Bacterial Destruction of Sodium Nitrite in Open CoolingWater Systems1,2

D. G. LUNDGREN AND A. KRIKSZENS

Microbiological and Biochemical Research Center and Department of Bacteriology and Botany, Syracuse University,Syracuse, New York

Received for publication Februiary 17, 1959

Corrosion is a problem that has plagued man eversince he turned to the use of metals; corrosion is definedas the destructive alteration of a metal by reaction withits environment. Corrosion, viewed in a broad sense,has two main causes; these are:

1. Chemical-a direct cause. In this instance, anattack occurs on refined metals by acids, chemicalfumes, moisture, and air.

2. Biological-an indirect cause. This too is mediatedby chemical action but, in itself, represents a uniquecase. Within this category are found corrosion prob-lems due to the presence of microorganisms in indus-trial systems; the microorganisms of major importanceinclude sulfur bacteria, iron bacteria, and ammonia,nitrate and nitrite utilizing bacteria. The presence ofbacteria can result in conditions under which chemicalcorrosion occurs, whereas, in the organism's absence, cor-rosion ordinarily would not be found. A prime exampleof this situation is the microbiological attack and break-down of a chemical substance used as an anticorrosionagent, which loss results in corrosion.The present paper reports on a problem of this type;

specifically, the destruction of sodium nitrite in opencooling water systems. This study investigated: (a) therelationship between bacterial activity and nitriteutilization, (b) techniques for mass propagation ofNitrobacter agilis for use both as a pure culture inocu-lum for controlled experimentation and as a source ofraw material for some basic physiological research, (c)chemical screening (laboratory-scale operation) as re-gards the compounds' effect on inhibiting nitrite oxida-tion by Nitrobacter agilis, and (d) the nitrite-stabiliz-ing capacity of various chemicals in circulating watersof pilot plant cooling systems.

EXPERIMENTAL METHODS

Culture Methods

Nitrobacter agilis strain ATCC 9482 was purified byrepeated transfers in an inorganic liquid medium fol-

1 These studies were under the direction of 1). G. Lundgreniand were aided by a contract between Solvay Process Division,Allied Chemical Corporation, and Syracuse University.

2 Portions of this investigation constitute part of a thesissubmitted by Arthur Krikszens to the Graduate School ofSyracuse University in partial fulfillment of the requirementsfor the Ph.D. in Microbiology.

lowed by selection of isolated clones, after plating outcells on either a silica gel inorganic medium or theinorganic medium containing Noble's agar. Pure cul-tures were maintained by repeated transfer in a liquidinorganic medium. The first experimental mediumused was that of Meiklejohn (1953). The medium com-position was: NaNO2, 1.0 g; NaCl, 9.6 g; MgSO4, 0.14g; KH2PO4, 0.27 g; CaCO3, 10.0 g; FeSO4, 0.03 g; dis-tilled water to 1 L. The pH of the medium was ad-justed to 7.5 and autoclaved at 15 psi for 15 min.

For mass propagation of Nitrobacter agilis, the syn-thetic medium of Alexander and associates (Alexanderet al., 1957; Aleem and Alexander, 1958; Alexander andAleem, 1958; Alexander and Engel, 1958) was em-ployed. The synthetic medium contained:

9A. NaNO2.. .0.3

MgSO4 ... 0.17 Sin 500 ml water-autoclavedNaCl ..0.17 J

B. FeSO4.... 0.015 in 100 ml water Seitz filteredC. K2HPO4. .0.17 in 200 ml water-autoclavedD. CaCl2 ...0.06 in 100 ml water-autoclavedE. KHCO3. .1.50 in 100 ml water Seitz filtered

Total. 1000 ml

The components of the medium were treated as follows:

A, C, D-adjusted to a pH of 8.0 before autoclavingB-not adjustedE-adjusted to pH 8.0 before filteringFinal pH of medium after autoclaving was 7.5.

