decolorized by 30% H2O2 or an hypochlorite solution. The addition of FeC13 to an aqueous solution of the pigment gave a dark flocculent precipitate. It was partly soluble in some organic solvents (butanol, ether, ethyl acetate) from acidified solutions but insoluble in less polar solvents such as hexane, it gave a positive reaction for polyphenols and it was retained on Amicon PM 10 membrane which is selective for compounds with molecular weights higher than 10,000 dalton. The color became lighter in acidic solution and the pigment precipitated at basic ph. When B-206 was grown on BP a similar black pigment was also formed but on benzoic acid there was no such pigment formation. Growth of strain B-206 on other aromatic compounds In shake flasks at 29 with a large inoculum size, strain B-206 could grow on 2-chlorobiphenyl (2CB) and 3-chlorobiphenyl (3CB) (Fig. 4) with the formation of colored intermediates. However, in both cases the growth was less than on 4CB. When mixtures of monochlorinated biphenyl compounds were used as growth substrates, there was an inhibition of growth, even if the final concentration of the mixture of these substrates was equal or less to that used when each was added individually to the culture. Hence the addition of BP (0.125%) plus 4CB (0.125%) as growth substrates using optimal growth conditions was inhibitory for the cell growth (Fig. 5). The same was true for the mixture 2CB+3CB -4CB.

8 68 SYLVESTRE and FAUTEUX VOL. 28 Fig. 4. Growth of strain B-206 on 2CB and 3CB. Strain B-206 was inoculated (1 x 108 cells/ml), as described in the text, in basal medium No. 30 containing 0.01 % yeast extract and 0.25% 2CB (---) or 3CB (-) as sole growth substrate. Aroclor 1254 also had an inhibitory effect on B-206 (Fig. 5). There was no growth and no biodegradation at low inoculum size, but at large inoculum size (ca. 10$ cells/ml), the culture turned yellow although there was no growth and it was shown by GLC that there was ten percent lost in the peaks height after 10 days. Growth occurred on benzoic acid (0.0250) in MM No. 30 with no colored intermediates released. In this same medium No. 30 plus glucose, the minimal inhibitory concentration (m.i.c.) for benzoic acid was 0.5 % and for the chlorinated benzoic acids it was 5 %. These concentrations were insufficient to bring any change in ph of the medium. However, no growth ever occurred on any of the three monochlorinated benzoic acid isomers, even at concentrations 1,000 times less than the m.i.c. DISCUSSION At first sight, from its phenotypes reported here, strain B-206 appeared close

9 1982 A New Facultative Anaerobe Capable of Growth on Chlorobiphenyls 69 Fig. 5. Growth of strain B-206 on mixtures of chlorinated biphenyls. The cultures were prepared as in Fig. 4 except that the growth substrates were either 0.125% 4CB % BP (0); 0.075% 2CB % 3CB % 4CB (s); 0.1 Aroclor 1254 mixture (R). to the bacterial group IVe as described by TATUM et al. (14). This bacteria, generally isolated from urine specimens are gram negative short rod; most of the strains are motile; they are oxidase and catalase positive and they give a strong urease reaction; they are able to reduce nitrate to gaseous nitrogen; growth on OF medium with added carbohydrates is very light and considered absent; indole is not produced and gelatine is not liquefied. However, we observed that upon appropriate transfers, strain B-206 was able to ferment and oxidize some carbohydrates, including glucose, xylose, sucrose, trehalose and maltose. There is no reported data on the ability of group IVe to ferment or oxidize any of these sugars. On the other end, strain B-206 appear different from all other facultative anaerobes described in the Bergey's Manual of Determinative Bacteriology. Thus, it might

