Isolation and characterization of surfactant degrading bacteria in a marine environment

FEMS MicrobiologyEcology73 (1990)50-68 Publishedby Elsevier

59

FEMSEC00234

Isolation and characterization of surfactant degrading bacteria in a marine environment Jean-Claude Sigoillot and Marie-H616ne Nguycn Lab~tatoire de Mwrohmlogie. Facult~ des Sciences et Techmq~es de Smm-Jdr~me, Marseille. France

Received28 October 1988 Rewsionreceived26 April1989 Accepted 5 May 1989 Key words: Surfactant: Marine environment: Biodegradation: Poflution; Aerobic heterotrophic bacteria

1. SUMM~RY Sea water a n d sediment samples taken near Ihe coasts of Hyeres bay (France) were used for anionic surfactant titradons with surface and bottom waters and the finest part of sediments. The capacity for surfactant degradation by the 'in situ' microflora was evaluated, By using a selective plating technique 26 strains able to utilize anionic surfactant were isolated from the selected bacterial communities. Their ability to degrade anionic surfactant was verified according to the bioticgradation standard method. Isolated strains were characterized by morphological and physiological properties using API 20 NE micro-method. All tested strains were Gram negative, strictly aerobic. rod or helical shaped. Their weak utilization of phenolic sobstrates suggests that they degrade preferenlially the alkyl chain of the surfaetant molecule. Biodegradation was more efficient with baclerial communities rather than with any iso-

Correspondence to," J.C. Sigoillot,Laboratoir¢de Microbiologi¢, Facult6 des Sciences ct T0chniques de Saint-J6rbmc, Avenue

F.scadfill~ Nor'a~ndie-Nicmcn, 13397 Marscillo Cedes 13, France.

lated strains, Such observations indicate that complete mineralization involves several other so far non-isolated strains which complete the degradation initiated by the isolated strains.

2, INTRODUCTION Anionic sudactants are widely used as the active part of washing products [1]. The most common types are alkylbenzene sulfonates which appear in urban sewage flowing to the sea {2]. These anionic sarfactants arc hazardous for the aquatic environment [31. On the basis of O.E.C.D, recommendations [4] currently commercialized products muSl show primary biodegradability higher than 80'~. However, biodegradability tests are condueled on river walers or activated sludges. They are carried out with microorganisms from waste water at high densities, which are then particularly adapted [5]. In the marine environment, except for work done on non-ionic dispersants used to fight occasional oil pollution [6], little has been done on the biodegradation of anionic surfactants in the sea. A study on the Cadix bay [7] showed degradation of these products during 3-week periods. Tempera-

0168-6496/89/$03.50 ~ 1989 Federationof European MicrobiologicalSocieties

lure was found to be the most important factor. Degradation is in fact totally stopped when the temperature falls under 12-1d°C in the surface waters. However, these authors give no indications on the degradation mechanism. As in river waters [8], chronic pollution can induce a microflora adapted to anionic surfactant biodegradation. Therefore, our study was done on a littoral area receiving urban sewage exclusively. In such an environment, the mieroflora can originate from the marine environment or sewage. According to Larsen [9], a marine origin of bacteria is charnotarized by their sodium requirement which is specific and independent of osmotic function because marine strains cannot grow in a medium where sodium is replaced by potassium o f equivalent molarity [10]. One of the currently accepted degradation mechanism of alkylbenzene sulfonates [ll] begins with a desulfonation reaction, which leads to a phenol, followed by the degradation of the alkyl chain. /3-Oxydation is the main mechanism and leads principally to various phenols substituted by a short acid chain in the paru position. Aromatic ring cleavage then occurs and represents a particularly important step for the complete elimination of the surfactant molecule. Beck and Stache [5], however, found that desulfonatiou often occurs after ring cleavage. 3. MATERIALS A N D METHODS 3,1. Surfactam titration In sea water and culture media, the classic colorimetric method of Longwell and Maniece [12], modified by Armangau [2], was used. This technique, recommended by O.E,C.D. for environmental determinations [4], gives the total amount of sulfonated alkylbenzenes and para sulfophenyl carboxylic acids with alkyl or carboxylic chains containing at least five carbons [13], In sediments determinations were made in the finest part ( < 200 p,m) of sediments in order to eliminate variations due to the coarse part, After sonication in 100 ml distilled water about 5 g of sediments was passed on a 200-/~m mesh sieve. Surfactant concentrations were then determined by the method indicated previously, -