The medium was compounded in 6-, 8- or 10-Lquantities in either a 3- or 5-gallon solution bottle-typefermentor equipped with a sparger (bacteriologicalfilter). Sterile air was supplied at a rate of 0.71 L per Lper min by serial passage through a Kelly bottle packedwith sterile glass wool and a humidifier containingsterile distilled water and fitted with a sterile porcelainfilter candle before dispersion into the medium througha second sterile filter candle. Culture temperature was26 C to 28 C. Inocula were built up from shake cul-tures, grown in 250-m] Erlenmeyer flasks containing 70ml of Alexander's medium, to a volume of 10 per centof the mass culture medium. Growth of N. agilis wasmaintained by a periodical recharging of the fermenta-

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BACTERIAL DESTRUCTION OF SODIUM NITRITE

tion medium with 300 ppm nitrite approximately every10 hr. The recharging with nitrite maintained activelygrowing cultures for 1 to 2 weeks or until another com-ponent of the medium became the factor limitinggrowth. Using this procedure, sizable yields were ob-tained of pure cultures of N. agilis.

Cells were harvested by continuous centrifugation ina Sharples3 centrifuge; the cells were resuspended in asmall volume of phosphate buffer (pH 7) and frozen.N. agilis stored in this manner remained viable for 14days; after 14 days viability dropped off rapidly. Thestored cells were used both for inoculation purposes andfor physiological studies.

Prior to the employment of Alexander's medium, at-tempts were made to modify Meiklejohn's nitrobactermedium to increase growth of N. agilis. The effects ongrowth of pH and of different phosphate sources werestudied. Growth and nitrite utilization were investi-gated in Meiklejohn's medium at adjusted pH levels,ranging from 2 to 11, and employing shake cultures ata temperature of 28 C. Three different sources of in-organic phosphates were studied; these were tribasicsodium phosphate, sodium pyrophosphate, and sodiummetaphosphate at concentrations ranging from 0.02to 0.04 g per L of medium.

Different organic supplements to Meiklejohn's me-dium were also investigated as regards growth promot-ing properties. The mineral medium was supplementedwith various intermediates (added singly) common tothe glycolytic cycle and the tricarboxylic acid cycle;these were (used at 1.0 g per L): calcium glycerophos-phate, fructose-6-phosphate, sodium pyruvate, potas-sium citrate, sodium succinate, sodium fumarate, L-malic acid, a-ketoglutaric acid, oxalosuccinic acid,sodium oxalacetate, glucose-6-phosphate, isocitric acid.

Chemical Screening for Nitrite Stabilizers UsingShake Cultures

A rapid screening test for detecting chemicals inter-fering with the oxidation of nitrite by N. agilis wascarried out in the following manner: Chemicals to betested were added in two concentrations (1 ppm and10 ppm active ingredients) to the nitrobacter mediumcontaining 300 ppm sodium nitrite, so that the finalvolume in 250 ml Erlenmeyer flasks after inoculationwas 50 ml. Each flask was inoculated with 1 ml of anitrobacter stock culture containing 1 X 109 cells perml. The flasks were shaken on a rotary shaker at roomtemperature. Nitrite levels were determined everythird day for a period of 12 days.Compounds inhibiting some growth of N. agilis were

also tested at 50 ppm in shaken flasks prior to an in-vestigation at the pilot plant level. The chemical com-

3 The Sharples Corporation, Philadelphia, Pennsylvania.

pounds screened included:2, 4-DinitrophenolSodium azideSodium fluorideSodium iodoacetateSodium malonaten-Butyl-p-hydroxybenzoateHydroxylamine hydrochloride8-HydroxyquinolineCopper-8-quinolinolateUrethan (ethyl carbamate)SulfanilamideOttacide (dichloro-m-xylenol [position of Cl groups

not knownl 3, 5-dimethyl-2, 4-dichlorophenol)p-NitrophenylhydrazinePyridylmercuric acetate (technical)4-ChlororesorcinolHyamine 2389 (alkyl tolylmethyl trimethyl am-monium chloride)

Winroc (methylalkylbenzyl trimethyl ammoniumchloride)

Roccal (alkyl dimethyl benzylammonium chloride)Versene TBionol A-50 (alkyl dimethyl ammonium chloride)Shirlan A (salicylanilide)Dowicide A (sodium o-phenylphenate)Dowicide B (sodium 2,4, 5-trichlorophenate)Agri-Mycin 100 (streptomycin 15.0 %-oxytetracy-

cline 1.5 %)Agri-Strep (streptomycin sulfate 37 %)Vancide 51 (sodium salt of dimethyldithiocarbamic

acid plus the sodium salt of 2-mercaptobenzo-thiazole 30 %)

