Evaluation of Factors Affecting Survival ofEscherichia coli in Sea Water

V. Studies with Heat- and Filter-sterilized Sea Wateri

A. F. CARLUCCI,2 P. V. SCARPINO, AND DAVID PRAMER

Department of Agricultural Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication December 9, 1960

ABSTRACT

CARLUCCI, A. F. (Rutgers, the State University,New Brunswick, N. J.), P. V. SCARPINO, AND DAVIDPRAMER. Evaluation of factors affecting survival ofEscherichia coli in sea water. V. Studies with heat-and filter-sterilized sea water. Appl. Microbiol. 9:400-404. 1961.-The bactericidal action of sea water wasmeasured as the difference in survival of cells of Escher-ichia coli in untreated and autoclaved portions of watersamples. The beneficial effect of sterilization by heaton the survival of E. coli in sea water varied withseason and was most marked during summer months,however, the magnitude of the effect differed greatlyfrom sample to sample. The more obvious and com-monly suggested explanations for the bactericidalaction of sea water were tested experimentally. ThepH and salinity of sea water were changed by auto-claving, but the direction of the former was detri-mental rather than beneficial and the significance ofthe latter was not clarified. The survival of cells of E.coli in filtered portions of some water samples wasgreater than that in untreated portions and equal tothat in autoclaved portions, indicating that predatorsand competitors removed by filtration had contributedsignificantly to the rapid death of the bacterium in theuntreated water. However, in the majority of samplestested, survival of E. coli in autoclaved water was con-siderably greater than survival in filtered water.The possibility that the beneficial effect of auto-

claving over and above that of filtration resulted frominactivation or destruction by heat of bacteriophagesand thermolabile toxic substances such as antibioticswas considered. Moreover, the suggestion was testedthat the increased survival of E. coli in autoclaved seawater was due to the ability of heat to disrupt anddegrade microbial cells and thermolabile compounds

I Paper of the Journal Series, New Jersey AgriculturalExperiment Station, Rutgers, the State University, Depart-ment of Agricultural Microbiology, New Brunswick, N. J.This investigation was supported in part by research grantE1437 from the National Institute of Allergy and InfectiousDiseases, National Institutes of Health, U. S. Public HealthService.

2 Present address: Research Department, United FruitCompany, La Lima, Honduras, C. A.

400

and, thereby, to cause an increase in concentration ofavailable nutrients in sea water. It was concludedthat the bactericidal action of sea water is not explic-able in terms of the destruction or inactivation by heatof bacteriophages or antibiotics. Although addedorganic matter influenced the survival of E. coli, thetest organism was not an effective competitor in seawater and the nutrient levels required to offset thebactericidal action were excessive.

Artificial sea water was demonstrated to exert abactericidal action comparable to that of natural seawater. Low levels of cysteine which favor survival ofE. coli in natural sea water had a similar effect inartificial sea water. Nevertheless, it is not at this timepossible to conclude that the factors responsible forthe bactericidal action of artificial sea water are iden-tical with those responsible in natural sea water.

It has been observed repeatedly that bacteria sur-vive to a greater extent in heat-sterilized sea waterthan in untreated sea water (Nicati and Rietsch, 1885;De Giaxa, 1889; Kiribayashi and Aida, 1934; ZoBell,1936; Krassilnikov, 1938; Ketchum, Carey, and Briggs,1949; Vaccaro et al., 1950; Nusbaum and Garver,1955; Richou, Neant, and Richou, 1955). This ther-molabile bactericidal action of sea water varied withseason of the year and was greatest during summermonths (Vaccaro et al., 1950). Furthermore, thesurvival time of bacteria in sea water has been ex-tended by filtration, chlorination, and treatment withorganic matter as well as by heating (Beard andMeadowcroft, 1935; Krassilnikov, 1938; Vaccaro etal., 1950; Williams, 1950; ZoBell, 1936). However, ourpresent knowledge provides little insight into thenature of the factors responsible for the phenomenonobserved. Although numerous explanations for thebeneficial effect of complete or partial sterilization onthe survival of bacteria in sea water have been sug-gested (Carlucci and Pramer, 1959), they have notbeen tested experimentally and the identity of thefactors responsible for the more rapid death of bacteriain natural than in sterilized sea water remains to beestablished.

