Appuzr MicaomouooGy, Feb. 1972, p. 407-414Copyright 0 1972 American Society for Microbiology

Vol. 23, No. 2Printed in U.SA.

Degradation of Alkyl Benzene Sulfonate byPseudomonas Species'

R. S. HORVATH2 AND B. W. KOFTDepartment of Bacteriology, Rutgers-The State University, New Brunswick, New Jersey 08903

Received for publication 3 November 1971

Pseudomonas sp. HK-1 showed a direct relation between the concentrationof alkyl benzene sulfonate (ABS) supplied and cell yields. Since growth onABS alone did not occur, it was necessary to correlate the total energy ob-tained by the cells to the ABS concentration when glucose was supplied in alimiting concentration. Several types of metabolic attack in addition to the sul-fonate removal were noted: (i) side-chain utilization as indicated by the pro-duction of tertiarybutyl alcohol and isopropanol and (ii) ring metabolism asindicated by the presence of phenol, catechol, mandelic acid, benzyl alcohol,and benzoic acid in spent growth media. Utilization of ABS was greatly en-hanced by the presence of phenol. This enhancement suggests co-metabolismand that limited concentrations of phenolic products derived from ABS mustbe accumulated to get active metabolism of the ABS molecule.

Alkyl benzene sulfonate (ABS) is generallyregarded as a biologically recalcitrant mole-cule. A steady increase in detergent use hasbeen accompanied by problems such asfoaming in sewage treatment plants and in thewaters receiving the effluent from these plants(24). The accumulation of ABS in water is dueprimarily to its resistance to biodegradation,and several factors have been suggested whichmight affect the biodegradability. Swisher (31)stated that among the different ABS, thebranched molecules are far more refractory inwater and sewage than the straight-chain ABS.McKenna and Kallio (23) noted that the intro-duction of a single methyl branch imparts re-sistance to saturated hydrocarbons. This isespecially true if the methyl branch is placedon the terminal carbon.

It was of biochemical interest to isolate anorganism capable of degrading ABS (7). Al-though oxidation of the alkyl moiety of ABSwould be expected to occur, attempts to iso-late organisms capable of metabolizing thishydrophobic structure had led to a very lim-ited success. This limited success did indicate,however, that organisms capable of utilizingthis alkyl chain do exist in nature.

Oxidation of the benzene ring of ABS

'Presented in part at the 68th Annual Meeting of theAmerican Society for Microbiology, 5-10 May 1968, Detroit,Mich. (Bacteriol. Proc., p. 52, 1968).

'Present address: Department of Biology, Bowling GreenState University, Bowling Green, Ohio 43403.

seemed to be a possibility, but this idea wasnot pursued initially. Kitagawa (21), studyingtoluene oxidation, showed the pathway to pro-ceed through benzyl alcohol, benzaldehyde,benzoic acid, and catechol. Also, p-cresol wasoxidized via the methyl group before attack onthe ring (9).House and Fries (20), while seeking informa-

tion on the interaction of ABS and activatedsludge, noted the production of significantamounts of inorganic 3'S compounds from 35S_ABS. In 1952, Leclerc and Beaujean reportedon the production of hydrogen sulfide by bac-teria metabolizing ABS (22). These observa-tions, although they were not pursued further,tended to support the rationale that organismscapable of removing this sulfonate shouldoccur in nature.As previously reported, an organism was iso-

lated from an enrichment culture, which wascapable of cleaving the sulfonate moiety fromalkyl benzene sulfonate and of using this sulfo-nate as its source of sulfur for growth (7). Ap-proximately 70% ABS reduction was measuredin 7 days in pure culture systems. The isolate,designated HK-1 and identified as a Pseudo-monas sp., did not grow to a visible turbidityin a sulfur-poor glucose-salts medium butshowed a good growth response to the additionof ABS to this medium. There appeared to bea direct relation between the ABS concentra-tion and the energetics of the cell. Because theexpected insoluble product, alkyl benzene,

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HORVATH AND KOFT

could not be detected in these studies, investi-gations were initiated to ascertain the meta-bolic products derived from either the benzenering or the alkane portion of the molecule.