Preventol GDC (2,2'-methylenebis[4-chlorophenol])Ottasept 430 (p-chloro-m-xylenol)Arquad S (alkyl trimethyl ammonium chloride,

technical)Milban D (zinc dimethyldithiocarbamate 71.2%)Nalco D-1486 (commercial corrosion inhibitor)Nalco D-1487 (commercial corrosion inhibitor)Nalco D-1488 (commercial corrosion inhibitor)Nalco 889 (commercial corrosion inhibitor)Butrol (phenylmercuric acetate and potassium

o-phenylphenate)Bufen 30 (phenylmercuric acetate)BSM-11 (phenylmercuric acetate and potassium

2,4, 6-trichlorophenate)Busani 881 (disodium cyanodithioimidocarbonate)Duponol ME (sodium lauryl sulfate)Daxad 11 (sodium alkylnaphthyl sulfonate)Arquad 12 (alkyl trimethyl ammonium chloride)Arquad T-50 (alkyl or dialkyl methyl ammonium

chloride)Duomeen C (N-alkyl trimethylamine)Arquad C-50 (quaternary ammonium chloride)Chromium chloride (CrCl3 7H20)

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D. G. LUNDGREN AND A. KRIKSZENS

Duromium (Cr2(SO4)3. K2SO4 24H20)p-NitroanilinePreventol 1 liquid (sodium trichlorophenate 50 %)Sterimine (2,4, 6-tris[chloroamino]triazine 55.5 %)Tin-San (tributyltin-abietylamine ethylene oxide ad-

duct)o-Dianisidine

Figur e 1. Photograph shows a front view of three pilotplant cooling towers with the cooling water flowing over thewooden baffles. The water level of the reservoir was main-tained by means of an auitomatic float valve located in thereservoir.

Figure 2. Photograph shows a close-up of the back of asingle pilot plant containing the heating element, water pump,and switch box.

Pilot Plant Studies

The pilot plants used in this project were modifiedmodels of the open cooling water pilot plant describedby Haering (1941). Modifications included the elimina-tion of the removable pipe corrosion study sectionsand construction of a single set of baffles rather thanthe double set so described. Six units were employedfor this investigation. Photographs of the units areshowni in figures 1 and 2.

Following development of mass culture techniquesfor growing nitrobacter, large enough numbers of cellswere available to make studies at the pilot plant levelmeaningful. Nitrite loss in the circulating water wasfirst investigated under normal conditions, that is,under conditions where no known bacterial inoculumwas added to the system. The pilot plants were chargedwith 9 L of water, and 500 ppm sodium nitrite. Nitrite,nitrate, and ammonia levels were checked daily usingthe methods of Snell and Snell (1954). Bacterial platecounts of the cooling water were made employing nu-trient agar for isolation of the heterotrophic popula-tions and either washed agar or Noble's agar supple-mented with Alexander's medium for isolation ofNitrobacter agilis.Sodium nitrite levels (500 ppm) in the circulating

water of the pilot plants were studied after inoculationof the water with known amounts of Nitrobacter agilis.The inoculum contained approximately 1 X 106 cellsper ml, which number was determined using a Petroff-Hausser4 counter; 20 ml of inoculum were used. Am-monium and nitrate levels were also checked throughoutthe run. Bacterial cell counts of the water were deter-mined as outlined above.

All pilot plants were exposed to normal atmosphericconditions. The water levels were automatically regu-lated by means of a float valve. Temperature was regu-lated by a thermostat heating element; the temperatureof the water of the reservoir was approximately 28 C.The effects of various chemical agents on stabilizing

the nitrite levels of the circulating water were studied.Some chemicals were tested without prior testing inshake culture; the remainder examined were demon-strated to have had some inhibitory effect upon nitro-bacter. Those commercial germicidal or bacteriostaticchemicals (stabilizers) tested without any previousscreening at the laboratory level were: sorbic acid, 200ppm; sodium propionate, 200 ppm; methyl-p-hydroxy-benzoate, 200 ppm; propyl-p-hydroxybenzoate, 200ppm; zinc sulfate, 200 ppm; ammonium sulfate, 200ppm; ammonium phosphate, 200 ppm; ammoniiumnitrate, 200 ppm; ammonium acetate, 200 ppm; am-monium molybdate, 200 ppm; streptomycin sulfate,100 ppm. When a sensitive analytical method wasreadily available, the level of the chemical stabilizer at

I C. A. Hauisser and Sons, Philadelphia, Pennsylvania.

