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Coagulation of Escherichia Coli by neutral salts
Water Research Pergamon Press 1969. Vol. 3, pp. 843-852. Printed in Great Britain.
COAGULATION OF ESCHERICHIA COLI BY NEUTRAL SALTS A. J. RUBIN,P. L. HAYDEN and G. P. HANNA,JR. Water Resources Center, College of Engineering, The Ohio State University, Columbus, Ohio 43210, U.S.A. (Received 2 May 1969)
Abstract An experimental procedure for the systematic study of microbial aggregation was developed and the coagulation of Escherichia coli by neutral salts was investigated. Changes in turbidity as estimated by absorbance measurements during settling were used as the criterion for coagulation. The effects of several parameters on the procedure and results are discussed. It was found that E. coli are coagulated by hydrogen ions and polyvalent cations at concentrations considerably lower than that of simple univalent cations. The effectivenessof divalent cations was related to their ionic radii. In general, the organisms followed the Schulze-Hardy rule for coagulation being similar in this respect to hydrophobic colloids. INTRODUCTION THE ARBITRARY selection of 0.1 # and 1 mp as the boundary limits of colloidally dispersed systems automatically excluded bacteria, algae and other microorganisms from this classification and placed them in the general grouping of the so-called "coarse dispersions." It has, however, been well established that bacteria exhibit certain colloid characteristics (LAMANNAand MALLETE, 1965). Indeed, they may well be included in a more general definition of a colloid being a dispersed phase more or less uniformly distributed in a continuous phase. The light scattering characteristics of bacterial suspensions and the surface charge on the microorganisms are examples of these colloid-like properties. Microorganisms exhibiting such properties have thus come to be known as "biocolloids." The colloid properties of microorganisms are of major concern to scientists and engineers attempting to culture them, to describe their behavior and to harness or control cell development. Early investigators recognized that bacterial suspensions responded to additions of various substances by forming aggregates (e.g., HODGE and MEICALFE, 1958), and that these formations were unique, depending on the type of bacteria and the specific additives. Thus this clumping of the cells or "agglutination" became useful in the identification of bacterial cultures, and was especially applied to the differentiation of certain pathogens. Further observations led to a recognition of the effects of various concentrations of electrolytes on the charge density of the cells and their aggregation or peptization. More recently, investigators working with mixed bacterial cultures have indicated the uncertainty of the mechanisms of aggregation and have suggested further study of the phenomena, as, for example the importance of microbial surface properties in removal techniques such as chemical coagulation and foam separation. The further consideration of bacterial suspensions as colloid systems appears to offer considerable promise. Most studies on the effects of electrolytes on bacteria have been concerned with the response to viability, turbidity, or electrophoretic mobility. For the most part these 843
844
A.J. RUBIN,P. L. HAYDENand G. P. HANNA,JR.
responses have neither been systematically studied, nor have the studies been unambiguous. For example, the survival of bacteria in sea water has been extensively investigated. Viability, however, has usually been indicated by plate counts even though salt solutions less concentrated than sea water lead to clumping and hence a reduction in colonies. The purposes of this investigation were to establish a procedure for systematically studying the aggregation of bacteria and to determine the concentrations of several selected neutral salts required to coagulate suspensions of Escherichia coli. EXPERIMENTAL Culture growth and preparation The tests organisms were two variants of E. coli, ATCC No. 11229 and Cincinnati Culture No. 198, obtained on Trypticase Soy Agar (TSA, Difco) slants from the Cincinnati Water Research Laboratory of the U.S. Department of the Interior. These will be identified as E. coli ATC and E. coli 198; most of the quantitative work was done with the latter variant and only its culturing and preparation is described in detail. The organisms were maintained by their transfer every 72 hr to fresh TSA slants. They were grown in batch shake cultures using 500-ml Erlenmeyer flasks containing 250 ml of 30 g/1 sterile Trypitcase Soy Broth (TSB, Difco). A growth curve for E. coli 198 plotted from 37 ° C. plate counts on selective Endo agar revealed that the log growth period was essentially complete at the end of 6 hr, reaching a stationary phase of about l 0 9 cells/ml, which persisted up to 48 hr. Most of the experiments were run using 12-hr cultures since this represented the early stage of the stationary phase which was easily reproducible. The flasks containing the TSB were incubated at 37 ° C in a constant temperature shaker bath set at 80 oscillations per rain. The bacteria were harvested by centrifuging, washing, and redispersing three successive times in distilled water. Each centrifugation was for a period of 20 min at 4000 rev/min. The final redispersion was made in carbonate free distilled water. The cell suspension was used immediately or was stored prior to its use for not more than 12 hr at 1-2 ° C. Comparative plate counts of refrigerated and non-refrigerated portions of the dispersion indicated no loss in culture viability after 24 hr refrigeration. Just before experimentation the dispersion was brought to room temperature in a water bath. The stock dispersion was diluted with carbonate-free distilled water to a transmittance of approximately 11 per cent as measured in a 19-mm round cuvette at a wavelength of 640 m/~ in a Colemen model 14 spectrophotometer. These dilutions were maintained at room temperature throughout the testing period and were never used for more than 6 hr. A constant E. coli 198 concentration was maintained for each test by diluting with the test reagents to a standard sample volume containing 2.5 x I0 a cells/ml.