MATERIALS AND METHODS

Purification of ABS. ABS as supplied by theAssociation of American Soap and Glycerine Prod-ucts contained approximately 48% inorganic sulfate.This necessitated a purification procedure of ABSwhich was carried out as previously reported (7).Dry cell weight determination. All dry weight

determinations were done by measuring optical den-sity and converting it to dry weight from a standardcurve.Media and inocula. The basal salts medium

employed in these studies consisted of 1.0 g of NH4,Cl, 1.0 g of Na2HPO4, 0.5 g of KCl, and 0.1 g ofMgCl,2, per liter of distilled water. ABS, glucose,inorganic sulfate, and various carbon and energy

sources were added to this medium in various con-

centrations and combinations. A 1% inoculum was

used in these studies and was taken from a 48-hrculture in basal medium plus 0.01% glucose and 0.1%ABS unless otherwise noted.Simultaneous adaptation growth responses.

Growth measurements for simultaneous adaptationgrowth responses were made by a Bonet, Maury, andJouan biophotometer. Adapted cells were taken froma 9-day-old culture grown in ABS-glucose-saltsmedium. Nonadapted cells were taken from a 48-hrculture grown in glucose-salts medium. An inoculumcontaining 107 bacteria/ml was used.

Identification of metabolic intermediates. Met-abolic intermediates were identified by physicalproperties of the isolated compounds or of deriva-tives of the compounds. Amide derivatives were usedfor the identification of carboxylic acids, 3,5-dini-trobenzoate derivatives were used for identificationof alcohols, and 2,4-dinitrophenylhydrazone deriva-tives and semicarbazone derivatives were used forthe identification of aldehydes and ketones. All de-rivatives were prepared by procedures outlined byShriner, Fuson, and Curtin (27). Molecular weightdeterminations were done by the freezing point de-pression method as described by Gattermann (13).Mixed melting point determinations using the iso-lated derivative and the same derivative of a

standard compound corresponding to the suspectedidentity of the isolated compound were used to con-

firm tentative identifications whenever possible.Quantitative and qualitative procedures. ABS

was meaured by the methylene blue procedure out-lined in Standard Methods for the Examination ofWater and Wastewater (3). Catechol was quantitatedby the method of Arnow (4), and phenol determina-tions were accomplished by the 4-aminoantipyrinemethod described by the United States PublicHealth Service (32). Benzaldehyde determinationswere carried out by a modification of the diphenyl-amine procedure (5): 1 volume of the test samplewas treated with 1 volume of a cold 20% trichloroa-cetic acid solution and filtered. Benzaldehyde was

quantitated as described by Ashwell (5). Qualitative

periodic acid tests were used for the detection ofepoxides and glycols according to Shriner, Fuson,and Curtin (27).

RESULTS AND DISCUSSIONPseudomonas sp. HK-1, the isolate em-

ployed in these studies, did not grow to visibleturbidity in a sulfur-poor glucose-salts mediumbut showed a good growth response with theaddition of ABS to this medium (7). This iso-late was also capable of using inorganic sulfateas its sulfur source for growth, but, as seen inTable 1, the growth response to inorganicsulfur was not as great as the response exhib-ited when ABS was supplied as the sole sulfursource. Sulfur was not a limiting factor inthese studies but glucose was limiting; there-fore, it appeared that the greater growth re-sponse shown when ABS was added to the glu-cose-salts medium indicated that a greateramount of energy was available to the cellwhen ABS was present.The tentative identification by ultraviolet

spectrophotometry of a phenol-like compoundand a catechol-like compound in spent mediaindicated that benzene ring oxidation was oc-curring in a manner similar to the oxidation ofthe aromatic ring structure studied in somedetail by Stanier (28, 29, 30). This determina-tion involved a counter-current distributiontechnique in conjunction with ultraviolet spec-trophotometry. The phenol-like compound wasidentified in 18-day-old spent media, and thecatechol-like compound, was identified in 30-day-old spent media.Compounds isolated from spent growth

media are shown in Table 2 with the meltingpoints, derivative melting points, and molec-ular weight obtained.To establish the origin of the isolated com-

pounds as ABS, quantitation of benzaldehydeby the diphenylamine procedure and phenol