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BACTERIAL DESTRUCTION OF SODIUM NITRITE

different times was quantitated along with the levels ofnitrite, nitrate and ammonia. Sorbic acid, zinc sul-fate, ammonium compounds and streptomycin sul-fate were quantitated; the anlytical methods wereMelnick and Luckman, 1954, Humphries, 1956, Snelland Snell, 1954, and Grove and Randal, 1955, re-spectively. Bacterial counts were checked daily. Of some54 compounds screened at the laboratory level, as re-gards their stabilizing effect on nitrite, 10 were selectedfor testing at the pilot plant level; these compoundswere: sodium iodoacetate, 10 ppm; sodium azide, 1 and10 ppm; Hyamine, 50 ppm; Winroc, 50 ppm; PreventolGDC, 50 ppm; Milban, 50 ppm; Butrol, 10 ppm;Bufen 30, 10 ppm; BSM 11, 10 ppm; Arquad S, 10 ppm.

Heterotrophic ContaminantsHeterotrophic contaminants found in the pilot plant

circulating water were isolated employing standardbacteriological methods. The isolates were maintainedas pure cultures. Isolates were identified using thestandard battery of biochemical tests and physiologicalreactions recommended in the Manual of Microbiologi-cal Methods (1957). All isolates were tested in staticculture in a synthetic medium as regards their abilityto utilize ammonia, nitrite, and nitrate as the solesource of nitrogen. The basal medium contained (percent): Na2HPO4, 0.3; KH2PO4, 0.1; K2HPO4, 0.05;MgSO4, 0.02; Na acetate, 0.16; glucose, 0.2; Na citrate,0.2; nitrogen source, 0.6; and trace elements of boron,5 X 10-6; calcium, 5 X 10-4; copper, 1 X 10-6; iron,2 X 104; manganese, 1 X 104; zinc, 2 X 10-4. ThepH of the medium was adjusted to 6.8 and autoclavedfor 15 min at 15 psi. Fifty ml of medium were dis-pensed in 250-ml Erlenmeyer flasks, inoculated with a1 per cent inoculum and stored at 28 C for 12 days.Nitrogen levels were determined every third day.

RESULTSCulture Studies

Purification of Nitrobacter agilis required as many as25 transfers in liquid medium, followed by streaking ona solid medium before single clone isolates gave purecultures. Criteria for purity were (1) no growth onnutrient agar after 14 days at 30 C, and (2) micro-scopic examination.Growth of N. agilis in Alexander's medium in the

fermentor was fairly heavy after 7 days' incubation.After the cells were harvested in the Sharples3 centri-fuge, a heavy paste of cells was obtained; upon resus-pending the cells in 100 ml of phosphate buffer (pH 7),the final cell count averaged 1 X 1012 to 1 X 1015cells per ml as counted with the Petroff-Hauser cham-ber.

Results of growth studies in shake culture usingAlexander's basic medium and employing pure cultureinocula showed a complete loss of nitrite by 6 days

(figure 3) with an increase in cell population. Ammoniaproduction was negligible, and the nitrate level rose inthe inoculated medium stoichiometrically. Nitrite lossin the absence of inoculum was negligible over a periodof 28 days.

Results of pH studies made with Meiklejohn'smedium indicated that the range of nitrite utilizationby nitrobacter was from pH 4 to pH 11; however, therange for optimum utilization was only pH 7.5 to pH 8.On either side of this optimum, nitrite utilization felloff rather rapidly as did growth.

Results of phosphate studies with Meiklejohn'smedium showed that the source of phosphate does playa role in growth of nitrobacter. A highly soluble, andtherefore available, source of inorganic phosphate suchas KH2PO4 must be available to support the growth ofnitrobacter. Sodium pyrophosphate (Na4P207. 10H20)and sodium metaphosphate (NaPO3) did not supportgrowth or nitrite utilization; tribasic sodium phos-phate (Na3PO4- 12H20) did support some nitrite utili-zation but very little growth.The additions of organic supplements to the medium

of Meiklejohn had no effect on enhancing growth.

500

450

400 -

350

300

E 250a.a.O

gm 200 -zIL0

ISO

z0

2 4 6 8 10 12 14 16 18 20 22 24 26 28TIME (DAYS)

Figure 3. Nitrite oxidation in cooling waters and in nitro-bacter medium. 0 = nitrite oxidation in shaken flasks inocu-lated with Nitrobacter agilis; 0 = uninoculated flasks (auto-oxidation); A = nitrite oxidation in the cooling water ofpilot plants inoculated with N. agilis; A\ = uninoculatedcooling water.