SURVIVAL OF E. COLI IN SEA WATER

The present report describes the influence of season,

pH, salinity, filtration, and organic matter on thebactericidal action of sea water. Moreover, artificialsea water was observed to exert a bactericidal actioncomparable to that of natural sea water.

MATERIALS AND METHODS

The bactericidal action of sea water was measuredas the difference in survival of cells of Escherichia coliin untreated and autoclaved portions of the same watersample. Detailed descriptions of the methods employedfor preparation and use of inocula, treatment andstorage of water samples, and of the enumeration andcalculation of survival of cells of E. coli in sea waterwere presented previously (Carlucci and Pramer,1960a).Sea water samples were heat sterilized by auto-

claving at 121 C for 15 min. Sterilization by filtrationwas performed using glass filter holders and type HAMillipore membranes3 having a pore size of 0.45 ,u.

RESULTS AND DISCUSSIONSeasonal variation. The bactericidal action of sea

water was determined at irregular intervals for 2 years

by measuring the survival of cells of E. coli in untreatedand autoclaved portions of each water sample. It was

first demonstrable in April, increased in magnitude toa maximum in July, then decreased, and was absentfrom water collected in November. The greatestbactericidal action was exerted by water sampledduring July 1957 when the ratio of per cent survivalafter 48 hr in autoclaved to that in untreated waterexceeded 24. These results were consistent with thoseof Vaccaro et al. (1950) who reported that the bacteri-cidal action of sea water was most pronounced duringthe summer months and absent in the winter. Watercollected during the winter months exerted no bac-tericidal action and it was not unusual for cells of E.coli to die more rapidly in autoclaved than in untreatedportions of these samples. This has been observed(Williams, 1950) but not commented on by previousinvestigators.pH and salinity. The influence of autoclaving on pH

and salinity and the possible contribution of thesefactors to the bactericidal action of sea water were

considered experimentally. Electrometric determina-tions demonstrated that the reaction of sea waterincreased from 0.5 to 1.0 pH unit during heat steriliza-tion and that equilibrium was not re-established for 24to 48 hr. It has been noted previously that the survivalof E. coli in sea water varied inversely with hydrogen-ion concentration (Carlucci and Pramer, 1960b) andon the basis of pH alone sterilization by heat would beexpected to have an adverse rather than beneficialeffect. Since the survival of E. coli in heat-sterilizedwater is increased rather than decreased the bactericidal

3 Millipore Filter Corporation, Bedford, Mass.

action cannot be attributed to the influence of auto-claving on pH alone.The effects of sterilization by filtration and auto-

claving on the salinity of sea water were examinedand although there was tendency for filtration to causea decrease and autoclaving to cause an increase insalinity, the magnitude of the changes never exceeded1.0%. The nature of the changes was not determinedand therefore their significance is not known.

Filtration. Water samples collected during July andAugust of 1957 were divided into three portions. Onewas sterilized by filtration, the second was sterilizedby autoclaving, and the third served as an untreatedcontrol. The survival of cells of E. coli in these waterswas determined for each of six different collections.The results listed in Table 1 show that in each of thesix samples tested E. coli died more rapidly in untreatedthan in autoclaved water but the magnitude of theeffect varied greatly. Likewise, the influence of filtrationwas not constant. In four of the six experiments survivalof the test organism was significantly greater in filteredthan in untreated sea water. If it is assumed that thefavorable effect of filtration resulted from removal ofpredatory and competitive organisms, then the numbersand activities of these organisms varied from sampleto sample. Waksman and Carey (1935) consideredpredation as a factor contributing significantly to thedeath of bacteria in sea water. The decreased survivalof E. coli in the filtered portion of water sample no. 5can be explained by the possible but improbable as-sumption that a beneficial substance or organism wasremoved during filtration.