Preparation of test samples Carbonate-free distilled water was prepared by distilling laboratory tap water using a Corning model AG-2 all glass still, deionizing through a Crystalab "Deeminizier" with MS-12 resin, and boiling. After boiling, the water was cooled and stored in polythylene bottles protected from the atmosphere by a vent tube filled with Mallinckrodt 30-50 mesh "Mallcosorb" indicating carbon dioxide adsorbent.
Coagulationof Escherichia coli by Neutral Salts
845
Stock solutions of the salts were prepared by dissolving the appropriate analytical reagent grade chemical in carbonate-free distilled water. These were stored in polyethylene bottles. Those salts not considered primary standards were standardized titrimetrically using EDTA (WELCHER, 1958). Reagent grade acetic acid-sodium acetate buffer solution was also prepared and stored as described above for use with some of the experiments. Five-ml aliquots of the standard bacterial dispersion were added to 19 x 105 mm round cuvettes just prior to experimentation. A second series, consisting of 5 ml of varying concentrations of salt solution, were prepared in small glass vials. The test sample was prepared by transferring the contents of a single vial to a cuvette containing the organisms. The cuvettes were stoppered, mixed vigorously for 20 sec and placed in the spectrophotometer for the first reading at I min. The samples were given no further agitation. The cuvettes were removed from the spectrophotometer after the individual readings and stored until the next measurement. All cuvettes were cleaned with chromic acid followed by washing in hot water, then rinsed in distilled water and allowed to air dry. Measurements and treatment of data After adding a certain critical concentration of coagulant, sols aggregate and increase their particle size. Although the number of particles then decreases, the turbidity usually increases and is indicative of coagulation. Thus the criterion for coagulation is particle growth; however, the settling of normally stable sols also is usually a direct result of coagulation. In this study changes in the turbidity of the test samples during settling were used as a measure of coagulation. Sample turbidities, expressed in absorbance units, were estimated by transmittance readings taken with a Coleman model 14 spectrophotometer. Preliminary results over the wavelength range 440-640 m# indicated that the most sensitivity occurred at the higher wavelengths and thus 640 m# was arbitrarily chosen for all subsequent work. A Sargent model DR pH meter and miniature combination electrode was used to measure the pH corresponding to each turbidity measurement. All values were between pH 6 and 7. The concentration of salt required to destabilize the bacterial suspensions was determined as a function of several parameters. The data at selected time intervals were plotted as absorbance against the independent variable, usually the logarithm of the applied salt concentration Typically, a sharp decrease in turbidity occurred over very narrow ranges of the independent variable. These steep portions of the curves were extrapolated back to the turbidity of a blank (control) bacterial dispersion of the same concentration as the test sample to give the critical coagulation concentration of the salt (RUBIN and HANNA, 1968). This critical value is the minimum concentration of salt required for coagulation to occur.
RESULTS AND DISCUSSION Determination of critical values The surface charge of microorganisms is known to vary both with the solution pH (HARDEN and HARRIS, 1953) and their culture age (MovER, 1936). The density of the cells, and hence their settleability, also varies with age. To establish the experimental regime the first series of experiments were run to examine the effect of pH and growth stage and to determine the critical time for taking the turbidity readings.