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D. G. LUNDGREN AND A. KRIKSZENS

TABLE 1

Effects of chemical stabilizers upon nitrite oxidation

N02 Level Days toCompound Tested Concen- _______Reach Fi-trationI naLe lInitial Final a ee

2, 4-Dinitrophenol ..........

Sodium fluoride.............

Sodium iodoacetate.

Sodium malonate....

Hydroxylamine*HCl.

Copper-8-quinolinolate...

Urethan.

Pyridylmercuric acetate...

Sodium azide.

n-Butyl-p-hydroxyben-zoate.....

8-Hydroxyquinoline.

Sulfanilamide..............

Ottacide...

p-Nitrophenylhydrazine

4-Chlororesorcinol .........

Hyamine..................

ppm

1050

Control1

1050

Control1

1050

Control1

1050

Control1

1050

Control1

1050

Control1

1050

Control1

1050

Control1

10Control

110

Control1

10Control

1

10Control

110

Control1

10Control

110

Control1

1050

Control

Ppm300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300

300300300300300300300300300300300300300300300300300300300300300300

ppm

0

0

0

0

0

0

0

0

0

1500

0

0

0

0

0

0

2600

0

0

0

0

0

0

0

0

0

0

0

2770

280300

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2802800

666666366121266636612366612666366612612126

6666126666666666666612123

TABLE 1-Continued

NO2 Level Days toCompound Tested Cne-_______Reach Fi-

tain

Initial Final nal Level

Winroc .....................

Roccal......

Versene T..................

Bionol A-50..

ShirlanA.

Dowicide A.

Dowicide B.

Agri-Mycin 100.............

Agri-Strep.........

Vancide 51.......

Vancide 32..

Preventol GDC.

Ottasept 430.

Arquad S.......

Milban D.... ..

Nalco D 1486.

Nalco D 1487.

Nalco D 1488.

ppm

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

1050

Control1

1050

Control1

1050

Control1

10Control

11050

Control1

10Control

11050

Control1

1050

Control1

10Control

110

Control1

10Control

ppm300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300

Ppm0

2509

289292

0000

275280

00

2500000000

61240295

00

15029000

265270

0000

275275275

00000

269270

00

245269

0000000000

61231212333312126312336333312121236121231012123610312121231010310121231012123333333333

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BACTERIAL DESTRUCTION OF SODIUM NITRITE

TABLE 1-Continued

NO2 Level Days toCompound Tested Concen- Initial_Final Reach Fi-

Initial Final nal Level

Nalco 889.

Butrol....

Bufen 30.

BSM-11.. .

Busan 881.

Duponol ME.

Daxad-l1.. .

Arquad 12.

Arquad T-50.

Duomeen C.

Arquad C-50..

Chromium chloride.........I

Duromium.................

Busan 586...............

o-Dianisidine...............

p-Nitroaniline.............

Preventol 1 liquid..........

Sterimine..................

Tin-San....................

ppm

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control1

10Control

110

Control

PPM300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300300

PPM000

287280

0288291

0286285

00000000000

2000

275300

029030000

2900000000000000000

280000

2950

2952950

33312123121231212339310101010101010121012121012121010121099691299969969991299012912126

Chemical Screening in Shake Cultures forNitrite Stabilizers

Table 1 shows the results of chemical screening tests.Results are expressed in terms of the change in thenitrite levels (ppm) and the time (days) needed for thechange to occur.

Pilot Plant StuudiesResults of pilot plant studies in which the cooling

water was charged with 500 ppm of nitrite and un-inoculated (under normal atmospheric conditions),showed the loss of nitrite to be slow. A loss of nitriteoccurred; however, the loss extended over a period of28 (or more) days. In plants charged with 500 ppm ofnitrite and inoculated with nitrobacter (20 ml of 1 X106 cells per ml suspension), the results showed a veryrapid and precipitous drop in the nitrite level; thezero level of nitrite was obtained by 6 days (figure 3).Nitrite loss in pilot plants receiving an inoculum closelyparalleled nitrite loss in inoculated shaken flasks,whereas nitrate and ammonia levels generally remainednegligible, never exceeding 5 to 10 ppm.