-

3.2. Bacterial cultures Liquid cultures were made with the basal medium (BM) described by Le Petit and Nguyen [14]: NHdCI (4 g); Tris buffer, pH 7.2 (Tri-hydroxymethyl aminomethane, 6.05 g; HCI 4N, 11.05 ml): phosphate buffer, 0.1 M, pH 7.0 (4 ml); 1 m g / m l FeSO4 - 7 H 2 0 (2 ml); sea water (1000 ml). The phosphate buffer and the iron sulfate were sterilized separately and added aseptically to the cold medium to prevent phosphate precipitation and iron oxidation. The tested carbon source was added aseptically i n the medium at the appropriate concentrations. 3.2.1. Trypcase.soyase medium (TSD). This medium was used for isolations and contained: trypcase (7.5 g), soyase (2,5 g), agar (15 g), seawater (1000 ml). For subcultures in tubes, 100 m g / I of anionic surfactant (Commercial linear dodecylbenzene sulfonate - trade mark: dobanic acid 83, mean relative molecular mass: 313 furnished by Procter and Gamble, France) was added before sterilization, This surfactant concentration was raised to 500 m g / I to obtain more visible degradation halos on Petri dishes, Surfactant purity was up to 97~. Major impurities were unsulfonated organic matter ( < 2.5~), free sulfuric acid ( < 0.7%) and water ( < 0.3~). 3.2,2. Sodium requirement medium. To obtain correct growth of isolated strains we adapted the method used by Baumann [10]. The medium used was" trypcase (7,5 g), soyase (2.5 g), half-strength synthetic seawater (104)0 ml), Final concentration: NaCI, 0.2 M: MgSO,~- 7 H20, 0.05 M; KCI, 0.01 M; CAC12.2 H20, 0.01 M, For sodium requirement tests, NaCl was replaced by KCI at the same molar concentration. In this case, the trypcase and soyasc supply of NaCI was only 3 mM. Tests were done in inverted T-shaped tubes, specially designed for vigorous shaking and aeration, containing 10 ml of medium, For each test, two tubes were inoculated with the strain to be tested, suspended in sodium - and potassium - free synthetic sea water (50~ v/v). The two tubes were put on a shaking table for 48 h at 30°C. Growth was recorded at the end of the experiments by optical density measurement at 450 nm, 3,2.3. Selection of bacterial communities degrading a commercial surfuctant. Eflenmeyer flasks

containing 50 ml of 4-fold concentrated BM (described before) were used. In each flask, 150 ml of sampled sea water (harvested in sterile bottles) was added. The commercial surfactant (dobanic acid 83) was added aseptically at 20 m g / I and flasks were incubated on an orbital shaker at 30 ° C. When surfactant degradation reached 50%, as verified by residual snrfactant titration, a subculture was inoculated by transferring 10 ml of the old culture into a new flask containing 190 ml of medium (at the appropriate concentration) and 50 x ~ / I of surfactant. The next subculture was initiated in the same way by using a culture medium containing 100 m $ / I of surfactant in order to verify the adaptation of the microfl'~ra to increasing quantities of surfactant. 3.2.4. I. elation of bacterial strains. Bacterial strains were isolated by spreading on Petri dishes 0.l ml of the last enrichment cultures (100 m g / l ) using dilution series. Isolation medium (BMA) was BM with agar (15 g / I ) and surfaetaut as sole carbon source (500 rag/I), The low solubility of dobanic acid 83 in sea water (about 100 rag/l) gave a homogeneous turbidity to the medium. After incubation, colonies surrounded by a transparent halo (indicating the disappearance of surfactant) were isolated in pure culture on TSD. 3,2,5. Strain characterization. Gram staining, flagella examination after Rhodes staining 115] and eytochrome oxidase testing with OXY-SWAB technique (Biological labs, 6620 Manor road, Austin, Texas 78723) were performed on all strains. As they were all Gram negative and oxidase positive a numerical profile was determined by using APi 20 NE strips (API System, 38000 La Balme les Grottes, France). For optimal development of saltwater strains, the API 20 NE strips were prepared by using 20 g / I NaCI solutions instead of the 8,5 8,4 recommended in the API procedure. The surfactant degradation experiments with isolated strains were done in inverted T-shaped tubes with 10 mi of BM containing 500 m g / I of dobanic acid 83 as sole carbon source. The amount of degradation was measured after 14 days of incubation by titration of the residual surfactant. The use of some model molecules as sole carbon source was verified in the same way: these pos-