846
A.J. ROBIN,P. L. HAYDENand G. P. HANNA,JR.
The pH of a series of E. coli 198 suspensions was varied with nitric acid and sodium hydroxide as previously described. The results are shown in FIG. 1. Coagulation and clarification started within 15 min at pH 5 below. After 24 hr the maximum pH of instability, designated as pH~ in FIG. 1 (see RUBIN and HANNA, 1968), was 5.65. This corresponds with a critical coagulation concentration (activity) for hydrogen ion of 2.24 x 10 -6 M. Thus, to determine unambiguously the critical coagulation concentrations of other cations the solutions must be kept at pH 5.7 or greater. Similar experiments were run on 12-hr cultures of E. coli 198 to examine the effect of varying concentration of neutral salt. The pH remained in the range 6-7. Since the organisms are negatively charged the coagulating ions are cations. Results with magnesium nitrate and sodium chloride are shown in FIGs. 2 and 3, respectively; the relative absorbance (ratio of the turbidities of the sample to that of a blank) is plotted as a function of the logarithm of the molar concentration of salt. 060
;
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i
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INITIAL TURBID~T~ 050
15 MINUTES "O'°
,~
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030
020
2 4 HOURS
0.10
0
I
I 3
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I 5
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f
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7
pH.
FIG. 1. Effectof pH on the turbidity of E. coli 198 suspensionsas a functionof time. For magnesium ion the turbidity of the suspensions decreased unvaryingly with increasing time until some critical salt concentration was reached. At this point, designated the "critical coagulation concentration" or "c.c.c.," the turbidity of the suspensions increased significantly and then decreased in time as the aggregated cells settled. The 1 min time curve illustrates the initial step in coagulation which is an increase in turbidity due to increase in particle size through clumping. These 1 min turbidities were the maximum observed for magnesium ion. Apparently the cells aggregated immediately upon adding the salt solution, and the coagulum remained in suspension for over 30 min without a decrease in turbidity. Essentially complete settling was observed within 10 hr which was chosen as the critical time because of the reproducibility of the steep slope of the curve. Notice that although settling continued for 24 hr that the c.c.c., determined from extrapolating the steep portions of the time curves back to the initial turbidity (relative absorbance of 1), does not vary as a function of time. Upon coagulation visible clumps were formed and after settling the coagulum was compact and dense. Experiments with magnesium nitrate were also run using E. coli ATC. The absorbances of the suspensions of these organisms also increased initially with concentrations greater than the c.c.e, but instead of settling, the turbidity remained high for over 24 hr. Apparently, the weight density of the coagulum was insufficient to allow settling. Extrapolation of the turbidity curves back to the initial, however, gave a c.c.c, value only very slightly greater than that for E. coli 198. The technique of determining c.c.c.
Coagulation of Escherichia coli by Neutral Salts
847
values by means of changes in turbidity after settling, then, is limited to those species which will indeed settle after coagulation. As shown in FIo. 2, no increase in absorbance was observed at magnesium nitrate concentrations less than the c.c.c. This was typical with all of the divalent cations studied. Conversely, the turbidity increased just before reaching the c.c.c, in all suspensions containing univalent cations. The maximum turbidity occurred at the c.c.c, and, as shown for sodium chloride in FIG. 3, the suspensions containing higher concentrations settled. Again the I0 hr curve was judged the most suitable experimentally for the critical time. It should be pointed out that the c.c.c, for univalent J I MINUTE 14 IHOUR 1.2 Z LO
~0.8
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0.2
~
24 HOURS
0 -25
-20 LOG
~15 MOLAR
-I0 CONe.