TABLE 1. Growth response of Pseudomonas sp. tothe addition of ABS or (NH4) 2S04 to a medium

containing basal salts plus 0.01% glucose

Amt (mmoles) Amt (mmoles) Amt (mg)/liter (dryof ABS/liter of SO,/liter cell wt)

2.870 0.000 378.001.430 0.000 243.600.715 0.000 151.200.287 0.000 92.400.000 0.000 50.400.000 0.287 151.200.000 0.715 193.200.000 1.430 193.200.000 2.870 193.20

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DEGRADATION OF ALKYL BENZENE SULFONATE

TABLE 2. Melting points and molecular weights ofcompounds and specific derivatives isolated fromspent ABS (0.1%)-glucose (0.01%)-salts culture

medium

Me-Deriv- Melting Mol diumative2 point wt age

(days)

Isopropanol B 120 60 2t-Butanol B 142 70 2Methylphenylcarbinol B 95 122 6Phenylethyleneglycol A 66-68 137 6

B 98 131Mandelic acid A 133 152 22

E 122 152Benzyl alcohol B 112-114 104 22Catechol B 149-153 105 32Benzoic acid E 126-130 120 22

a A = pure compound; B = 3, 5-dinitrobenzoate; C =

semicarbazone; D = 2, 4-dinitrophenylhydrazone; E =

amide. Reference standard MP - 10, 12, 25, 27.

by the 4-aminoantipyrine procedure was car-ried out in ABS-glucose-salts media 3, 7, 10,and 18 days after inoculation and in glucose-salts media lacking ABS on days 3 and 18. Theamount of ABS reduced was also determinedby the methylene blue procedure. As seen inTable 3, from 76% (day 3) to 94% (day 18) ofthe reduced ABS could be accounted for asbenzaldehyde-like and phenol-like compoundsin the ABS-glucose-salts medium. No benzal-dehyde-like or phenol-like compounds could bedetected from the culture in the glucose-saltsmedium without ABS.

In view of the energy derived from oxidationof ring compounds by pseudomonads asstudied by Stanier, it was of interest to deter-mine the relation of degraded ABS to the ener-getics of the cell. Because an energy sourcesuch as glucose was required to allow growth,it was necessary to correlate the cell yield withthe ABS utilized when the energy source, glu-cose, was present in limited concentration. Bythe method of calculation of Bauchop andEldsen (6), the possible energy yield from ABSwas determined. It was calculated that 40.6 mg(dry weight) was formed for each millimole ofABS degraded. Energy calculations yielded avalue of 4 mmoles of adenosine triphosphate(ATP) per mmole of ABS degraded.Growth experiments and energy calculations

were also employed to determine possiblemechanisms for the reactions involved in thedegradation of ABS (Table 4). Specific stand-ards corresponding to compounds identified asintermediates of ABS degradation were usedas the sole source of carbon and energy in abasal salts medium. The energy available to

the cell from each standard compound utilizedwas calculated on the basis of maximum cellyield obtained. The amount of ATP producedby a growing culture was not measured di-rectly. The ATP yields were approximated onthe basis of total cell yield produced per milli-mole of substrate used. Measurement of ABSconcentration, by the methylene blue proce-dure, before and after growth allowed calcula-tion of ATP yields from ABS on the basis ofthe amount of ABS actually degraded. For allother substrates, the ATP yields calculatedwere related to the amount of substrate addedto the medium.The order of appearance of compounds in-

volved in this sequence should follow Stanier'spathway for ring oxidation (28, 29, 30). Calcu-lations based on bond energies were done tomake correlations with the results obtainedfrom growth studies. The reactions of benzylalcohol to benzaldehyde and benzaldehyde tobenzoic acid (Fig. 1) were consistent with reac-tions proposed for the metabolism of aromaticcompounds by P. fluorescens. The conclusionsbased on cell yield data and bond energy cal-culations were the same as those based on res-pirometry data (28, 29). The formation ofphenol by isolate HK-1 seems to be an impor-tant reaction in ABS degradation. The co-metabolism of the ABS molecule with glucoseand the disappearance of ABS after phenol hasbeen detected in the ABS-glucose mediumsuggest an active role for a phenolic compoundin the degradation of ABS.The proposed reaction for the oxidation of