Nitrobacter cell counts of the inoculated water of thepilot plants increased with time, going from approxi-

10-

9-

8-

7-

6-

5-

4-

E3-

9LiUI.0

C 2-

m

z

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

TIME (DAYS)Figure 4. Bacterial growth curves of cooling water charged

withl nitrite. 0 = heterotrophic cells; * = Nitrobacter agilis.

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D. G. LUNDGREN AND A. KRIKSZENS

mately 1 X 103 cells per ml to 1 X 105 cells per ml.Heterotrophic populations increased from about 1 X102 cells per ml to 1 X 106 cells per ml (figure 4).

Sorbic acid had little stabilizing effect on nitrite, andwas lost from the uninoculated nitrite-charged systemrather rapidly; exhaustion of the acid was complete by8 days. Nitrite levels dropped slowly and evenly thefirst 8 days until a low level of sorbic acid was reached;after this, nitrite levels dropped more rapidly, reach-ing zero by 22 days. With the addition of nitrobacterto the circulation water, the nitrite level dropped tozero in 16 days; the level fell from 400 ppm to zero inthe last 8 days, at which time sorbic acid levels were

zero. The net effect of sorbic acid was to extend thenitrite utilization time 6 days.The effect of sorbic acid on growth of heterotrophs

as well as autotrophic nitrifiers was negligible and didnot cause aniy major deviations from the growth curves

shown in figure 4.The majority of other stabilizing compounds tested

without previous laboratory screening showed the same

general nitrite patterns. Generally, the stabilizerscaused a short lag which was followed by a rapid lossin nitrite; nitrite exhaustion from the cooling water oc-

curred by 20 days. Exceptions to the general picturewere noted in the case of zinc sulfate, streptomycinsulfate, and ammonium phosphate. Zinc sulfate inthree concentrations (100, 200, 400 ppm) had a stabiliz-ing effect upon nitrite for 44 days. Streptomycin sul-fate prevented complete nitrite loss for 40 days. Am-monium phosphate had an enhancing effect on nitriteloss, where the nitrite level fell to zero by 6 days.The ammonium compounids caused a very rapid rise

in the heterotrophic population (1 X 105 cells per mlby 5 days). Nitrobacter counts rose slowly in the pres-

ence of high levels of ammonium ions; however, thecell numbers increased as the ammonium ion level de-creased. The addition of ZnSO4 to the circulatingwater checked bacterial growth for 23 days, whereassodium propionate, methyl-p-hydroxybenzoate, andpropyl-p-hydroxybenzoate had no demonstrable effectupon the bacterial growth.

Table 2 lists 10 compounds, previously tested at thelaboratory level, which retarded nitrite oxidation.Only two of these when tested in the pilot plants were

able to stabilize the nitrite in the cooling waters formore than 20 days. Pilot plants inoculated with N.

agilis had no nitrite after 6 days (control) whereasuninoculated uniits showed zero nitrite levels by 28days (figure 3). Sodium azide (10 ppm) in the coolingwater retarded nitrite utilization considerably. Thenitrite zero level was not reached until the 43rd day.Preventol GDC stabilized nitrite in the cooling waterfor 31 days.The effects on growth of microorganisms in the cool-

ing water by the ten compounds shown in table 2 were

very similar; exceptions were noted with sodium azideand Preventol GDC. All eight compounds caused a lagin the growth of both heterotrophic and autotrophicpopulations, although the results of cell counts indi-cated that both populations eventually reached num-

bers approaching those found in the cooling watercharged with only nitrite. The azide prevented growthin the cooling water for 7 days; thereafter, total growth

Nitrite stabilizingTABLE 2

ability of compounds inof cooling systems

circulating water

NOs LevelConcen- N02__Level_ Days to ReachCompound Tested tration Final Level

Initial Final

ppm ppm ppm

Sodium iodoacetate.. 10 500 0 6Control 500 0 6

Sodium azide. 1 500 0 2010 500 0 43

Control 500 0 6Hyamine ............. 50 500 0 20

Control 500 0 6Winroc ............... 50 500 0 20

Control 500 0 6Preventol GDC. 50 500 300 31

(Experimentstopped)

Control 500 0 6Milban D. 50 500 0 10

Control 500 0 6Butrol. .. .. 10 500 0 10

Control 500 0 6Bufen 30. 10 500 0 20

Control 500 0 6BSM 11. 10 500 0 8

Control 500 0 6Arquad-S. . 10 500 0 8

Control 500 0 6

TABLE 3Utilization of nitrogen sources for growth by microorganisms

isolated from cooling water

Nitrogen SourceOrganism

NO2 NOs NH2

Sarcina lutea . - +Micrococcus pyogenes var. albus (Sta-

phylococcus aureus).- __Micrococcus epidermidis (Staphylococcus

epidermidis).- -_Bacillus sphaericus .......................