In two of the samples tested (no. 1 and 6) the bacteri-cidal action of the water could have been fully ac-counted for by predatory and competitive organismsthat were eliminated by filtration; survival of E. coliwas the same in filtered and autoclaved water. In twoother samples (no. 3 and 5) filtration had no influenceon survival of the test organism and it appeared un-likely that predation and competition contributedsignificantly to the rapid death of E. coli in the un-treated water. In four of the six samples tested (no. 2,3, 4, and 5), predation and competition were not anadequate explanation for the rapid death of E. coli inuntreated water, since survival in autoclaved water wasconsiderably greater than survival in filtered water.

TABLE 1. Survival of Escherichia coli in untreated, filtered, andautoclaved portions of six sea water samples collected during

July and August, 1957

Survival after 48 hr in sea water sample no.

Treatment1 Sot2 3 4 5 6

None 2.2 0.7 4.6 22.8 4.4 2.7Filtered 8.3 3.8 4.8 30.1 0.6 39.6Autoclaved 8.4 30.9 64.6 53.6 69.6 38.5

1961] 401

A. F. CARLUCCI, P. V. SCARPINO, AND D. PRAMER

The beneficial effects of autoclaving over and abovethose resulting from filtration may have been due toany one or a combination of possibilities that include:(i) destruction of bacteriophages; (ii) inactivation ofthermolabile toxic substances such as antibiotics; (iii)increase in available nutrients.The influences of bacteriophages and antibiotics

on the survival of E. coli in sea water were tested anddiscussed previously (Carlucci and Pramer, 1960c, d).The effectiveness of bacteriophages depends on growthand multiplication of host cells. Since sea water doesnot support development of cells of E. coli, coliphageshave no significant influence on survival of the testbacterium and it is doubtful that they contribute tothe bactericidal action of sea water. The biologicaleffects of antibiotics in sea water vary with the natureand concentration of the antibiotic. It was observedthat, although some antibiotics adversely influencedthe survival of cells of E. coli in sea water, the resultwas of academic interest only since relatively highconcentrations were required and there is no evidencethat antibiotics are produced under natural condi-tions by marine microorganisms.

Nutrients. Since heat kills and disrupts microbialcells and degrades thermolabile compounds the thirdpossibility listed above appeared reasonable, namely,that the concentration of available nutrients in seawater was increased by autoclaving. Previous studies(Vaccaro et al., 1950; Orlob, 1956; Carlucci andPramer, 1960b) have shown that the addition of organicmatter to sea water which was otherwise untreatedincreased the survival of E. coli. The effect varied withthe composition and concentration of the substancetested. Relatively high levels were required and it wasdemonstrated that the ability of E. coli to utilizeorganic matter added to sea water was limited by pHand salinity, as well as by competition from the in-digenous micropopulation (Carlucci and Pramer,1960b). Since sterilization by heat would simultane-ously eliminate competitors and increase the amount ofavailable organic matter in sea water, lower nutrientlevels may be required for a beneficial effect in auto-claved than in untreated sea water. Therefore, theinfluence of concentration of peptone on the survivalof cells of E. coli in untreated and autoclaved portionsof the same water sample was determined at dailyintervals for 4 days. The results are illustrated in Fig. 1.The bactericidal action of the water was apparent

from the more rapid death of cells of E. coli in the un-treated than in the autoclaved unsupplemented portionof the water sample. Peptone at levels of 1 and 10 ppmfavored survival of the test organism in both untreatedand autoclaved sea water but the effect was greaterin the autoclaved portions. A peptone concentrationof 100 ppm supported growth of E. coli in both un-treated and autoclaved water and the inerease in cellnumbers was greater in the sterilized water. The re-

duced benefit derived by cells of E. coli from peptoneadded to water which was otherwise untreated was duein part to competition, since turbidity measurementsand plate counts showed that the indigenous microbialpopulation of the sea water increased with an increasein the amount of peptone added.The survival of cells of E. coli in sea water that was