05
FIG. 2. Effect of magnesium nitrate concentration on the turbidity of E. coli 198 suspensions as a function of time. salts is significantly greater than for multivalent ions. Thus, the organisms in such solutions are subjected to large osmotic forces. When coagulating E. coli 198 at pH 6 with low concentrations of aluminum no increase in turbidity was observed at the c.c.c. At pH 2 a much higher concentration of aluminum nitrate was required for coagulation and a very large increase in turbidity occurred upon approaching the c.c.c. (RtmIN and HANNA,1968). Of course, both very low pH and large concentrations of aluminum nitrate result in a large ionic strength. An increase in absorbance upon adding neutral salts to suspensions of gramnegative bacteria was attributed by MAGER et aL (1956) to be an osmotic phenomenon. Similar conclusions were reached by BATEMAN(1968) and LOVETT (1965) from their light scattering studies. It is evident, then, that increase in turbidity is not an adequate criterion for coagulation of biocolloids. The effects of culture age on the coagulation and subsequent settling of E. coli 198 suspensions were investigated. Magnesium nitrate was the coagulant and 3, 5, 12, 26, 48, and 72 hr cultures were used. Typical results after 10 hr of settling are shown in FIG. 4. The 5 and 48 hr curves were very similar to those shown for the 12 and 72 hr cultures, respectively. The age of the cultures influenced both the c.c.c, and the settleability of the suspensions. The older cultures, although settling at difi'erent rates,
848
A.J. RUBIN,P. L. HAYDENand G. P. HANNA,JR.
had approximately the same c.c.c. The rate o f settling of the 3-hr culture, however, was apparently less than that for the 5 and 12-hr cultures and its c.e.c, was about half. These observations reflect both the change in charge density and size o f the organisms with age. For example, electrophoretie measurements indicate that after 5 hr of growth the surface charge o f bacteria does not vary, and hence the concentration o f f MINUTE 1.4
I HOUR 1,2 = o
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24 HOURS 02
0 -I.5
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FIG. 3. Effect of sodium chloride concentration on the turbidity of E. coli 198 suspensions as a function of time. ~o
o o6
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0 -25
-2.0
-15 LOG
MOLAR
-I0
-05
CONC.
FIG. 4. Effect of culture age on the turbidity of E. coil 198 coagulated with varying concentrations of magnesium nitrate. Results after 10 hr of settling. salt required for coagulation should also remain constant. On the other hand, their size or weight density would be expected to vary continuously with age. Coagulation with neutral salts Having established the experimental procedure, studies with several salts were undertaken to determine their critical coagulation concentrations. The turbidities of 12-hr cultures redispersed in distilled water were measured after 10 hr of settling. The suspensions were maintained at a pH of about 6.
Coagulation of Escherichia coli by Neutral Salts
849
The first studies in this series were performed using NaCI, Na2SO4, NaNO3, and LiNO3. Since bacteria and all other known biocolloids are negatively charged, the effect of the anion associated with the coagulating cation should be very slight at best. For example, it has been demonstrated with negative silver halide sols that the sulfate coagulates at slightly higher concentrations than the nitrate of the same counterion (TE~'AK et al., 1955). The results with E. coli 198, as shown in FIG. 5, would indicate
I0 LITHIUM NITRATE NITRATE
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Fio. 5. Coagulation of E. coli 198 with sodium nitrate, lithium nitrate, and sodium sulfate. Results after 10 hr of settling. ~_018 x
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9,0 HYDRATED
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Fro. 6. Relationship between critical coagulation concentration and the hydrated radii of divalent counterions. Ten hour settling data for E coli 198.
the opposite effect. The difference, however, between the critical values, 0.32 N and 0.34 N for sodium sulfate and sodium nitrate, respectively, is not significantly different. The results with lithium nitrate were similar to those for sodium chloride (FIG. 3) having c.c.c, values of 0.45 and 0.42 N, respectively. Comparing NaNO 3 and LiNO3, the results are as expected since sodium ion has the larger crystallographic radius and therefore should be a more effective coagulant than lithium ion (TE£AK et al., 1955). To examine the effect of the counterion size more closely, several experiments were run with the nitrates of barium, calcium, magnesium, and strontium. The age of these salt solutions did not appear to affect their c.c.c, values. The results are summarized in FIG. 6 showing the critical coagulation concentrations as a linear function of the hydrated radius of the coagulating cation. Since the hydrated radii of simple ions is
850
A.J. RUBIN, P. L. HAYDENand G. P. HANNA, JR.