Bacillus brevis. . + +Bacillus subtilis..+ +Pseudomonas putrefaciens.. - +Escherichia freundii . ......... + +Flavobacterium aquatile.- - _Flavobacterium diffusum.- __Alcaligenes viscosus.- -_Rhodotorula sp.+ + +

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was sluggish and nitrobacter cell counts never exceeded1 X 103 cells per ml. Preventol GDC had a similareffect on bacterial growth, highest nitrobacter countrecorded being 1 X 104 cells per ml.

Heterotrophic Contaminants

Contamiinants isolated from the pilot plant coolingwaters were identified as bacteria except for one yeast.The yeast was tentatively identified as a Rhodotorulasp. The genera of bacteria represented were Escherichia,Bacillus, Pseudomonas, Sarcina and Alcaligenes.

Table 3 shows the different heterotrophic contami-nants isolated and also their ability to use nitrite,nitrate or ammonia as a nitrogen supply for growth inthe experimental synthetic medium.

DIscussIoNBiological oxidation of nitrite was investigated in the

circulating water of cooling systems; the mechanismfor nitrite loss was the oxidation of nitrite to nitrate.This fundamental reaction is one carried out by thechemoautotrophic bacterium Nitrobacter agilis, and isthe reaction from which energy is derived for growthand/or respiration. Results of experimental work re-ported here demonstrate that the genus Nitrobacter isat least one of the major causes of nitrite depletion incooling water systems.

It was apparent from a survey of the literature thatbasic knowledge was limited pertinent to the biologyand biochemistry of nitrobacter. Consequently, re-search ramified into a program encompassing theapplied aspects of the problem as well as into somebasic areas, for the applied problem was predicatedupon certain basic information. To study the relation-ship between bacterial action and nitrite oxidation,large quantities of nitrobacter (in pure culture) had tobe propagated. The use of classical media reported inthe literature and direct modifications thereof gave un-satisfactory growth results. The development of asuccessful growth medium by Alexander and his groupand the application of this medium proved fruitful tothe solution of our problems of culture purification andmass propagation of Nitrobacter agilis.

There were several striking differences in Meikle-john's medium from that of Alexander's; the mostnotable difference was the concentration of nitrite.Meiklejohn's medium contained 1000 ppm nitrite,whereas that of Alexander's contained only 300 ppm.The lower concentration employed in Alexander'smedium apparently allows the organism to establishitself in the medium. A second difference in the mediawas in the concentration of iron (FeSO4); Alexander'smedium contained one-third the concentration of iron.Iron, in increasing concentration, does stimulate nitriteoxidation but only up to a certain level; beyond a con-centration of 10 ppm iron appears to become inhibitory.

The pH of the medium employed by Alexander's groupwas 7.5 to 8.0 whereas that of Meiklejohn's mediumwas 7.0. The higher pH seems to enhance growth andnitrite utilization.

After the successful development of techniques formass propagation of pure cultures of Nitrobacter agilis,a chemical screening was feasible at both the laboratoryand pilot plant levels for nitrite oxidation inhibitors.The chemical screening program employed was simpleand direct. It was felt that if a chemical stabilizer insmall concentrations could slow down or inhibit theutilization of nitrite by nitrobacter in shake culture,then it should be tested at the pilot plant level.

It was apparent from the results that most of thebacteriostatic and bacteriocidal agents used commer-cially or in research laboratories (as competitive or non-competitive inhibitors) proved ineffective againstnitrobacter. The chemical compounds tested under thescreening program fell into one of two general cate-gories: (1) compounds such as sodium azide, whichinhibited or slowed down utilization of nitrite, and (2)compounds such as the ammonium derivatives, whichdid not inhibit oxidation. Those compounds that didinhibit nitrite oxidation most likely did so by directlyinhibiting important metabolic processes of this micro-organism. No attempt was made to uncover the mecha-nism of action of any of the stabilizers. The fact thatsome compounds did not stop nitrite loss is best ex-plained by the conception that these compoundsfailed to inhibit because of the particular metabolicmosaic of nitrobacter or because of permeability proper-ties. Some test compounds even enhanced nitrite loss,which action was by eitheI direct chemical action withthe nitrite (oxidation or reduction) or through sometype of a supplementary biological action directly orindirectly linked to nitrite oxidation.Whereas the laboratory screening program allowed an

