APPLIED MICROBIOLOGY, Jan. 1968, p. 62-68 Vol. 16, No ICopyright © 1968 American Society for Microbiology Prinited in U.S.A.

Activated-Sludge Nitrification in the Presence ofLinear and Branched-Chain Alkyl

Benzene SulfonatesCHARLES R. BAILLOD AND W. C. BOYLE

Department of Civil Engineering, The University of Wisconsini, Madison, Wisconsin 53706

Received for publication 18 July 1967

The effects of biodegradable linear alkyl benzene sulfonate and branched-chainalkyl benzene sulfonate detergents on activated-sludge nitrification were investi-gated by administering a synthetic waste containing up to 23 mg of each detergentper liter to eight bench-scale, batch, activated-sludge units. It was found that bothdetergents tended to promote complete oxidation of ammonia to nitrate, whereascontrol units produced approximately equal amounts of nitrite and nitrate. Varioushypotheses are offered to explain the phenomenon.

Within recent years, biodegradable linear alkylbenzene sulfonates (LAS) have replaced theolder branched-chain alkyl benzene sulfonates(ABS) in nearly all household detergent formu-lations. It is apparent that these surfactantsreach biological waste treatment plants wherethey come into contact with the biota of theplant. Small concentrations of certain organicsubstances are known to exert inhibitory effectson nitrification (3, 6, 14, 17), and surface-activeagents have been reported to produce physio-logical effects on bacteria (6). The purpose ofthis study was to investigate the effects of anionicdetergents on nitrification occurring in the ac-tivated-sludge process. Particular attention wasdevoted to determining what, if any, differencesexist between the effects of LAS and ABS onactivated-sludge nitrification.

In fresh sewage, most nitrogen is in the formof organic nitrogen (protein, urea, and aminoacids) with some ammonia. During biologicalstabilization of the waste, protein proteolysis anddeamination produces ammonia, and urea ishydrolyzed to form additional amounts of am-monia. Ammonia may be utilized in bacterialcell synthesis, or it may be oxidized to nitrite.Downing et al. (2) indicated that, in heavily loadedactivated-sludge plants, the rate of sludge syn-thesis is high, and the relatively slow-growingnitrifying bacteria are literally "squeezed out" bythe heterotrophic sludge mass; consequently,nearly all of the ammonia is utilized in sludgesynthesis. On the other hand, in lightly loadedplants the net rate of synthesis is low, and theslow-growing nitrifiers can become established in

the sludge, resulting in the conversion of am-monia to nitrite. The nitrite is usually rapidlyoxidized to nitrate; in the absence of dissolvedoxygen, nitrite may be denitrified to nitrogen gas.Denitrification has been proposed as an efficientmeans of removing nitrogen in waste treatment(9).

Nitrification may or may not be desirable, de-pending on the biota and dissolved-oxygen levelsof the receiving stream. Nitrate tends to encouragealgal blooms, whereas ammonia nitrogen servesas a nitrogen source for many bacteria. Nitratemay also act as a hydrogen acceptor through itsreduction to nitrite. Consequently, an effluentcontaining 10 mg of nitrate nitrogen per litercould supply an oxygenation capacity equivalentto 11.4 mg of dissolved oxygen per liter throughnitrate reduction. Furthermore, nitrification cancause a significant oxygen depletion when it takesplace in receiving waters. Thus, the advantagesand disadvantages of nitrification in waste treat-ment take on a quantitative significance only whenjudged in relation to the problems likely to beencountered in a particular receiving stream.