inversely p r o p o r t i o n a l to their crystallographic (naked) radii it is a p p a r e n t that the c o a g u l a t i n g power of the c o u n t e r i o n is p r o p o r t i o n a l to its crystallographic radius. The experiments with b a r i u m , m a g n e s i u m , a n d calcium were repeated a n d the results r e p r o d u c e d within a few per cent. The c.c.c, for b a r i u m was also determined at p H 6.0 using s o d i u m acetate-acetic acid buffer a n d again the results were in excellent agreement. U s i n g the p H 6 acetate buffer the critical c o a g u l a t i o n c o n c e n t r a t i o n s were deter= m i n e d for the salts Zn(NO3)2, AI(NO3)3, Al2(SO4)a, a n d La(NO3)3. The results, a n d TABLE 1. TYPICAL VALUES OF MILLIMOLAR CRITICAL COAGULATION CONCENTRATIONS FOR NEGATIVE SOLS
Electrolyte
Gold*
Silver iodide*
Silver bromidet
E. coli
Univalent cations LiNOa NaCI NaNO3 KNO3 1/2 K2SO, 1/2 Na2SO, Mean
-24 -25 23 -24
165 -140 136 --142
------180
450 415 340 --320 381
Divalent cations Mg(NO3)2 Ca(NO3)2 Sr(NOa)2 Ba(NO3)2 Zn(NO3)2 Mean
---0.35 -0.38
2.60 2.40 -2.26 2.50 2.43
-----2.65
Trivalent cations AI(NOa)3 1/2 A12(SO4)a La(NOa)3 Mean
---0.006
0.067 -0.069 0.068
---0.0367
0.0218 0.0190 0.026
Tetravalent cations Th(NOa), A18(OH)2o 4+
0.0009 __
0.013 __
-0.0010
-0.00272
15 9.5 9.0 7.0 7.0 9.4
Data from: * KRUYT, 1952, and t MATU~VI6et al., 1959. those for the n e u t r a l salts, are s u m m a r i z e d i n Table 1. These metals hydrolyze even in solution o f low p H f o r m i n g m o n o n u c l e a r a n d p o l y n u c l e a r species whose charge depends u p o n their c o n c e n t r a t i o n a n d the p H of the solution. Thus at p H 6 zinc would n o t be expected to be 2 + a n d a l u m i n u m a n d l a n t h a n u m would n o t be 3 + . F o r a l u m i n u m nitrate the c.c.c, was 2.15 x 10 -5 M. O f the various soluble polymeric h y d r o x o species that have been proposed for a l u m i n u m , the most reasonable at p H 6 appears to be the octamer, AIs(OH)2o 4+, suggested by Matijevi6 a n d co-workers (e.g., MATIJEVI6 et aL, 1966). A s s u m i n g this species, its c.c.c, then is one-eighth of the applied dose or 2.7 x 10 - 6 M. This value a n d others for several negative sols are also listed for c o m p a r i s o n in TABLE 1.
Coagulation of Escherichia coil by Neutral Salts
851
Coagulation is effected by ions of opposite charge to that of the sol, that is, by counterions. This is the Schulze-Hardy rule, which further states that the coagulating power of a counterion increases greatly with its charge. FIGURE7 shows that a plot of the logarithm of the critical coagulation concentrations of the counterions against their charge yields a straight line giving a quantitative expression of the rule. Thus, the
z 0 u
-2
,< 0 u
I-= u a:
0 -4
B -5
-6 I
2
3
COUNTERION CHARGE
FIG. 7. Relation between critical coagulation concentration and the charge of the coagulating cations. Ten hour settling data for E. coli 198 using average values from TABLE1.
coagulation of E. coli 198 follows the Schulze-Hardy rule as for the negative hydrophic sols. It is interesting that the slopes of the lines for E. coli 198 and silver bromide are similar and that the difference between the curves is well within an order of magniture in c.c.c. CONCLUSION
A method for the systematic study of the aggregation of settleable bacterial suspensions has been described. The results are reproducible provided a pure and viable culture is maintained. Factors affecting settleability include culture age, solution pH, and the type and concentrations of ions present. It was demonstrated that E. coli 198 are readily coagulated in salt solutions. The critical coagulation concentrations for a series of salts was determined on 12 hr cultures after a 10-hr settling period. The amount of neutral salt required to coagulate the bacterial suspensions varied both with the charge and ionic size of the counterion as predicted by the Schulze-Hardy rule and the lyotropic series of ions. Graphical representations of these rules correlated very well with published data on hydrophobic colloids. The organisms were coagulated by hydrogen ion at concentrations considerably below that of other univalent cations. Apparently, since coagulation by this ion does not follow the Schulze-Hardy rule, it is preferentially adsorbed by the organisms (see McCALLA, 194 I).