tulated intermediary products represent degradation end products after a and fi-oxidation. These commercially available products were 4-hydroxyphenyl acetic (4HPA), 3-(4-hydroxypbenyl) propionic (4HPP), 4-hydroxybenzoie (4HB) and 4-hydroxypbenyl pyruvic (4HPPy) acids. Molecules representing ortho substituted products (2-hydroxyphenyl acetic and salicylic acids) were also tested. All these products were of analytical grade.

4. RESULTS A N D DISCUSSION 4.L Sampling Five sampling sites were chosen (Fig. 1): L Miramar beach; 2, the mouth of the Gapeau river; 3, Saint-Pierre de lamer harbour; 4. Cape Esterel; and 5, Porquerolles island harbonr. Sampling site 2 (Gapeau mouth) represents the main pollution source. Sampling was carried out in the beginning of June by gentle ESE wind which induced a surface current following the coasts in the N E - S W direction. On each sampling site two samples of surface water, two samples of bottom water and one sample of superficial sediment were harvested by diving. 4.2. Titration results The results listed in Table 1 show a weak surfactant content for all sampling sites. June is probably a period of minimal tellaric pollution with regard to surfactants, as it is after spring rains and before summer touristic affluence. However, significant concentrations were found at practically all the sampling sites. The maximum concentration was expectedly found at the Gapeau mouth, and the quantities decreased regularly toward Porquerolles island. Surface concentrations were greater than the bottom one, except for sampling site 2 where the slight depth allowed only one sampling. Analyses done on waters may be subject to large variations due to bulk water displacement and irregularity of sewage contribution (i.e. daily pollution peak). However, a considerable part of surfactant is adsorbed on particulate matters. Titration with the finest part of the sediment gives an average representation of this kind of pollution

_

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~.. ,,,

.,~.:.~;'~._.~-

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"" i ,.:,,

L.__", ~

i

43~N

i

(frOm BLANC.1975I '"~ ' ~ ' ~ " • 0

,/~1~_~.~'"

sampling Sits I

2 km

Fig, 1, Map of Hyercsnay showinBIhc p~iLionsof s~mplin~lsilts f~m the moutho[ Ih~Gapcauriver Io Po~qu©ro|l~sisland. [16], As a rcsull, surfactant quantities are in such a case clearly g~ater than those in the water, They vary inversely: the smaller concentrations at sam-

piing site 2, the highest in front of Porquerones island harbour. These results may be explained by the sedimentation of current-borne particles con-

Table 1 Surfactanl concentrations found in water and sedimeats of the five sampling sile~ Sampling ~te

Depth (m)

Surface water concentration ( / t g / I DBSA)

BOttom water conoentration ( tt g,,'l DBSA)

Sedim~:nt concentration ( m g / k g DBSA)

A m o u n t of ~diments < 200 ~ m (%)

I 2 3 4

6 2- 3 8 13-14

3+0 11.8 6.9 2.4 0.0

0.O

0.7 0.2 0.6 0.9 3.0

81

6

5

1.5 0.0 0.0

57 26 71

Concentrations are expn~sed in P8 of dode~'ylbenzenc sulfonat¢ per litr¢ for surface and bottom waters and in m g / k g for the sediment fractions smaller than 200 Mm. Welghl ratios (%) of the latter versus total sediment are also indicated. Table 2 I~gradation of surfactanls in cultur©s inoculated with various water samples Sampling