Nitrification is defined as the biological oxida-tion of ammonia to nitrite and the subsequentconversion of nitrite to nitrate according to thestoichiometric equations:

(1) 2NH4+ + 302 - 2NO2- + 2H20 + 4H+

(2) 2NO2-+ 02 - 2NO3-These oxidations are believed to be carried outprimarily by genera of the bacterial family Nitro-bacteriaceae, notably Nitrosomonas and Nitro-

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bacter. In view of the well-documented ability ofcertain heterotrophs to produce nitrite (4, 5, 7)and the reported ability of certain fungi to pro-

duce both nitrite and nitrate (13, 15), it is possiblethat at least a portion of the nitrification takingplace in activated sludge could be due to or-

ganisms other than the Nitrobacteriaceae. Sinceno isolation nor identification of nitrifying or-

ganisms was attempted in this work, organismswhich produce nitrite will be referred to collec-

tively as the nitrite-forming group; those whichproduce nitrate will be referred to as the nitrate-forming group.

MATERIALS AND METHODS

Eight 2-liter Erlenmeyer flasks were operated asbench-scale, batch, activated-sludge units. Aerationwas accomplished by passing unmetered air throughcylindrical diffuser stones at rates high enough tokeep the mixed-liquor solids suspended. With thisaeration procedure, the mixed-liquor dissolved oxygenlevels were maintained between 8 and 9 mg/liter.Each unit was inoculated with 200 ml of returnsludge obtained from the Nine Springs Sewage Treat-ment Plant, Madison, Wis. A substrate was thenadded and aeration was begun. Later, in order tosupply a more adequate inoculum of nitrifying organ-isms, each unit was inoculated with 100 ml of ahighly nitrified, unsettled, trickling-filter effluent(also from the Nine Springs Plant).The volumetric operating parameters for each

batch unit were maintained as follows: total volumeunder aeration, 1,430 ml; volume of substrate added,760 ml; volume of return sludge, 670 ml; and intervalbetween feedings, 24 hr. At the end of the aerationperiod, the sludge was allowed to settle for approxi-mately 30 min, and the supernatant fluid was siphonedoff. Fresh substrate (760 ml) was added to each unitand aeration was resumed. To minimize foaming, a

coating of Dow-Corning antifoam emulsion wasapplied to the necks of all units. Throughout theentire study, the mixed-liquor temperature rangedfrom 24.5 to 27 C.The substrate consisted of nonfat dry milk solids,

with ammonium phosphate, phosphate buffer, andtap water added to provide trace metals. A detailedcomposition and analysis of the substrate withoutadded detergents is as follows. Composition: nonfatdry milk solids, 500 mg/liter; (NH4)jHPO4, 10mg/liter as N; K2,HPO4, 348 mg/liter; tap water,14% (v/v); and distilled water, 86% (v/v). Analysis:pH, 7.85; chemical oxygen demand (COD), 500mg/liter; biochemical oxygen demand (BOD), 330mg/liter; organic nitrogen, 28 mg/liter as N; am-monia nitrogen, 10 mg/liter as N; nitrite, 0.0 mg/liter;nitrate, 0.0 mg/liter; alkalinity, 200 mg/liter asCaCO3; and methylene blue active substance (MBAS),0.1 mg/liter.

The two types of anionic detergents used werestandard reagents of known composition and per-centage of methylene blue active substance, obtainedfrom the Assoc. of American Soap and Glycerine

Producers, Inc. The composition of the reagents wasas follows. ABS reagent: ABS, 54.8%; Na2SO4,40.3%; free oil, 0.5%; and Na2CO3, 0.7%. Equivalentweight, 348. LAS reagent: sodium dodecyl benzenesulfonate, 42.1%; NaCl, 1.0%; alcohol insolubles(mainly Na2SO4 with some sodium tripolyphosphate),50.5%; and unsulfonated oils, 0.6%.

In the case of ABS, "alkyl" merely signifies arandomly branched hydrocarbon chain averaging12 carbon atoms in total length, whereas LAS denotesa homogeneous compound with a linear 12-carbonalkyl side-chain. Standard solutions of 4,000 mg ofMBAS per liter were prepared for each reagent andwere added to the substrate in order to obtain thedesired concentrations of detergents.