852
A.J. RUBIN, P. L. HAYDENand G. P. HANNA,JR.
Acknowledgement---The work described in this paper was conducted at, and supported in part by the College of Engineering, University of Cincinnati, Cinicinati, Ohio. REFERENCES BATEMANJ. B. (1968) Osmotic responses and light scattering of bacteria. Natn. colloidSymp. Preprints 42, 178-194. HARDENV. P. and HARRISJ. C. (1953) The isoelectric point of bacterial cells. J. Bact. 65, 198-202. HODOEH. M. and M~TCALrS. N., Jr. (1958) Flocculation of bacteria by hydrophilic colloids. J. Bact. 75, 258-264. KRUYTH. R. (1952) Colloid Science Vol. 1, Elsevier, New York. LAMANNAC. and MALLETTEM. F. (1965) Basic Bacteriology and its Biological and Chemical Background 3rd Edn. Williams & Wilkins, Baltimore. LOVETTS. (1965) Rapid changes in bacteria following introduction into hypertonic media. Proc. Soc. exp. biol. Med. 120, 567-569. MAGER J., KUCZYNSKIM., SCHATZBERGG. and AvI-DoR Y. (1956) Turbidity changes in bacterial suspensions in relation to osmotic pressure. J. gen. Microbiol. 14, 69-75. MATLIEVI~ E., BROADHURSTD. and KERKER M.(1959) On coagulation effects of highly charged counterions. J. phys. Chem. 63, 1552-1557. MATIJEVI(~E., JANAUERG. E. and KERKER M. (1964) Reversal of charge of lyophobic colloids by hydrolyzed metal ions--I. Aluminum nitrate. J. colloid Sci. 19, 333-346. MCCALLAT. M. (1941) The adsorption of H + by bacteria as measured by the glass electrode. J. Bact. 41, 775-784. MOYERL. S. (1936) Changes in the electrokinetic potential of bacteria at various phases of the culture Cycle. J. Bact. 32, 433-464. RUBIN A. J. and HANNA G. P. (1968) Coagulation of the bacterium Escherichia coli by aluminum nitrate. Environ. Sci. TechnoL 2, 358-362. TE~'.AKB., MATUEVI6E. and SCHULZK. F. (1955) Coagulation of hydrophobic sols in statu nascendi-IIL The influence of the ionic size and valency of the counterion. J. phys. Chem. 59, 769-773. WELCHER F. J. (1958) The Analytical Uses of Ethylenediaminetetraacetic Acid Van Nostrand, New York.
COAGULATION OF ESCHERICHIA COLI BY NEUTRAL SALTS A. J. RUBIN,P. L. HAYDEN and G. P. HANNA,JR. Water Resources Center, College of Engineering, The Ohio State University, Columbus, Ohio 43210, U.S.A. (Received 2 May 1969)
Abstract An experimental procedure for the systematic study of microbial aggregation was developed and the coagulation of Escherichia coli by neutral salts was investigated. Changes in turbidity as estimated by absorbance measurements during settling were used as the criterion for coagulation. The effects of several parameters on the procedure and results are discussed. It was found that E. coli are coagulated by hydrogen ions and polyvalent cations at concentrations considerably lower than that of simple univalent cations. The effectivenessof divalent cations was related to their ionic radii. In general, the organisms followed the Schulze-Hardy rule for coagulation being similar in this respect to hydrophobic colloids. INTRODUCTION THE ARBITRARY selection of 0.1 # and 1 mp as the boundary limits of colloidally dispersed systems automatically excluded bacteria, algae and other microorganisms from this classification and placed them in the general grouping of the so-called "coarse dispersions." It has, however, been well established that bacteria exhibit certain colloid characteristics (LAMANNAand MALLETE, 1965). Indeed, they may well be included in a more general definition of a colloid being a dispersed phase more or less uniformly distributed in a continuous phase. The light scattering characteristics of bacterial suspensions and the surface charge on the microorganisms are examples of these colloid-like properties. Microorganisms exhibiting such properties have thus come to be known as "biocolloids." The colloid properties of microorganisms are of major concern to scientists and engineers attempting to culture them, to describe their behavior and to harness or control cell development. Early investigators recognized that bacterial suspensions responded to additions of various substances by forming aggregates (e.g., HODGE and MEICALFE, 1958), and that these formations were unique, depending on the type of bacteria and the specific additives. Thus this clumping of the cells or "agglutination" became useful in the identification of bacterial cultures, and was especially applied to the differentiation of certain pathogens. Further observations led to a recognition of the effects of various concentrations of electrolytes on the charge density of the cells and their aggregation or peptization. More recently, investigators working with mixed bacterial cultures have indicated the uncertainty of the mechanisms of aggregation and have suggested further study of the phenomena, as, for example the importance of microbial surface properties in removal techniques such as chemical coagulation and foam separation. The further consideration of bacterial suspensions as colloid systems appears to offer considerable promise. Most studies on the effects of electrolytes on bacteria have been concerned with the response to viability, turbidity, or electrophoretic mobility. For the most part these 843
844
A.J. RUBIN,P. L. HAYDENand G. P. HANNA,JR.