10 70 SYLVESTRE and FAUTEUX VOL. 28 be a variant of the bacterial group VIe that is able to grow in anaerobic conditions after an adaptation period. YAGI and SUDO (8) postulated that, besides a meta cleavage between the 1 and 2 positions of the PCB molecule, already observed by FURUKAWA et al. (7), ortho cleavage between the 2 and 3 positions or 3 and 4 positions may also occur in Alcaligenes leading to chlorinated phenylacetic acids. However beside the pink colored pigment, 4CBA was the only metabolic intermediate detected in our pink cultures which suggests a meta cleavage between the 1 and 2 positions. An explanation for the initial drop in the viable count of the pink culture type would still be speculative, but it is most probably linked to 4CB itself or the production of a toxic metabolite of 4CB that appears faster and/or in larger amounts in aerobic conditions. The presence of 4CBA in the yellow type of culture, also suggest that 4CB is cleaved by a meta fission between the 1 and 2 positions. Like the yellow compounds isolated by FURUKAWA and MATSUMURA (16), two of our yellow intermediates had an absorption maximum between 300 and 350 in acidic solutions and between in basic solutions. Yet, we have however no further evidence that one of these compounds is the 4-chloro-2-hydroxy-6-oxo-6-phenylhexa- 2, 4-dienoic acid which is the expected intermediate produced from the meta cleavage between the 1 and 2 positions (17, 18). There are several lines of evidence which indicate that 4CBA is the ultimate metabolic product of this degradative pathway. Hence, all attempts to have 4CBA degraded by strain B-206 were unsuccessful; even a concentrated cell suspension previously grown on 4CB could not degrade it. Moreover, we were unable to detect any deshalogenase activity of cells grown on 4CB using BERGMANN and SANIK (19) chloride assay or a chloride electrode. It can be further noticed that 4CBA concentration in the growth medium never decreased during growth on 4CB even after 48 hr, at the time of appearance of the yellow intermediates. Strain B-206 also produced a black pigment in the yellow cultures. This pigment possesses all the characteristics of pigments resulting from the oxidative polymerization of diols as described by NICOLAUS et al. (20) and by HALLER and FINN (15). It is likely that the monomers, which polymerized to produce the black pigment in the yellow culture, are intermediates between 4CB and 4CBA rather than chlorocatechol since B-206 produced this pigment when grown on 4CB or BP but not on benzoic acid and that we have no evidence that the degradation goes further than 4CBA. For this same reason it does not seem to be protocatechuate or chloro-protocatechuate which might be expected as intermediates in bacteria metabolizing benzoate by the meta fission pathway (21) and which do polymerize readily to a black pigment (22). Moreover strain B-206 was unable to grow on protocatechuate which should be the case if this compound was an intermediate, while it did grow on 2, 5-dihydroxybenzoate.

11 1982 A New Facultative Anaerobe Capable of Growth on Chlorobiphenyls 71 It was noticed that strain B-206 could degrade Aroclor 1254 mixture very slowly but that it could not grow on this substrate. It is likely that this observation is due to a toxicity of Aroclor 1254 mixture for that strain. As a matter of fact, we showed that simple mixture of monochlorinated biphenyls where inhibitory for the growth of the cells. This observation is interesting with respect to the development of mutants with greater capacity to degrade PCB's. Such an inhibitory effect of mixture of chlorinated biphenyls has not yet been reported for any other bacterial strain able to degrade PCB's. This behavior would be explained if there were several pathways involved in the degradation of these compounds. In this respect, DORN and KNACKMUSS (23) described two types of pyrocatechase in Pseudomonas sp. B13. The first one being specific for catechol, the second, inducible by 3CBA was active on catechol or chlorocatechol. No studies of the enzymes involved in the PCB degradation pathway have yet been reported, but the recent work of YAGI and SUDO (8) seems to indicate that the Alcaligenes strain they studied did possess several degradation pathways, which is coroborated by the works of TRECANNI et al. (24) and BAILY and MCKENZIE (25) who showed the presence of both 1, 2 and 2, 3-catechol dioxygenase in two species of Pseudomonas. The unexpected loss of the ability of strain B-206 to grow on 4CB after a series of transfers on this substrate could also be explained by the existence of different pathways for the degradation of 4CB and BP. There is only one example in the literature (26), still unexplained, whereby adaptation of a bacterium to a substrate would lead to the loss of the ability of that bacterium to degrade it, and it concerns the biodegradation of 3CBA. This work was supported by a grant from the Institut Armand-Frappier. 1) 2) 3) 4) S) 6) 7) 8) 9) 10) 11) 12) REFERENCES M. GILBERTSON, A report of task force, to the Canadian Dept. of Environment and National Health and Welfare (1976). 0. HUTZINGER, S. SAFE, and V. ZITKO, The Chemistry of PCB's, CRC Press, Cleveland (1974). R. W. RISEBROUGH, P. RILCHE, D. B. PEAKALL, S. G. HERMAN, and M. N. KIRVEN, Nature (London), 220, 1098 (1968). R. A. BAXTER, P. E. GILBERT, R. A. LIDGETT, J. H. MAINPRIZE, and H. A. VODDEN, SCI. Total Environ., 4, 53 (1975). G. S. SAYLER, M. SHON, and R. R. CoLWELL, Microbial Ecol., 3, 241 (1977). M. AHMED and D. D. FOCHT, Can. J. Microbiol.,19, 49 (1973). K. FURUKAWA, K. TONOMURA, and A. KAMIBAYASHI, Appl. Env. Microbiol., 35, 223 (1978). 0. YAGI and R. SUDO, J. Water Poll. Cont. Fed., 52,1035 (1980). M. SYLVESTRE, App. Env. Microbiol., 39, 1223 (1980). R. Y. STANIER, N. J. PALERONI, and M. DOUDOROFF, J. Gen. Microbiol., 43, 159 (1966). R. HUGH, Tech. Improv. Serv., No. 17, p. 48 (1974). G. SIERRA, Antonie van Leeuwenhoek J. Microbiol. Serol., 23, 15 (1957).

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