Chemical analyses were performed according tothe procedures outlined in Standard Methods for theExamination of Water and Wastewater (AmericanPublic Health Assoc., Inc., New York, 1960). Nitratewas determined by the brucine procedure, whereasammonia was determined by both direct nesslerizationand titration. Detergents remaining in the effluentwere determined according to the methylene blue pro-cedure proposed by Longwell and Maniece (11).

For a period of 2 months, the eight experimentalunits were operated on a substrate free of detergents.During the first month, the substrate compositionand volumetric loading parameters were varied inorder to develop a substrate composition and de-tention time which were both realistic and conduciveto nitrification. At the end of the first month, themixed liquor was homogenized and was redispensedto the units. During the second month, all units wereoperated identically on the detergent-free substratedescribed previously. Sludge was wasted occasionallyduring this period in order to maintain the mixed-liquor suspended solids in the neighborhood of 1,500to 2,000 mg/liter. After the acclimation period, thebatch-activated sludge units were subjected to threeconcentrations of both ABS and LAS detergents.The type and concentration of detergent fed to eachunit are shown in Table 1.

RESULTS

The initial-shock effects of the detergents wereobserved by sampling each unit at intervals during

TABLE 1. ABS and LAS concentrations present infeed solutions

Unit Type of detergent (mg/liter)

1 LAS 6.62 LAS 13.83 LAS 23.04 ABS 6.65 ABS 13.86 ABS 23.07 Control 0.08 Control 0.0

a MBAS = methylene blue active substance.

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the 23-hr aeration period immediately followingthe initial addition of detergents. Each samplewas analyzed for nitrite, nitrate, and COD. Or-ganic nitrogen and ammonia determinations wereperformed on a portion of the samples. Duringthis initial period, the average organic nitrogenand ammonia concentrations decreased from ap-proximately 10 to less than 5 mg/liter, whereasthe average oxidized nitrogen concentrations in-creased from initial values of less than 1 to 4 mgof nitrite per liter and 3 mg of nitrate per liter.It was not possible to demonstrate any effect ofdetergents on nitrification during this initialperi6d, since four out of six units produced efflu-ent COD, nitrite, and nitrate concentrationswithin the range established by the two controls.Some sorption of detergents was apparentlytaking place, because samples from units 3 and6 showed 61.5 and 57% removal, respectively,after 2 hr of aeration (calculated on the basis ofinitial MBAS concentration in the mixed liquor).After 23 hr of aeration, MBAS removal forunits 3 and 6 was 71 and 75%, respectively.

Immediately after the initial-shock effect in-vestigation, substrate containing detergents wasfed daily over a 29-day period. The levels of de-terizent annlied to each unit and the oneratineparameters were the same as thosNo solids were wasted during tifor the portion lost in the effluentThe effluents of all units weretrite, nitrate, pH, and COD,

z

co

15

E

zw

10

z

aw 5

0x0

5 10 15TIME, DAYS

FIG. 1. Oxidized nitrogen and3, 23 mg ofLAS per liter.

0 5 10 15 20 25TIME, DAYS

FIG. 2. Oxidized nitrogen and pH profiles. Unit 6,23 mg ofABS per liter.

leoutlinedabove, liquor suspended solids were determined at in-his period, except tervals over the 29-day period. Occasional organict and in sampling, nitrogen and ammonia analyses were performedanalyzed for ni- on the effluents.and the mixed- Typical oxidized nitrogen and pH profiles ob-

served during this period are shown in Fig. 1 and2 for units 3 and 6, which were subjected to 23mg of LAS and ABS per liter, respectively. Thecorresponding profiles for the control units 7and 8 are shown in Fig. 3 and 4. The profiles ofall units demonstrated the same pattern as thosereported by Downing et al. (2), with nitritebuilding up first, followed by a subsequent in-crease in nitrate. The effluent ammonia and or-ganic nitrogen concentrations were generally be-tween 1 and 5 mg/liter during this period. Adefinite relationship may be observed betweenthe oxidized nitrogen and the pH profiles, as anincrease in total oxidized nitrogen is normallyaccompanied by a marked decrease in pH. Thisis caused by the liberation of hydrogen ions duringthe conversion of ammonia to nitrite, accordingto equation 1.