responses have neither been systematically studied, nor have the studies been unambiguous. For example, the survival of bacteria in sea water has been extensively investigated. Viability, however, has usually been indicated by plate counts even though salt solutions less concentrated than sea water lead to clumping and hence a reduction in colonies. The purposes of this investigation were to establish a procedure for systematically studying the aggregation of bacteria and to determine the concentrations of several selected neutral salts required to coagulate suspensions of Escherichia coli. EXPERIMENTAL Culture growth and preparation The tests organisms were two variants of E. coli, ATCC No. 11229 and Cincinnati Culture No. 198, obtained on Trypticase Soy Agar (TSA, Difco) slants from the Cincinnati Water Research Laboratory of the U.S. Department of the Interior. These will be identified as E. coli ATC and E. coli 198; most of the quantitative work was done with the latter variant and only its culturing and preparation is described in detail. The organisms were maintained by their transfer every 72 hr to fresh TSA slants. They were grown in batch shake cultures using 500-ml Erlenmeyer flasks containing 250 ml of 30 g/1 sterile Trypitcase Soy Broth (TSB, Difco). A growth curve for E. coli 198 plotted from 37 ° C. plate counts on selective Endo agar revealed that the log growth period was essentially complete at the end of 6 hr, reaching a stationary phase of about l 0 9 cells/ml, which persisted up to 48 hr. Most of the experiments were run using 12-hr cultures since this represented the early stage of the stationary phase which was easily reproducible. The flasks containing the TSB were incubated at 37 ° C in a constant temperature shaker bath set at 80 oscillations per rain. The bacteria were harvested by centrifuging, washing, and redispersing three successive times in distilled water. Each centrifugation was for a period of 20 min at 4000 rev/min. The final redispersion was made in carbonate free distilled water. The cell suspension was used immediately or was stored prior to its use for not more than 12 hr at 1-2 ° C. Comparative plate counts of refrigerated and non-refrigerated portions of the dispersion indicated no loss in culture viability after 24 hr refrigeration. Just before experimentation the dispersion was brought to room temperature in a water bath. The stock dispersion was diluted with carbonate-free distilled water to a transmittance of approximately 11 per cent as measured in a 19-mm round cuvette at a wavelength of 640 m/~ in a Colemen model 14 spectrophotometer. These dilutions were maintained at room temperature throughout the testing period and were never used for more than 6 hr. A constant E. coli 198 concentration was maintained for each test by diluting with the test reagents to a standard sample volume containing 2.5 x I0 a cells/ml.
Preparation of test samples Carbonate-free distilled water was prepared by distilling laboratory tap water using a Corning model AG-2 all glass still, deionizing through a Crystalab "Deeminizier" with MS-12 resin, and boiling. After boiling, the water was cooled and stored in polythylene bottles protected from the atmosphere by a vent tube filled with Mallinckrodt 30-50 mesh "Mallcosorb" indicating carbon dioxide adsorbent.
