Bacterial Utilization of Dodecyl Sulfate and Dodecyl BenzeneSulfonate

W. J. PAYNE AND V. E. FEISAL

Department of Bacteriology, University of Georgia, Athens, Georgia

Received for publication 18 February 1963

ABSTRACT

PAYNE, W. J. (University of Georgia, Athens) ANDV. E. FEISAL. Bacterial utilization of dodecyl sulfate anddodecyl benzene sulfonate. Appl. Microbiol. 11:339-344.1963.-Two unknown bacterial isolants (C12 and C12B)were obtained from enriched soils and cultured on mediacontaining detergent compounds as sole sources of carbon.Either isolant destroyed the foaming capacity of culturesconitaining dodecyl sulfate; but C12B, which could growon dodecyl benzene sulfonate (DBS) whereas C12 couldnot, did not destroy the foaming capacity of this sur-factant. The source of DBS available in quantity was amixture of isomers derived from kerosene, and the bacteriautilized only one-fifth to one-fourth of this material duringgrowth. Both isolants grew on short- or long-chainedorganic acids, and resting cells of both rapidly oxidizedseveral long-chain acids and alcohols. Three of five phenyl-placement isomers of DBS (with the phenyl group atcarbon 2, 3, or 6 on the alkyl chains) were excellent sub-strates for growth of C12B, but isomers with phenylplacement at carbon 4 or 5 were toxic and killed the bac-teria.

The persistence of synithetic detergent compounds intreated sewage effluents and waterways has become anincreasingly serious problem. There have been severalstudies, using colorimetric and manometric analyticaltechniques, of the biodegradation of these materials by themixed bacterial flora in sewage and river water (Boganand Sawyer, 1954, 1955, 1956; Sawyer, Bogan, and Simp-son, 1956). Recently, Swisher (J. Water Pollution ControlFederation, in press) and Huddleston and Allred (Develop.Ind. Microbiol., in press) have shown that gas chromatog-raphy provides a very effective analytical method fordetermining intermediates in, and the time required for,degradation in such systems.Bogan and Sawyer (1954) classified the organic com-

pounds incorporated into commercial detergent productsas "soft" and "hard," according to the ease with whichthey are biodegraded. In their scheme, alkyl sulfates,esters, and amides are "soft," whereas alkyl benzenesulfonates, alkyl phenoxy polyethylene glycols, and poly-ethylene glycols are "hard." We recently isolated a bac-

terium which can utilize oligoethylene glycols up to amolecular weight of 400 (Fincher and Payne, 1962). Wehave now turned our attention to bacteria which canutilize other surfactants, and the purpose of this paper isto describe the isolation from soil of bacteria which canutilize dodecyl sulfate and dodecyl benzene sulfonate(DBS) as sole sources of carbon. One isolant was able touse only the straight-chain compound, whereas a secondgrew on both.

MIATERIALS AND METHODS

Bacteria. Samples of soils obtained near the outfall fromthe local sewage disposal plant were mixed into slurries

MOLARITY OF DODECYL BENZENE SULFONATE

E0N

C-CMm

y4wa.

0 0.025 0.05 0.075 0.10MOLARITY OF SODIUM LAURYL SULFATE

FIG. 1. Influence of concentration of carbon substrates on growthof isolants C12 and C12B in minimal media. 1, C12B on SLS (pH7.5); 2, C12 on SLS (pH 7.5); 3, C12B on DBS (considering thematerial to be 90% DBS with molecular weight of 315). Temperature,30 C.

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with 1 %c solutions of 95 % sodium dodecyl sulfate (alsocalled sodium lauryl sulfate, SLS; Matheson, Coleman &Bell, East Rutherford, N.J.) or DBS (Nacconol SZA,kindly provided by National Aniline Co., New York,N.Y.) and incubated for 1 week. Inocula from these slurrieswere then pipetted into synthetic media containing NH4Cl,0.5 g; (NH4)2S04, 0.5 g; Na2HP04, 3 g; KH2PO4, 2 g;MgSO4 7H20, 0.01 g; and either SLS, 7.2 g, or DBS, 7.8 gper liter (pH 7.0). The crude cultures were incubated at30 C in agitated flasks until turbidity was seen, and inoculafrom these were streaked and restreaked for pure cultureisolation on nutrient agar. Use of minimal salts-detergentagar for isolation or storage of stocks was unsatisfactorydue to rapid death of the isolants, and, therefore, stockcultures were maintained oni nutrient agar slants storedin the cold and transferred weekly.Two isolants were chosen for extended studies. The

first, designated C12, grew on SLS but not DBS; thesecond, called C12B, grew on both. These organisms areaerobic, gram-negative rods and are as yet unidentified.