Incubalion lime

sile

0

1A b

1B b

2A

+ 7 days

+ 14 days

+ 21 days

+ 28 days

a b c

17.50 48.01 83.2

18,55 20.76 36.3

6,87 * 19.50 29.7

18.43 ~

12.2

a b C

17,07 47.82 951~

18,27 17.27 ~ 92.15

63.44

6215

24.8

a

16.76 46.66 92.34

2.68 a 7,86 ~ 31.82

13.58

b c 3A

7,64 a

9.51

b c a

17.39

b

47,43

26,09

c

89,63

31.82

16,68

71L8

4A

a b c

17,05 47,53 91.96

] 7.89 16.68 ~ 25.03

9.62 ~ 14.36 26.0

6,75

4B

a b c

17,94 4~.|1

18.47 52,67

19.48 60,43

5Pt

~t h c

1'1.81

48,50 86.33

Ig,90 40.45 57.42

6.79 ~ 11.64 ~ I 16

~

17.87

b C

47.14 91.96

18.74 16.30 ~ 40,93

7.18 ~ 12.71 24,64

3B

5B

18,04 13.0 56,65

+ 42 days

15.33

17.11 40.74 10t),49

a

+ 35 days

4.85

7.87

114 ~ 17.07

8.92

I 1.8

6158 a 9.22 ~b

9.51

8,3 19.21

11.3

7.95

2

20.8

10.5

7.37 a

18.2

Starting lime of n~xt subculture when more than 50~ of the initial surfa¢tant quantity was degraded. b A, surface water~ B, bottom water, c a, starlin 8 cultur~ on 20 m g / l surfactant; b and c. next subcultures with increasing concentrations o f surfactant in the medium (resp. 50 and 100 m 8 / l surractam).

taining adsorbed surfactants and originating from the Gapeau river. 4.3. Microbiological study Only sample,s 2 and 3 (bottom water) allowed a rapid surfactant degradation (Table 2). These waters have a well adapted microflora and correspond to the zone polluted by the Gapeau river. In other samples there was no detectable surfactant degradation after an incubation time of 1 week. The slight increase in values is due to medium evaporation resulting from the 30°C incubation. After 2 weeks of incubation, surfactants were degraded in all culture flasks, except those corresponding to sampling site 4 (bottom water). However, degradation did not reach 100% because of the presence in the commercial surfactant of different branched chain isomers with degradation times longer than those of the linear chain products [7]. This may indicate that bacteria potentially able to degrade surfactants are always present along the littoral coasts. This microflora can be progressively adapted to degrade increasing quantities of surfactant, it must be noted, however, that the adaptation time of such microflora is at least 1 week or more, with the exception of sampling sites exposed to marked chronic pollution. On the other hand, the degradation test only represents a degradation potential. Its 'in-situ' expression depends, among other things, on the availability of nitrogen and phosphorus, added in excess in our experiments, and on the temperature, regulated here at 30°C. 4.4. Strain isolation After 21 days of incubation, colonies showing a distinct transparent halo on BMA (Fig, 2) were isolated in pure culture on TSD which allows faster growth. With this technique 26 strains were isolated. They were distributed as follows: Sampling site 1, 6 strains for surface, 3 from bottom water, Sampling site 2, 4 strains, - Sampling site 3 , 3 from surface, 7 from bottom water. Sampling site 4, no strains. - Sampling site 5, 3 from surface.

Fig, 2, a, D~grc~dationhalos in a8ar mediumaround bacterial coloni~ isolated [rom dilutiontubes, b, Pure culture c,btained by spn:adingon the same m~liuma colonyshowinga marked transparent halo indicatingsurfactant degradation, as verified by titration.

Strains were isolated from the most active cultures originating from waters containing the highest concentrations of surfactant.