It is evident from Fig. 1-4 that nitrification wasnot taking place to any great extent at the be-ginning of the 29-day test period. This was prob-ably because the suspended solids were main-

20 25 tained at approximately 1,500 mg/liter by occa-sionally wasting sludge during the acclimation

pH profiles. Unit period. At lower suspended solids concentrations,the net rate of sludge synthesis tended to increase;

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LAS AND ABS IN SI

consequently, more nitrogen was used in synthesisand less was available for nitrification.The most obvious differences between the oxi-

dized-nitrogen levels of the controls and theoxidized-nitrogen levels of the units fed the twotypes of anionic detergent were that, after 29days of operation, (i) almost all (98%) of theoxidized nitrogen in the effluent of the units fedthe ABS detergent was in the form of nitrate,whereas only about half of the oxidized nitrogenin the control units was in the form of nitrate,and (ii) the percentage of oxidized nitrogen ap-pearing as nitrate varied directly (but not lin-early) with the concentration of LAS detergentapplied to the units.A nitrite and nitrate analysis of the effluents

after 29 days of operation using the substrates

LUDGE NITRIFICATION 65

containing detergents is shown in Table 2. Fig-ures 5 and 6 show the levels of oxidized nitrogenplotted as a function of substrate-detergent con-centration for the LAS and ABS detergents, re-spectively. The mixed-liquor suspended solidsconcentrations are plotted in the lower portionof these figures. It is evident that the oxidized-nitrogen concentrations generally increase withincreasing detergent concentrations up to a pointand then decrease. This indicates that, below agiven concentration, detergents may actuallystimulate nitrification; in higher concentrations,they inhibit nitrification.Throughout this study, the sludges exhibited a

predominance of rotifers of the two genera,Philodina and Lecane. The sludge of unit 6, fed23 mg of ABS per liter, exhibited lower protozoan

I

0 5 10 15 20 25TIME, DAYS

FiG. 3. Oxidized nitrogen and pH profiles. Unit 7,control.

5 10 15TIME, DAYS

20 25

FIG. 4. Oxidized nitrogen and pH profiles. Unit 8,control.

TABLE 2. Oxidized nitrogen concentrations present in the effluents on day 29

Type of detergent

Substrate LAS ABS Controls

Unit 1 Unit 2 Unit 3 Unit 4 Unit 5 Unit 6 Unit 7 Unit 8

Methylene blue active substance 6.6 13.8 23 6.6 13.8 23 0(mg/liter)

Nitrite (mg/liter) 13.0 7.5 0.8 0.6 0.2 0.3 12.0 11.0Nitrate (mg/liter) 18.0 24.4 22.2 23.4 16.2 11.4 12.5 12.5Total oxidized N 31.0 31.9 23.0 24.0 16.4 11.7 24.5 23.5Per cent as nitrate 58 74 97 98 99 98 51 53

0 NITRITEa NITRATE

I7.0~

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30

Z 25I

4

20E

zw 15

0

0

w

N

5

0

_

* TOTAL OXIDIZED NITROGENO NITRATE

O

3500 -

3000< -

en 2500 @E 2000 l

0 5 10 15 20 25

LAS CONCENTRATION ¶,Y MBAS

FIG. 5. Oxidized nitrogen and mixed-liquor sus-

pended solids as a function of LAS concentration.Data ofday 29.

0 5 10 15 20 25

ABS CONCENTRATION mY MBAS

FIG. 6. Oxidized nitrogen and mixed-liquor sus-

pended solids as a function of ABS concentration.Data ofday 29.

and rotifer activity. No filamentous organismswere noted at any time.