phenol to catechol is shown in Fig. 2. Sincegrowth of isolate HK-1 in the absence of glu-cose occurred on phenol only in the presence ofABS, a coupled reaction could be postulated.This reaction could involve the desulfonationof ABS with a simultaneous oxidation ofphenol to catechol. Energy calculations basedon growth data indicated a net gain of 2.98kcal per mmole of phenol oxidized by the iso-late in the presence of ABS. However, the oxi-dation of phenol to catechol, in the absence ofABS, would be expected to release 31.7 kcalbased on bond energy calculations. Appar-ently, the energy from this exergonic reactionis utilized to drive the endergonic desulfona-tion of ABS. Thus, bond energy calculationsbased on this proposed coupled reaction yielda value of 3.0 kcal of energy per mmole of ABSand phenol.To obtain quantitative results in the deter-

mination of inorganic sulfate resulting fromthe degradation of ABS, it was necessary tooxidize inorganic sulfur products in spent

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TABLE 3. Quantitation of benzaldehyde-like and phenol-like compounds and ABS recovered in spent growthmedia of Pseudomonas sp.

ABS ABS present Benzal- ABS present ABS presentMedia" Age (days) reduced Phenol as phenol dehyde as benzal- as benzal-

(mM) mm(%) (mM) dehyde (%) phe (phenol(%

A 3 0.000 0.000A 18 0.000 0.000B 3 1.305 0.688 52.4 0.311 23.8 76.2B 7 1.545 1.297 84.0 0.151 9.8 93.8B 10 1.940 1.376 71.1 0.245 12.6 83.7B 18 2.154 1.755 81.7 0.074 3.4 85.1

a A = 0.01% glucose-salts; B = 0.1% ABS + 0.01% glucose-salts.

TABLE 4. Twenty-four-hour growth response ofPseudomonas sp. to the addition of compounds,which are possibly related to ABS degradation, to

the basal salts mediuma

Amt [mg Amt (mmole)(dry cell wt)] of ATP/

Medium supplement per mmole mmole ofof substrate substrate

added added

Benzyl alcohol 2.01 0.201Benzaldehyde .......... 9.68 0.968Benzoic acid.32.48 3.248Phenol.0.00 0.000ABS.0.00 0.000Phenol + ABS 2.48 0.248Glucose.80.80 8.080Glucose + ABS .121.40 12.140Isopropanol.18.87 1.887t-Butanol.0.00 0.000

a Compounds were added at 2.4 mmoles/liter.Growth at the expense of glucose + ABS minusgrowth at expense of glucose = growth attributableto ABS. Values were 40.60 mg (dry cell weight) permmole of substrate added and 4.060 mmole of ATPper mmole of substrate added.

media to sulfate by using hydrogen peroxide(7). This test required the generation of inor-ganic sulfur compounds either as sulfate or asthose oxidizable to sulfate by addition of hy-drogen peroxide. The sodium sulfite resultingfrom the proposed coupled reaction would ex-plain the required oxidation by hydrogen per-oxide to determine sulfate.

Cain and Farr (8) also reported the release ofthe sulfonate moiety of benzenesulfonate, p-toluene sulfonate, and benzene sulfonate deter-gent homologues as sulfite by P. aeruginosagrowing at the expense of these compounds.However, phenol was not formed duringgrowth of this organism on the benzene sulfo-nate substrates, and desulfonation appeared to

HO

± Hg0~ ' H2

Y 1/20Q( + C02

OOH OH

KCAL.

(1IC.

410.4

'CAL.

+8.1S

+25.4 421.5

-34.5 -33.2

FIG. 1. Proposed pathways for the oxidation ofbenzyl alcohol to benzaldehyde, benzaldehyde tobenzoic acid, and benzoic acid to phenol by isolateHK-i.

+ H20+

+ NaHS03 4 H

FIG. 2. Mechanism of desulfonation of an alkylbenzene sulfonate intermediate by isolate HK-1.

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DEGRADATION OF ALKYL BENZENE SULFONATE

involve the incorporation of molecular oxygeninto the aromatic nucleus to yield catechol.