Utilization. Test media for determining effects of varyingconcentrations of SLS or DBS, and varying pH values,on growth were filter-sterilized and dispensed in 25-mllots in flasks with optical cuvettes attached. The inocula

1.0

0.8-E0

I-i0.

04

wa.

0.23

6.0 6.5 7.0 7.5 8.0pH

FIG. 2. Influence of initial pH of minimal media on gr owth ofisolants C12 and C12B. 1, C12 on 0.025 M SLS; 2, C12B on 0.01 m'

DBS; 3, C12B on 0.02 m SLS. Temperature, 30 C.

were cells in 0.1-ml samples from 24-hr nutrient brotlhcultures, volumes sufficient to give "zero hour" counts ofapproximately 106 bacteria per ml. Identical cultures inmedia with various carbon sources were prepared, and allthe flasks were incubated at 30 C on a rotary shaker.Periodic estimates of increases in turbidity at 420 m,u orviable counts, with nutrient agar as the plating medium,were obtained to determine rates of growth. Yeast extract(0.05 %) was added to the media in certain experiments.In addition, sodium salts of several organic acids, otherlong-chain carbon compounds, or benzene sulfonate weresubstituted for SLS or DBS in the basal medium to deter-mine the versatility to the bacteria.Manometric determinations of the ability of resting

cells from 18-hr cultures of both C12 and C12B to oxidizeSLS, DBS, and related compounds were obtained. Weused "de-adapted" bacteria which were obtained by tenlsuccessive transfers on nutrient agar. Afterwards, thesebacteria were grown on either nutrient broth or basalmedium containing either SLS or DBS as the sole carbonsource. The insoluble alcohols (dodecyl and cetyl) werehomogenized when needed as substrates in the appropriateconcentrations in water in a Waring Blendor for 30 min.This provided dispersions stable for 2 days.

-J

0 500

0~~~~~~~wN

w 25E I4~~~

z

0

0.05 0.1 0.2

MOLARITY OF DBSFIG. 3. Effects of varying concentrations of DBS on the quantity

of substrate utilized for growth by isolant C12B. The organic materialremaining in the cultures was determined by drying to constant-weight 10-ml samples of media after passing cultures through Seitzfilters, then incinerating, and subtracting the weight of ash. Ap-propriate control determinations u'ith uninoculated media wereobtained.

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V3ACTE]RIAL UTILIZATION O.lFl DETERGENT CONIPOU"NDS

RESULTS

We observed that bacterium C12 grew optimally in.media con-tainiing 0.025 M SLS, whereas 0.015 M was opti-mal for growth of C12B (Fig. 1). A much greater coniceni-tration of DBS (0.1 M) was required for optimal growtlof the latter bacterium. C12 did niot utilize DBS for growth.At pH 71.5, the most rapid growth of both isolants oIn SLSwas observed (Fig. 2); whereas on DBS bacterium C12Bgrew to greatest turbidity at pH 6.8, although the peakwas oilly slightly salienit.

Since the product we designiated I)BS in this study is amixture of dodecyl beiizenie sulfoinate isomers, some ofwhich the bacteria were unilikely to be able to utilize, wedetermined thie percenitage of organic material in thecultures that was actually degraded by the bacteria in32-hi' growth periods. The amounlt of DBS conisumedvaried with the coincenitrationi provided (Fig. 3). At thehalf-optimal concentrationi for growth, 26 '/ was removed;

_A-E=:~~~~FIG. 4. Effects of growth of isolant C12 on foaming in cultures

containing SLS as the sole soznrce of carbon. A, freshly inoculated,0.015 if SLS; B, identical cllture after 96 hr- of growth; C, cultureafter 96 hr in 0.025 mv SLS. Each flask was shaken vigorouisly im-mediately before the photographs were made.