not sterilized but contained 10 ppm of peptone did notdiffer significantly from that in unsupplemented auto-claved sea water; the addition of 10 ppm of peptone tosea water that was otherwise untreated eliminated thebactericidal action of the water. However, the effectlasted only 48 hr, after which cells of E. coli were ob-served to die off more rapidly in the peptone supple-mented unsterilized water than in the unsupplementedautoclaved water. It appeared that survival of thetest organism in natural water would have been com-parable to that in autoclaved water for the duration ofthe experiment if the former had been supplementedwith more than 10 ppm but less than 100 ppm of pep-tone. Although available organic matter in the form ofpeptone favored survival of E. coli in sea water themagnitude of the effect was not great. The number ofcells that persisted in sterilized sea water supplementedwith 10 ppm of peptone was approximately 10 timesthat in sterilized but unsupplemented water 4 daysafter treatment, whereas the latter was more than1,000 times the number of cells that survived in waterwhich was not sterilized or supplemented. If thefavorable effect of autoclaving on the survival of cellsof E. coli in sea water is to be explained on the basis ofan increase in concentration of available nutrients,it is necessary that the heat treatment release morethan 10 but less than 100 ppm of organic matter, andthis is unlikely, since the required range exceeds theorganic matter content of sea water (ZoBell, 1946).To better evaluate the importance of competition

and predation, the survival of E. coli was measured in

DAYSFIG. 1. Influence of concentration of peptone on the survival

of Escherichia coli in untreated and autoclaved sea water.

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SURVIVAL OF E. COLI IN SEA WATER

untreated, filter-sterilized, and autoclaved portionsof sea water samples with and without addition of 200ppm of glucose, peptone, and sewage volatile solids.The results summarized in Table 2 show that suirvivalof the test bacterium varied with the composition ofthe organic material tested and the treatment to whichthe water sample was subjected. In all but one case,survival of E. coli was greater in supplemented than inunsupplemented water. The exception was autoclavedwater that received sewage. E. coli multiplied in seawater supplemented with 200 ppm peptone. The addi-tion of glucose and sewage to sea water that was other-wise untreated increased survival but did not result inmultiplication of the test organism. Removal by filtra-tion of predators and competitors caused an increasein growth of the test organism in water treated withpeptone and permitted multiplication in water thatreceived glucose. The survival of E. coli in water sup-plemented with sewage was increased by filtration.Development of the test organism was similar in pep-tone-supplemented, autoclaved, and filter-sterilizedwater, which might suggest that competitors and pred-ators were of primary importance in limiting utiliza-tion of peptone by E. coli. The same explanation mayTABLE 2. Influence of organic matter on the survival of Escherichia

coli in untreated, filtered, and autoclaved sea water

Survival after 48 hr afterwater treatment:

Supplement

None Filtered Autoclaved

None 4.6 4.8 64.6Peptone, 200 ppm 272.7* 2,873.6* 2,631.2*None 2.2 8.3 8.4Glucose, 200 ppm + 17.6 712. 1* 3,305.9*(NH4)2HPO4-N, 4 ppm

None 0.7 3.8 30.9Sewage (volatile solids), 200 5.0 18.6 20.2ppm

* E. coli multiplied.

TABLE 3. Cosnposition of four artificial sea waters

ZoBell Tomlinson Sverdrup MacLeodConstituent (1946) and MacLeod et al. and Onofrey(1957) (1942) (1956)

NaCl 2.4 2.4 2.7 2.4MgCl2-6H20 1.1 1.1 0.5 1.1Na2SO4 0.4 0.4 0.4CaCl2-2H20 0.1 0.2 0.2KCI 0.07 0.07 0.07 0.07NaHCO3 0.02 0.02 0.02 0.02KBr 0.01 0.01 0.01SrCl2*6H20 0.004 0.004 0.004H3BO3 0.003 0.003 0.003Na2SiO3,9H20 0.0005NaF 0.0003NH4NO3 0.0002FePO4-4H20 0.0001MgSO4-7H20 0.7NaBr 0.008

be offered for the comparable survival of E. coli insewage-supplemented, autoclaved, and filter-sterilizedwater. On the other hand, growth of E. coli in auto-claved sea water supplemented with glucose was muchgreater than that in filtered water that received thesame supplement, suggesting that development of thetest organism in sea water containing glucose waslimited not only by predators and competitors, but alsoby other factors not removed by filtration but destroyedby the autoclaving process.From these and other studies it is apparent that