DIscussIoNAlthough the oxidized nitrogen and pH pro-

files never exhibited steady-state behavior fromday to day, the random, high-frequency fluctua-tions seemed to disappear toward the end of the29-day period. Thus, the oxidized nitrogen con-centrations produced at the end of the periodwere considered to be a valid measure of thesurfactant effects.The results shown in Fig. 5 and 6 could be ex-

plained by a number of hypotheses, one of whichis stated in terms of the effects of surface-activeagents on cell permeability, where permeability isdefined as the sum of all characteristics associatedwith the transfer of substances across cell mem-branes. According to the selective-solubilitytheory of cell permeability (10), the lipid contentof the cell membrane determines its permeability.Since lipids, like the hydrocarbon portion of adetergent molecule, are hydrophobic and areinsoluble in water, they will be solubilized bydetergents. Thus, it is possible that detergentsmay increase cell permeability by solubilizationof the lipid material in the cell membrane. Sinceincreased permeability will effect a higher rate ofsubstrate transfer into the cell, it may produce ahigher rate of metabolism. However, as the con-centration of detergent increases, it is possiblethat the permeability of the cell membrane 'is in-creased to a point where it can no longer functionto retain the vital protoplasmic constituents (8).At this point, cell metabolism would be impairedas vital constituents would be lost to the medium.This hypothesis is supported by reported stimu-latory effects of low concentrations of anionicdetergents (6) and is further reinforced by thewell-known inhibitory effects of very high con-centrations.

Concentrations of up to approximately 10 mgof LAS per liter stimulated both nitrite and ni-trate formation, whereas higher LAS concen-trations produced a decrease (Fig. 5). Accordingto the hypothetical mechanism of action of de-tergents described above, concentrations of upto approximately 10 mg of LAS per liter wouldstimulate ammonia transfer into cells of thenitrite-forming group, causing this group to be-come established in the sludge. Higher LAS con-centrations would impair the transfer rate, re-sulting in diminished numbers of ammonia-oxidizing organisms. Since the transport of sub-strate ammonia into the nitrite-forming group isprobably connected with active transport or with

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facilitated diffusion, substrate transfer would beimpaired if detergents increased cell permeabilityto a point where the enzymes needed for thetransport mechanism would be lost to the me-dium by ordinary diffusion.The decrease in nitrate formation exhibited at

the higher surfactant concentrations (Fig. 5 and6) was a result of the limited amount of nitriteavailable for oxidation to nitrate. Thus, in thepresence of a sufficient nitrite concentration, theestablishment of the nitrate-forming group of or-ganisms in the sludge might be facilitated by thesame concentrations of surfactants which inhibitestablishment of the nitrite-forming group. Thishypothesis would account for the extremely loweffluent nitrite concentrations produced in thepresence of surfactant concentrations equal to orin excess of 23 mg of LAS per liter and 6.6 mgof ABS per liter.Another hypothesis would be that the surfac-

tants facilitated the establishment of differentspecies of nitrifying organisms through a processof selection. However, as previously stated, littleis known regarding which species of nitrifiers areresponsible for the bulk of nitrification both inactivated sludge and in natural waters.A third hypothesis could be stated in terms of

the surfactant effects on the net suspended-solids accumulation. Figures 5 and 6 show thatthe units fed the higher concentrations of bothsurfactants accumulated more suspended solids.Since no solids were wasted over the test period(except for those lost in the effluent and in sam-pling), and since there appeared to be no sig-nificant difference between the solids content ofthe effluents, the accumulated suspended solidsrepresent a qualitative measure of sludge growth.Downing et al. (5) derived an equation defining

the minimum period of aeration which must beexceeded in order to obtain nitrification consist-ently under steady-state conditions. This valueis given approximately by:

AS(2) tm =kSkmS

tm = Minimum period of aera-tion which must be ex-ceeded to obtain nitrifica-tion consistently

AS = Increase in suspended solidsconcentration producedover the aeration periodtm

S = Suspended solids concen-tration

km = Specific growth-rate con-stant of the nitrite-form-ing group

According to this relationship, nitrificationwould be favored by increasing suspended-solidsconcentrations, provided that the sludge pro-duction did not increase with increasing solidsconcentrations. Normally, higher sludge concen-trations and the concomitant lower sludge loadingratios will effect an increase in the rate of auto-oxidation, thus causing a decrease in the net rateof sludge production.