6S Table 3 Identification and clustering of the: 26 isolated strains (all Gram - . oxidase+ ) by using the API 20 NE code numbers in addition to cultural and morphological characters Flageliar arrangeraent: Perilrichous Cell form: Rod Rod Station IS ISA2 0~4)~4)04 ISBI 0000024 ] SB2 (3000024 l SB3 0000024

Monomchous Helical Helical ISA1 0047744

S

Curved

Rod

] SB" 3 0 0 ~ 0 2 4

Station 1P

t PA (10~004 I PB 00~004 I PC [ ] 0 ~ 4

Station 2S

2SA 0020024 2SC 0420024 3SC 0420004

Station 3S

3SBI 0400004 3SB2 040~()4

Station 3P

Station 5S

2SBI 1044655 2SB2 1044655

3PD30000024 3PD21447444 3PB21044455 3PD4 0000024 3PB'3 1144455 3PD5 04)(0024 3PB"3 1044455 $SAI 0000024 5SB 0420004 5SA3 0000024

Sodium required

-

+

Genus assigned IO

Alcaligenes

De[eva

+

+

Aquozptrillum Octanespirillum

4+5, Strain characterization Morphological characters, biochemical tests and sodium requirement made it possible to classify the isolated strains into seven homogeneous and well separated groups, whatever the sampling site was (Table 3). Essentially we found (1) peritrichous bacteria similar to AIcaligenes (ISA2, ISB, 1SB2, ISB3, ISB'3, 5SAI. 5SA3). showing no sodium requirement, and marine bacteria similar to Deleya (2SC, 5SB) but differing from the type strain by the absence of glucose assimilation (18); (2) polady flagellated bacteria showing both curved and helical cells which could be either related to the genera Aquaspirillum (ISA1), Oceanospirillum (1PA, 1PB, IPC, 3SB, 3SB2), Alteromonas or Pseudomonas (3PD2); (3) the group containing strains 2SA, 2SC which showed a relatively weak growth on sodium as well as on potas-

Aquaspirillum Aiteromonas

Pseudomonas

slum medium. By this characters, they are similar to strain ISA1 and may be grouped with the genus Aquaspirillum; (4) polarly flagellated bacteria with rod-shaped cells belonging to the genus Pseudomonas ( | w e polar flagella, 3PB2, 3PB'3. 3 P B " 3 ) particularly P. sturzeri (l polar flagellum. 2SBI, 2SB2). All isolated strains were G r a m negative heterotrophic bacteria with strictly aerobic metabolism. Baleux [19] found that anionic surfactant degradation in Rh6ne water promotes growth of heterotrophic bacteria with oxidative or inert glucose metabolism and loss of fermenting bacteria. This seems to confirm that surfactant biodegradation is essentially an strictly aerobic phenomenon as it is generally due to Pseudomonas related bacteria [20], which were frequently found in our experiments. However, in a marine environment, other G r a m

negative oxidase positive bacteria, related to the genus Oceanospirillum seem to have an essential role in surfactant biodegradation. They are also strictly aerobic. It should be pointed out that Wagoner and Schink [21] have recently reported an anaerobic degradation of anionic and non-ionic surfactants, but aikylbenzene sulfonates still appear to be recalcitrant under these conditions.

4.6. Surfactant degradation The ability of each strain to use dobanic acid 83 as sole carbon source is expressed (Table 4) by growth results and the average % of surfactant degradation after 14 days of incubation. The utilization by these strains of model molecules allows determination of their ability to cleave the aromatic ring of the surfactant molecule. Only one strain (5SA3) out of the 26 that were isolated did not degrade the surfactant appreciably. However, it can be pointed out that results are generally weak as many strains did not degrade more than 20 to 30% of .the surfactant in 14 days. These strains showed very weak growth on the isolation medium (Fig. 2a). The very slight proportion of unsulfonated matter ( < 2.5%) seems insufficient to support growth of these strains. The best results were obtained with slowly growing strains, related to the genus Aquaspirillure. and not of obligatory marine origin since sodium improves but is not required f,~r ~,owth. These strains do not use any of the proposed intermediary products and seem to be able to degrade only the alkyl chain without further cleavage of the aromatic ring. This behaviour has been described for mutant strains but never for wild type strains I20]. Strains from the most active cultures give the best degradation ratios, which confirms the presence of adapted strains in the most contaminated sampling sites, However, initial bacterial communities give better degradation than do isolated strains, as can be seen by comparing results listed in Tables 2 and 4. Some strains show a low surfactant degradation but better growth on intermediary products, This suggest that some strains of the communities grow on metabolic end products of the other strains. A key step seems to be the aromatic ring cleavage. Among the proposed