Neither Focht and Williams (11) nor Ripin,Noon, and Cook (26) were able to detect anyaccumulation of sulfite during metabolism ofp-toluene sulfonate by Pseudomonas sp. How-ever, Ripin et al. (26) suggested that this mightbe due to the rapid enzymatic conversion ofsulfite to sulfate. The direct production of sul-fate as described in these reports (11, 26),without the intermediate formation of sulfite,is therefore questionable.Phenol was not reported to be an interme-

diate product of p-toluene sulfonate degrada-tion in either of the investigations (11, 26).However, Cain and Farr (8), Focht and Wil-liams (11), and Ripin et al. (26) were con-cerned with the complete metabolism of arylsulfonates by organisms capable of utilizingthese compounds as sole sources of carbon andenergy, rather than wtth co-metabolism ofthese aromatics in the presence of additionalcarbon and energy sources. Results obtainedby Horvath (15, 17) and Horvath and Alex-ander (18) indicated that the co-metabolic at-tack of chloro-aromatic compounds differedfrom the complete metabolic attack (16) inthat co-metabolism was not inhibited by meta-substitution whereas normal metabolism wasinhibited (2). It is not surprising therefore, thatthe co-metabolism of aryl sulfonates wouldalso differ from the mode of normal metabolicdegradation of these substrates and thanphenol was found to be an intermediate in thedegradation of ABS by the process of co-me-tabolism.

Since benzyl alcohol, benzaldehyde, andbenzoic acid are present in the ABS-glucose-salts spent media, it was postulated that adap-tation of the isolate to these compounds shouldoccur in the growth medium. Simultaneousadaptation growth response experiments wereemployed to test this hypothesis. The adaptedcells were grown in ABS-glucose-salts me-dium, and the nonadapted cells were grown inglucose-salts medium lacking ABS. Growth ofthe adapted strain began immediately at theexpense of benzyl alcohol, benzaldehyde, andbenzoic acid. Significantly, the nonadaptedstrain exhibited a lag period of 45 to 75 minwith these substrates, which was in agreementwith the manometric results of Stanier, whoreported a lag period of 30 to 80 min with non-adapted cultures (28, 29).

Accumulation of stoichiometric amounts ofcatechol in media containing ABS was notconsistent with the ability of the isolate to uti-

lize benzoic acid as a sole source of carbon andenergy for growth in a medium not containingABS. However, further metabolism of catecholappeared to be inhibited in the presence of thedetergent. The Pseudomonas was capable ofgrowth in a catechol-basal salts medium onlyin the absence of ABS. Growth was completelyinhibited in the presence of ABS.

Initial accumulation of catechol could resultfrom a lag in the appearance of an oxygenaserequired to further oxidize this compound, fol-lowing the first sequential inductive steps assuggested by Hegeman (14). After 32 days,catechol was present at a 2.0 to 2.4 mM con-centration, which equalled the amount of ABSdegraded. With 2.0 mmoles of catechol andonly 0.25 mmole of ABS, growth would notoccur at the expense of added glucose, indi-cating the presence of a toxic environment.The combination of accumulated catechol andnondegraded ABS seemed to produce a toxicenvironment, thus preventing further metabo-lism of the dihydroxylated benzene compound.This may also account for the fact that degra-dation of ABS never exceeded 75 to 80% of theinitial ABS supplied.The growth of isolate HK-1 in basal medium

supplemented with isopropanol occurred to theextent of 18.9 mg (dry cell weight) per mmoleof isopropanol in the absence of glucose. Thereactions involved in the utilization of isopro-panol appear to be those shown in Fig. 3. Allylalcohol, compound no. 3 [melting point (mp)of 3, 5-dinitrobenzoate derivative = 47 C; mo-lecular weight = 58]; glycidol, compound no. 4(mp of 3,5-dinitrobenzoate derivative = 94 C;molecular weight = 74); and acetol, compoundno. 5 (mp of semicarbazone derivative = 195,mp of 2, 4-dinitrophenylhydrazone derivative= 129; molecular weight 73 to 75) were identi-fied as products resulting from metabolism ofisopropanol by isolate HK-1.The formation of glycidol from allyl alcohol

indicated that a monooxygenase might func-tion in this system. A monooxygenase couldalso operate in the conversion of methylphen-ylcarbinol to phenylethyleneglycol as well as inthe formation of 8-phenyl-2, 4,6, 8-tetra-methyl-2-octanol from alkyl benzene. Thisreaction is shown in Fig. 4 (ABS consists of acomplex mixture of 100 or more compounds,and the structure given is speculative and mayrepresent only a small percentage of the ABSsupplied). The proposed pathways are con-sistent with experimental results to date andshow the general degradative scheme.