In this study, the data indicated that, at theend of the experimental period, the units withthe higher sludge concentrations were also exhibit-ing relatively high rates of sludge production;consequently, more ammonia nitrogen wouldhave been channeled into sludge synthesis andless would have been available for nitrification.This would account for the decreasing values oftotal oxidized nitrogen produced by increasingsludge concentrations. However, the mixed-liquorpH varied markedly with the degree of nitrifica-tion, so that it is difficult to distinguish the effectsof surfactants on sludge production from thoseof nitrification-induced pH changes.A comparison of Fig. 5 and 6 shows that ABS

exhibited proportionately greater effects on theoxidized nitrogen levels than did similar concen-trations of LAS. Although low concentrationsof ABS did not appreciably stimulate the forma-tion of total oxidized nitrogen, concentrationsof from 6.6 to 23 mg of ABS per liter inhibitedformation of total oxidized nitrogen considerablymore than did similar concentrations of LAS.In short, 6.6 mg of ABS per liter produced verynearly the same effects on the oxidized nitrogenconcentrations as did 23 mg of LAS per liter.The differences between the effects of LAS and

ABS on nitrification could be due, at least inpart, to the greater biodegradability of LAS, sinceoccasional effluent MBAS analyses showed anaverage of 95.1% removal for LAS and only88% removal for ABS. However, since the physi-cal properties of surfactant solutions are influ-enced by the nature of the alkyl side chain of thedetergent molecule, it seems likely that the ob-served differences in effects on nitrificationbetween LAS and ABS were not entirely dueto the difference in biodegradability between thetwo types of surfactants. Furthermore, sincedetergents are known to sorb onto biologicalfloc, it may be concluded that an effluent deter-gent analysis is not a quantitative measure ofbiodegradability. It is possible that the improve-ment in biodegradability of LAS over ABS was

greater than the effluent detergent analysis indi-cated.

Manganelli and Crosby, in 1953 (12), foundnitrification in activated sludge to be inhibited

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completely by 10 mg of anionic detergent perliter. In light of the results reported by others(1) regarding the effects of detergents on oxygentransfer, it appears that the results whichManganelli and Crosby obtained from shake-flaskstudies were due chiefly to the effects of the deter-gent on oxygen transfer. Since the critical oxygentensions for nitrifying bacteria are appreciablyhigher than those for other aerobic organisms(16), the nitrifiers are especially sensitive to lowoxygen concentrations. It has been shown (1)that the diffused-air absorption coefficientdecreased drastically when 10 mg of anionic deter-gent per liter was added to a detergent-freesewage, thereby causing nitrification to cease.Wuhrmann (18) reported that the level of oxidizednitrogen in activated sludge depended primarilyon the hydraulic loading and solids concentration.However, at certain loadings, dissolved oxygenvalues were found to affect nitrification greatly,as a mixed-liquor dissolved-oxygen concentrationof 1 mg/liter produced an effluent in which only5% of the inorganic nitrogen was oxidized;whereas, at the same loading, 85 and 90% of theeffluent inorganic nitrogen was oxidized at mixed-liquor dissolved-oxygen values of 4 and 7 mg/liter, respectively.