Table 4 Surfaetant degradation(mean valueof three experiments) and growth of the 26 isolatedstrainson variousaromaticsubstrates used as solecarbon source (initialeoncemration: los ma/I) Strain

Surfactant degraded(%)

Growthwith Dobanic 4HB acid 83

4HPPy

ISA1 ISA2 ISBI tsB2 lsn3 1SB'3 IPA IPB 1Pc 2SA 2S111 2SB2 2SC 3SBI

23.8 9.6 17.4 36.0

+ +

+

(+l

(+) (+)

(+) (+)

(+) (+)

(+)

(+)

t+)

3S132 3SO 3PB2

20.6

(+) (+) I+) (+)

1+) (+) ++

(+) (+1 (+) (+)

(+) (+) (+)

++ ++ +

1+) (+) (+)

3PB'3 3PB"3 3PD2 3PD3 3PD4 3PD5 SSAI SSA3 5se

(÷)

19.2

21.9 15.6 23.8 to.s 27.7 13.7 27.8 73.7 16.1 16.6 34.4 32.5 27.5 24.2 55.2 48.3 40.7 8~5 o.o 4.8

+ + +

(+) (+1

Growth was checked by opucal eensay measurements at 4~O nm, - opticaldensityis similarto cor,trol; ( + ) opticaldensity is more than control; + opticaldensity is two timesmore than control; + + opticsI density is three limos or mo¢¢ than cor.~toL I'~o growth was found with any strain on 4HPA, 4HPP, or onho-substitutcdphenols(2HPA and salicylicacid), intermediary producls only 4HB and 4HPPy were effectively used as sole carbon source. As a general rule, the behaviour of different strains with regard to these products confirms the distribution on Table 3, Aromatic substrates are used by more specific marine bacteria, as indicated by their sodium requirement, Bacteria seem to be highly adapted to their substrate because they use only well defined molecules, In particular no strain is able to cleave ortho substituted phenols. As concorns para substituted phenols, although some bacteria use 4HB and 4HPPy, they cannot use

67 4 H P A , 4 H P P is probably degraded via 4 H B by ~-oxydation as obser~'ed by Willets [111, and a r o m a t i c ring cleavage t e e m s to be easier if the lateral chain is shorter. T h e purpose of the isolation melhod was to select in bacterial communities only the strains able to ]nil]ate the surfactant degradation (25 strains versus 26 isolated). It appears t h a t strains thus selected preferentially grow o n 1he alky] chain of 1he surfactant. Subsequently, the aromatic ring should be cleaved b y other non-isolated strains of the communilies which cannot use 1he entire molecule. Study of surfactant biodegradalion in Ih¢ marine environment should take into account not only the disappearance of the initial molecule, as pointed out by WiUets [11], but also 1he fate of 1he phenolic products of degradation, which depends u p o n the presence of bacterial strains particularly a d a p t e d to such suhstrates.