HOURS

FIG. 5. Stimnulation of growth of isolant C12 on SLS by the addi-tion of a minimal qutantity of yeast extract to the basal medium.

0 6 12 Is 24 30HOURS

FIG. 6. Stimulaiion of growth of isolant C12B on SLS and DBSby the addition of minimal quantities of yeast extract to the basalmedium.

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at the optimal growth concentration, 20 % was utilized.Growth of the bacteria did not destroy the ability of anyof these DBS cultures to become sudsy if vigorouslyshaken.

However, Fig. 4 shows that growth of bacterium C12on suboptimal concentrations of SLS did remove thefoaming capacity of this surfactant. A freshly inoculatedculture in a medium which was 0.015 M with respect toSLS foamed significantly when agitated (Fig. 4A). Afterincubations for 72 to 96 hr, however, identical agitationdid not produce sudsing (Fig. 4B). This was a phenomenondependent on concentration, for Fig. 4C shows that eventhe considerable growth observed in the culture did notdestroy foaming in a medium containing 0.025 M SLS.Bacterium C12B produced identical results when culturedin the media with varied concentrations of SLS.

TABLE 1. Growth of bacteria on several organic acids and alcohols

Growth within 5 daysSubstrate

C12 C12B

Acetate ......................................

Glyoxylate .................................... + +Pyruvate......................................Propionate .................................... + +Citrate ......................................

Malate ...................................... + +Oxalate ...................................... - -Caprylate ..................................... + +Palmitate ..................................... + +Dodecyl alcohol ............................... + +Cetyl alcohol .................................. + +Benzene sulfonate ............................. +

0 60 120 0 60 120 0 60 120MINUTES

FIG. 7. Oxidation of several alkyl compounds by resting cells of isolants C12 and C12B cultured in various media. (large open circles)SLS; (small closed circles) dodecyl alcohol; (large closed circles) sodium caprylate; (small open circles) DBS. Each cup contained 2ml of a suspension of twice-washed cells in 0.067 M phosphate buffer (3 mg dry weight/ml); 2 ,umoles of each substrate were provided in1.0 ml. The center wells each contained 0.2 ml of 15% KOH. Temperature, 30 C.

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BACTERIAL tUTILIZATION OF DETERGENT CO-MPOUNDS

We found that inclusion of a minimal amount of yeastextract in the mineral salts-surfactant media greatlystimulated the rate of growth of the bacteria at the expenseof the synthetic compounds and shortened the lag periodssignificantly (Fig. 5 and 6). Both C12 and C12B grewmore extensively on SLS when provided with this nutri-tional supplement, but C12B apparently grew no moreextensively, only more rapidly, on DBS.

These bacteria were able to grow on saturated fattyacids and alcohols with chain lengths varying from 2 to 16carbon atoms (Table 1). C12B, but not C12, was addi-tionally versatile in using benzene sulfonate as a growthsubstrate in minimal medium.Enzymes for the oxidation of the various fatty acids

and alcohols by C12 were induced by growth on SLS (Fig.7A). Resting cells from nutrient broth did not oxidize thesecompounds. However, resting cells of C12B harvestedfrom broth rapidly oxidized SLS, dodecyl alcohol, andsodium caprylate without a lag (Fig. 7B). Growth ofC12B on DBS provided cells that oxidized SLS morerapidly but were not significantly changed with respect tothe other substrates (Fig. 7C). DBS was oxidized onlyslightly by the bacteria. It seems likely that the washedcells were sensitive to DBS. We had great difficulty getting

BASE10

£3,

- DBS7 GENERATIONS

30 9 1

I S~~~~~~~-J /B

,F 27 8 A /4 A j

AI

o237 8 1*

202 x \14and5+

BASE 5 15 25 35 452 HOURS

FIG. 8. Growth of bacteriumi. C12B on several phenyl-placementisomers and a complex isomneric mixture of DBS as sole sources ofcarbon.