investigation of the merits of various stabilizers underrestricted conditions, the pilot plant tests did permitan investigation of these compounds under conditionsapproaching those of industry. In the cooling water,the chemical compound was required to stabilize thenitrite under most complex conditions. Some of thevariables common to pilot plants were agitation, aera-tion, pH, and biological and chemical contamination.Contamination included that of the ubiquitous micro-organisms of air, soil, and so forth, where chemicalcontamination of the cooling water came from contactwith metal and wood. To require a compound to sta-bilize the nitrite level as well as remain stable itselfunder the constant fluctuations of these many condi-tions is a rigorous demand.

It might be emphasized that the conditions underwhich a compound was tested at the pilot plant levelwere somewhat controlled, that is, controlled in thesense that a known inoculum of Nitrobacter agilis was

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added to the circulating water. The autotroph addedcertainly exceeded by far the number of nitrobacterorganisms one would expect as contaminants in a sys-tem under normal industrial operation conditions. Inthis regard, it was felt that this (inhibiting nitriteoxidation by nitrobacter) was the most stringent of therequirements set up as the criteria for screening thesecompounds.

Further, it was felt that any compound which couldstabilize nitrite levels, even partially, under thesepilot plant conditions merits further investigation asit has potential value to industry.

ACKNOWLEDGMENTS

The authors wvish to thank Dr. A. P. Julien and Mr.R. Graham, Solvay Process Division, Allied Chemicaland Dye Corporation, for their suggestions during theinvestigation. Also, the helpful assistance of Dr. T.Demny, Merck & Company, Inc., is acknowledged.

SUMMARY

Preliminary studies are discussed as regards thephysiology of Nitrobacter agilis. The relationship of thepresence of nitrobacter as a contaminant in circulatingwater of cooling systems and the stability of nitrite asan antioxidant was studied. Flask-scale screening testsof compounds for their ability to stabilize nitrite incooling systems against oxidation by microorganismswas presented and results discussed. Those compounds

showing promise at the bench-level were further testedat the pilot plant level.

REFERENCESALEEM, M. I. H. AND ALEXANDER, M. 1958 Cell-free niitri-

fication by Nitrobacter. J. Bacteriol., 76, 510-514.ALEXANDER, M. AND ALEEM, M. I. H. 1958 Cell-free ntitri-

fication by Nitrobacter agilis. Bacteriol. Proc., A-7.ALEXANDER, M., ALEEM, M. I. H., AND ENGEL, M. S. 1957

The inhibition of nitrification by ammonia. Bacteriol.Proc., A-2.

ALEXANDER, M. AND ENGEL, M. S. 1958 Culture of Nitro-somonas europoea in media free of insoluble constituients.Nature, 181, 136.

GROVE, D. C. AND RANDALL, W. A. 1955 Assay methodsof antibiotics: a laboratory manual. Medical Encyclo-pedia, Inc., New York, New York.

HAERING, D. W. 1941 Experimental cooling systems.Design, construction and operation. Ind. Eng. Chem.,33, 1360-1365.

HUMPHRIES, E. C. 1956 Mineral components and ash anal-ysis, In Modern methods of plant analysis, Vol. 1, pp. 468-502. Edited by K. Paech and N. V. Tracey. Spriniger-Verlag, Berlin, Germany.

Manual of microbiological methods. 1958 By the Society ofAmerican Bacteriologists, Committee on BacteriologicalTechnic. McGraw-Hill Book Company, Inc., New York,New York.

MEIKLEJOHN, J. 1953 Iron and the nitrifying bacteria.J. Gen. Microbiol., 8, 58-65.

MELNICK, D. AND LUCKMAN, F. 1954 Sorbic acid as a fungi-static agent for foods. III. Spectrophotometric deter-minations of sorbic acid in cheese and in cheese wrappers.Food Research, 19, 20.

SNELL, F. D. AND SNELL. C. T. 1949 Colorimetric methodsof analysis, 3rd Ed., vol. 2. D. Van Nostrand Company, Inc.New York, New York. pp. 785-819.

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