REFERENCES Ill Karsa, D, |19861 New applicalions boost surfactants use. Perform Chem. 1.4-9, [2] Arnmngau, C. 0967) Tentative d'milisation des d~tergents aninniques comm¢ trac~urs d©pollution f~caie. Rg~.Tray. Inst, P~ches Marit. 31,417-424. [3] GledhiU, W.E. 0975) Linear aikylbenzene saifnnate: bi¢degradation and aquatic interaclions. Appl. Microbiol. 17. 265-293, [4] O.E.C.D. (19761. Propo~'d method for the determination of the biodegradability of surfaclants used in synthetic d¢lerg~nls, Paris. [5] Bock, KJ. and Slaehe, H, (19821Surfaclanls. in Handbook o1 Environm¢:ntai Chemist~ (O. Hutzin$cr, Ed,), pp. 169-199. Springer, Berlin, 161 Vasque~ U,G. and Nunez, G.M.J. (1983) Biodegredation of non-innic dispersants in sea-water. Appl. Microbiol. BinteghnoL 18, 315-319, 171 Sales, M,D,, Quiroga. A.J.M.. Gomez, P.A., Establier, T.R, and Finres, L. (19841cmrtica de la biodegradacion de n.Dod¢cil-henoeno-sulfonato sodico en aguas y sedimentc~ de la bahia de Cadiz, Cornea, Join, Com. Esp, Delerg. 15, 89-102.

[8] Baleux, EL (1977) A computer study of the evolurion of hctcrotrophic bat:tcriai population in sewage and river waters. Microb. Ecol. 4, 53-65. [9l Larsen, H. 119861 Halophilic and haloroterant microorganisms - an overview and historical perspective. FEMS Microbiol. Rev. 39, 3-7. [101 Baumann. P., Baumanr~ L., MandeL M. (19711Taxonomy o1 marine bacteria: the genus Beneckea. J. BacterioL 10"7, 268-294. {111 Willens, AJ. (19731How detergents are degraded biologi. c,al[y, Environ. Change 2, ll0-116. [12] Longw¢11.J. and Man]nee, W.D. (19551Determination of anionic detergents in sewage, sewage e f f l ~ t s and river waters. Analyst. 80. 167-171. [13] Kimede, R.A. and Swisher. R.D. (19771 Reduction of aquatic toxicity of linear aikylbcnzen¢ sulfonate (LAS) by bio~gradatinn. Water Res. 11.31-37. [141 Le Peril & a,qd Nguyen. M.H. (19761 Besoins en phosphore de ba¢trdes m~abolisant les hydroearbures en mer. Can. J. MicrobioL 22.1364-1373. i 15] Rhodes, M.E. (1955).The fylology of Pseuc?omon~ spogies as revealed by a silver plating staining method. J. Gen. MierobinL 18, 639-648. [161 Auclair-Dessemon, D. (19731 Etude de la pollution des ¢onds manns dans le secteur de Cortiou. Th~se. Facult~ de Pharmacie. MarseiUe,France. [17] Sigoillol,J.C. and Nguyen, M.H. (1987) Degradation d'un tensio-actif commercial en prt~senc¢ d'un¢ source ¢omplrmemaire de carbone, par une ¢ommunaut6 bact~rienne s~lectionn~een milieu matin. Can. J. Microbiol. 33. 929-932. [18[ Baumann. L.. Bowditeb, R.D.. Baumann, P. (19831 Descnptinn of Deleya Ben. nov. created to accommodate the marine species Altar]genes aeslus, d. pactflcus. A. cupidus. A. v e n m t ~ and Pseudomonas marina. Int. J. Syst. Bacleriol. 33. 793-g02. [19] Baieux. B. (19761 Drgredation biologiqu¢ des agents de surface: ~tude chlmique et microbiologique. Thee d'Etat. Universit~ des Sciences el Techniques du Languedoc. Montpcllier, France. 120] Cain, R.B. (19811Micorbial degradation of surfa¢tants, in Microbial Degredalion of Xenobiorigs and Rt:calaitranl Compound• FEMS Symposium n°12 (Leisinger, T.. Cook, A.M., Hi~tter. R., Niiesch. J., Eds.). pp. 325-370. Academic press. London. [21] Wagener. S. and Schink. B. (1987) Anaerobic degradation of noaionic and anionic surfactams in enrichment cultures and fixed-bed reaclors. Water Re& 21. 615-622. [22| Blanc, J.J. 0975) Rechetches de srdimentologie appliqur¢ au littoral rocheux de la Provence. C N.F_X.O. 75.