these results because the cells harvested from DBS cultureslysed in the washing buffer on many occasions.As we previously noted, the commercial DBS prepara-

tions are complex mixtures of isomers, varying in thebranching in the alkyl chains and in the placement of thephenyl group. Three of the straight-chained dodecylbenzene sulfonate isomers, varying only in the placementof the phenyl group, were excellent growth substrates forbacterium C12B (Fig. 8). The other two remaining isomerswere initially effective substrates, but apparently toxicintermediates accumulated and killed the bacteria after25 to 30 hr. The growth of C12B on commercial DBSoccurred after a much longer lag than was observed withthe single isomers, which permitted very rapid initiationof growth.The phenyl-placement isomers are not available com-

mercially but were the very kind gift of R. D. Swisherof the Monsanto Chemical Co., St. Louis, Mo. The quan-tities of each that we had did not permit any latitude inour experiments and were used up in the work reportedhere.

DISCUSSION

The filnding that SLS is easily biodegraded by the micro-organisms in sewage (Bogan and Sawyer, 1954) was sub-stantiated by our studies. Either of our isolants destroyedthis straight-chained surfactant's capacity to producefoam unaided by other bacteria and without any othersource of carbon available.The ability of these bacteria to dissimilate many of the

detergents provided is apparently due to the capacity forproducing enzymes for the oxidation of any of a series ofwater-soluble or dispersible compounds with hydrocarbonchains. If, as Bogan (1958) suggested, the hydrocarbonchains of SLS and DBS are degraded by shortening themtwo carbon units at each step ((-oxidation), we haveshown that our isolants have the mechanisms for growingon two-carbon compounds (acetate and glyoxylate) andcould thus be induced to grow on the intermediates of,B-oxidation. Bacterium C12B displayed additional versa-tility in growing on the ring structure, benzene sulfonate.Our findings that a single bacterial isolant (C12B)

degraded at most only one-fourth of the material providedin a commercial DBS, and that yeast extract nutrientsincreased the rate but not extent of utilization of thesesubstrates, suggest that very likely no single bacterialspecies will be found capable of dissimilating all the isomersin these complexes. Bacterium C12B could be considered,however, a potential test organism for determining therelative biological "softness" of a DBS product whichmight be developed in future for the market.We are planning now to attempt enrichment isolations

with the filtrate from DBS cultures after bacterium C12Bhas grown, to determine the possibility of getting bacteriawhich can then utilize the residual compounds.One interesting problem arising from this study is the

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toxicity of some of the isomers in the DBS mixture whosepresence extended the lag period greatly before growthof C12B on the assimilable isomers began. If it is possibleto obtain resolved samples of some of the DBS isomers,studies of competitive effects between pairs or amonggroups may be fruitful. We are interested as well in thepossibility of isolating toxic intermediates which appar-ently arise from partial degradation of certain isomers,such as 4- and 5-phenylalkyl benzene sulfonates, if thenecessary compounds can be gotten in greater quantity.

ACKNOWLEDGMENTSWe are grateful for the technical aid provided by Nor-

man Dice whose work was supported during the summerof 1962 by the National Science Foundation ResearchParticipancy for High School Teachers program. V. E.Feisal received support during the same period from theprogram of the National Science Foundation ResearchParticipancy for College Teachers.

LITERATURE CITEDBOGAN, R. H. 1958. Biochemical degradation products-a new

dimension in stream pollution. Sewage Ind. Wastes 30:208-214.

BOGAN, R. H., AND C. N. SAWYER. 1954. Biochemical degradationof synthetic detergents. I. Preliminary studies. Sewage Ind.Wastes 26:1069-1080.

BOGAN, R. H., AND C. N. SAWYER. 1955. Biochemical degradationof synthetic detergents. II. Studies on the relation betweenchemical structure and biochemical oxidation. Sewage Ind.Wastes 27:917-928.

BOGAN, R. H., AND C. N. SAWYER. 1956. Biochemical degradationof synthetic detergents. III. Relations between biologicaldegradation and froth persistence. Sewage Ind. Wastes 28:637-643.

FINCHER, E. L., AND W. J. PAYNE. 1962. Bacterial utilization ofether glycols. Appl. Microbiol. 10:542-547.

SAWYER, C. N., R. H. BOGAN, AND J. R. SIMPSON. 1956. Biochem-ical behavior of synthetic detergents. Ind. Eng. Chem. 48:236-240.

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