organic matter can influence the survival of E. coli insea water. However, the concentration required toobtain an effect of sufficient magnitude to overcomethe bactericidal action is in the range of 10 to 100 ppmand exceeds the total organic matter content of pelagicwater that is free of pollution. The report of Johan-nesson (1957) is unique in that the amino acid cysteinewas demonstrated to be particularly effective, and tofavor survival of E. coli in sea water at organic matterlevels that are conceivable under natural conditions.The beneficial effect of cysteine on survival of cells ofE. coli in sea water was confirmed (Carlucci and Pramer,1960b) and studies of the mode of action of cysteineare in progress.On the basis of the present investigations it is con-

cluded that the bactericidal action of sea water asmeasured by the difference in survival of cells of E.coli in untreated and autoclaved portions of water isnot explicable in terms of the destruction or inactiva-tion by heat of bacteriophages or antibiotics. Althoughautoclaving kills and disrupts microbial cells and de-grades thermolabile compounds, the resulting increasein nutrient level is not adequate to explain the favorableeffect of heat sterilization. Further evidence in supportof this conclusion was obtained from studies withartificial sea water.TABLE 4. Survival of Escherichia coli in untreated and autoclaved

portions of four artificial sea waters

Survival after 24 hrArtificial sea water

Untreated Autoclaved

ZoBell (1946) .................<........ 0.01 2.3Tomlinson and MacLeod (1957) ....... <0.01 0.7MacLeod and Onofrey (1956) ......... <0.01 5.1Sverdrup et al. (1942) ....... ......... <0. 01 0.1

TABLE 5. Influence of cysteine on the survival of Escherichia coliin artificial sea water

Treatment Survival after 24 hr

Autoclaved .............................. 13.6Untreated .........................<...... 0.01Untreated + cysteine, 0.035 ppm .. <0.01Untreated + cysteine, 0.35 ppm .......... 0.3Untreated + cysteine, 3.5 ppm ........... 20.3

1961] 403

A. F. CARLUCCI, P. V. SCARPINO, AND D. PRAMER

Artificial sea water. Several formulations of artificialsea water have been used for various purposes bynumerous investigators (ZoBell, 1946). To determineif artificial sea water exerted bactericidal action, thesurvival of E. coli in autoclaved and untreated por-tions of four different formulations was determined.The composition of the waters employed is presentedin Table 3. The simplest formulation contained 7 salts,whereas the most complex contained 13. They differedprimarily in the composition and concentration oftrace constituents.The results listed in Table 4 show that survival of

the test organism was consistently greater in auto-claved than in untreated water but the magnitude ofthe effect varied. Artificial sea water prepared accord-ing to MacLeod and Onofrey (1956) had the greatestbactericidal action and the formulation of Sverdrup,Johnson, and Fleming (1942) had the least effect. Thewater chosen for further use was that of MacLeodand Onofrey (1956). It was more convenient to preparethan that of ZoBell (1946), more complete in majorelements than that of Tomlinson and MacLeod (1957),and demonstrated a marked and consistent bactericidalaction. Artificial sea water is a more simple system forstudy than natural sea water. It is of known com-position, and lacks bacteriophages, predators, com-petitors, antibiotics, and organic nutrients that are ormay be present in natural sea water. However, thebactericidal action of artificial sea water may be causedby factors which differ from those responsible for theeffect in natural sea water and, in such a case, identi-fication of the former will not clarify the latter.

Evidence that the bactericidal action of artificialand natural sea water have a common basis was ob-tained from studies with cysteine. Since this com-pound is known to offset the bactericidal action ofnatural sea water (Johannesson, 1957; Carlucci andPramer, 1960b), its effect on the survival of E. coli inartificial sea water was determined. The results listedin Table 5 show that survival of the test organism wasgreater in autoclaved than in the untreated artificialsea water. Cysteine at a concentration of 0.035 ppmwas without effect, but survival was increased by 0.35ppm and in water that received 3.5 ppm of cysteinebut was otherwise untreated, survival of the testorganism exceeded that in autoclaved water. Furtherstudy is required to identify the factors responsiblefor the bactericidal action of artificial sea water andto establish that the same or different factors areresponsible for the more rapid death of E. coli in un-treated than in autoclaved natural sea water.

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