In this study, mixed-liquor dissolved-oxygenconcentrations were maintained between 8 and9 mg/liter. The dissolved-oxygen concentrationof the units fed detergents was virtually equalto that of the controls. However, in treatmentplants, it would be both difficult and uneconomi-cal to maintain this dissolved-oxygen concentra-tion. Consequently, depending on the aerationcapacity, detergents might inhibit nitrification intreatment plants by decreasing the rate of oxygentransfer.Although it would have been desirable to strike

a nitrogen balance, it was not feasible in thisstudy, since the data suggested that some nitro-gen was being lost as nitrogen gas through deni-trification. This was especially evident during thesettling and feeding periods, as the measurednitrate concentrations immediately after feedingwere less than the calculated values.As pointed out in the preceding discussion,

several hypotheses could account for the effectsof detergents on activated-sludge nitrificationobserved in this study. Consequently, as in anyinvestigation of a mixed-culture system, cautionmust be exercised in drawing inferences regardingthe direct effects of detergents on any single groupof organisms.

ACKNOWLEDGMENTThis work was supported by the National Science

Foundation.

LITERATURE CITED

1. DOWNING, A. L., AND A. G. BOON. 1963. Oxygentransfer in the activated sludge process, p.131-148. In W. W. Eckenfelder and B. J.McCabe [ed.], Advances in biological wastetreatment, vol. 3. Pergamon Press, New York.

2. DOwNING, A. L., H. A. PAINTER, AND G.KNOWLES. 1964. Nitrification in the activatedsludge process. Inst. Sewage Purif. J., p. 130-153.

3. DOWNING, A. L., T. G. TOMLINSON, AND G. A.TRUESDALE. 1964. Effect of inhibitors onnitrification in the activated sludge process.Inst. Sewage Purif. J. Proc. Part 6, p. 531-554.

4. DOXTADER, K. G., AND M. ALEXANDER. 1966.Nitrification by heterotrophic soil microor-ganisms. Soil Sci. Soc. Am. Proc. 30:351-358.

5. EYLAR, 0. R., AND E. L. SCHMIDT. 1959. A sur-vey of heterotrophic micro-organisms fromsoil for ability to form nitrite and nitrate.J. Gen. Microbiol. 20:473-481.

6. GLASSMAN, H. N. 1948. Surface active agents andtheir application in bacteriology. Bacteriol.Rev. 12:105-148.

7. HIRSCH, P., L. OVERREIN, AND M. ALEXANDER.1961. Formation of nitrite and nitrate by acti-nomycetes and fungi. J. Bacteriol. 82:442-448.

8. HOTCHKISS, R. D. 1946. The nature of the bac-tericidal action of surface active agents. Ann.N.Y. Acad. Sci. 46:479-494.

9. JOHNSON, W. K., AND G. J. SCHROEPFER. 1964.Nitrogen removal by nitrification and denitri-fication. J. Water Pollution Control Federa-tion 36:1015-1036.

10. LAMANNA, C., AND M. MALLErrE. 1963. Basicbacteriology, p. 292. The Williams & WilkinsCo., Baltimore.

11. LONGWELL, J., AND W. D. MANIECE. 1955. De-termination of anionic detergents in sewage,sewage effluents, and river water. Analyst 80:167-171.

12. MANGANELLI, R., AND E. S. CROSBY. 1953. Effectof detergent on sewage microorganisms. Sew-age Ind. Wastes 25:262-276.

13. MARSHALL, K. C., AND M. ALEXANDER. 1962.Nitrification by Aspergillusflavus. J. Bacteriol.83:572-578.

14. QUAESTEL, J. H., AND P. G. SCHOLEFIELD. 1949.Influence of organic compounds on nitrifica-tion in soil. Nature 164:1068- 1072.

15. SCHMIDT, E. L. 1954. Nitrate formation by a soilfungus. Science 119:187-189.

16. SCHOBERL, P., AND H. ENGEL. 1964. Das Verha-lten der nitrifizierenden Bakterien gegenubergelostem Sauerstoff. Arch. Mikrobiol. 48:393-400.

17. THIMANN, K. V. 1963. The life of bacteria, p. 399-423. Macmillan Co., New York.

18. WUHRMANN, K. 1963. Effect of oxygen tension insewage purification plants, p. 27-40. In W. W.Eckenfelder and B. J. McCabe [ed.], Advancesin biological waste treatment, vol. 3. PergamonPress, New York.

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