After 8-phenyl-2, 4,6, 8-tetramethyl-2-oc-

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4HORVATH AND KOFT

CH3-CHOH-CH3 (1)

1/2 02

+ CHT~.-..cI143

N-CHTCHOHH+ CH3-CHOH

FIG. 4. Proposed pathway for oxidation of the(4) alkyl side chain of ABS by a Pseudomonas.

qH3 vHH--CH H;H

6.CH3-C-CH OH

0

FIG. 3. Metabolism of isopropanol, an interme-diate product in the degradation of ABS, by a Pseu-domonas.

tanol is formed, t-butyl alcohol could resultfrom hydrolysis of the bond between the a andfB carbons to the first branch point. Hydrolysisof the bond between the a and is carbons tothe next branch point would then explain thepresence of isopropanol and 4-phenyl-2-pen-tanol. A repeat of the hydrolysis describedabove would yield a second mole of isopro-panol and methylphenylcarbinol as shown inFig. 5. Methylphenyl-carbinol could then un-

dergo oxidative conversion to phenyl-ethyleneglycol. Formation of mandelaldehydeand mandelic acid would occur as does theformation of benzaldehyde and benzoic acidshown previously (Fig. 1). Bond-energy calcu-lations yield an overall value of 4.04 mmoles ofATP per mmole of ABS for this reaction se-

( 5) TH3HH.

H3CH3 eHOH

11/°22H20H

OHA

|-2H

HO

n ~~~H2O

OOH

|tH2O '6

1HO

I-2H

CH20H

I H2iOOHsHOH

FIG. 5. Proposed pathway for oxidation of thealkyl side chain and the aromatic ring of ABS by aPseudomonas.

ries. This is to be compared to the value of 4.0mmoles of ATP per mmole of ABS obtainedfrom growth studies.

(2)

(3)

CH3- CH2-CH20H

I-2H

CH2 CH-CH20H

11/202

CH2 -bH-CH20H

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DEGRADATION OF ALKYL BENZENE SULFONATE

One possible means of desulfonation of theABS molecule is by way of a coupled reactionwith phenol (Fig. 2), and since we were unableto find any dodecyl benzene this must occur

later in the degradation pathway.The formation of significant amounts of

phenol resulting from ABS degradation couldoccur directly by decarboxylation of benzoicacid or could result from a reduction of cate-chol formed from benzoate oxidation. Phenolcould be detected in the supernatant fluidstaken from the growth media 3 days after inoc-ulation. Catechol concentrations did not reachsignificant levels until 30 days after inocula-tion. The order of appearance of these prod-ucts would tend to support the pathway pro-

posed for the formation of phenol from benzoicacid. The activation of growth in an ABS-saltsmedium by phenol, an apparent product ofABS metabolism, is in itself an interestingobservation.

Results of this work and that reported byHorvath (15-17) and Horvath and Alexander(18, 19) indicate that the concept of molecularrecalcitrance (1) may no longer be valid. Al-though those compounds previously consideredto be nonbiodegradable may not supportgrowth of microorganisms when supplied as

sole sources of carbon and energy, they are

subject to a co-metabolic attack, resulting inconsiderable alteration of the chemical andphysical properties of the original molecule.The theory of molecular recalcitrance (1)should be reconsidered in terms of co-metabo-lism as well as complete mineralization.

ACKNOWLEDGMENT

This work was supported by Public Health Service grantGM-T-505 from the National Institute of General MedicalSciences.

LITERATURE CITED

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26. Ripin, M. J., K. F. Noon, and T. M. Cook. 1971. Bac-terial metabolism of arylsulfonates. I. Benzene sulfo-nate as growth substrate for Pseudomonas testosteroniH-8. Appl. Microbiol. 21:495-499.

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APPL. MICROBIOL.

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