Coagulationof Escherichia coli by Neutral Salts
845
Stock solutions of the salts were prepared by dissolving the appropriate analytical reagent grade chemical in carbonate-free distilled water. These were stored in polyethylene bottles. Those salts not considered primary standards were standardized titrimetrically using EDTA (WELCHER, 1958). Reagent grade acetic acid-sodium acetate buffer solution was also prepared and stored as described above for use with some of the experiments. Five-ml aliquots of the standard bacterial dispersion were added to 19 x 105 mm round cuvettes just prior to experimentation. A second series, consisting of 5 ml of varying concentrations of salt solution, were prepared in small glass vials. The test sample was prepared by transferring the contents of a single vial to a cuvette containing the organisms. The cuvettes were stoppered, mixed vigorously for 20 sec and placed in the spectrophotometer for the first reading at I min. The samples were given no further agitation. The cuvettes were removed from the spectrophotometer after the individual readings and stored until the next measurement. All cuvettes were cleaned with chromic acid followed by washing in hot water, then rinsed in distilled water and allowed to air dry. Measurements and treatment of data After adding a certain critical concentration of coagulant, sols aggregate and increase their particle size. Although the number of particles then decreases, the turbidity usually increases and is indicative of coagulation. Thus the criterion for coagulation is particle growth; however, the settling of normally stable sols also is usually a direct result of coagulation. In this study changes in the turbidity of the test samples during settling were used as a measure of coagulation. Sample turbidities, expressed in absorbance units, were estimated by transmittance readings taken with a Coleman model 14 spectrophotometer. Preliminary results over the wavelength range 440-640 m# indicated that the most sensitivity occurred at the higher wavelengths and thus 640 m# was arbitrarily chosen for all subsequent work. A Sargent model DR pH meter and miniature combination electrode was used to measure the pH corresponding to each turbidity measurement. All values were between pH 6 and 7. The concentration of salt required to destabilize the bacterial suspensions was determined as a function of several parameters. The data at selected time intervals were plotted as absorbance against the independent variable, usually the logarithm of the applied salt concentration Typically, a sharp decrease in turbidity occurred over very narrow ranges of the independent variable. These steep portions of the curves were extrapolated back to the turbidity of a blank (control) bacterial dispersion of the same concentration as the test sample to give the critical coagulation concentration of the salt (RUBIN and HANNA, 1968). This critical value is the minimum concentration of salt required for coagulation to occur.
RESULTS AND DISCUSSION Determination of critical values The surface charge of microorganisms is known to vary both with the solution pH (HARDEN and HARRIS, 1953) and their culture age (MovER, 1936). The density of the cells, and hence their settleability, also varies with age. To establish the experimental regime the first series of experiments were run to examine the effect of pH and growth stage and to determine the critical time for taking the turbidity readings.
846
A.J. ROBIN,P. L. HAYDENand G. P. HANNA,JR.
The pH of a series of E. coli 198 suspensions was varied with nitric acid and sodium hydroxide as previously described. The results are shown in FIG. 1. Coagulation and clarification started within 15 min at pH 5 below. After 24 hr the maximum pH of instability, designated as pH~ in FIG. 1 (see RUBIN and HANNA, 1968), was 5.65. This corresponds with a critical coagulation concentration (activity) for hydrogen ion of 2.24 x 10 -6 M. Thus, to determine unambiguously the critical coagulation concentrations of other cations the solutions must be kept at pH 5.7 or greater. Similar experiments were run on 12-hr cultures of E. coli 198 to examine the effect of varying concentration of neutral salt. The pH remained in the range 6-7. Since the organisms are negatively charged the coagulating ions are cations. Results with magnesium nitrate and sodium chloride are shown in FIGs. 2 and 3, respectively; the relative absorbance (ratio of the turbidities of the sample to that of a blank) is plotted as a function of the logarithm of the molar concentration of salt. 060
;
I
i
k
i
J
INITIAL TURBID~T~ 050
15 MINUTES "O'°
,~
040 ~~ 040
o
s:
•
-
~
030
020
2 4 HOURS
0.10
0
I
I 3
I
I 5
I
f
I
7
pH.
FIG. 1. Effectof pH on the turbidity of E. coli 198 suspensionsas a functionof time. For magnesium ion the turbidity of the suspensions decreased unvaryingly with increasing time until some critical salt concentration was reached. At this point, designated the "critical coagulation concentration" or "c.c.c.," the turbidity of the suspensions increased significantly and then decreased in time as the aggregated cells settled. The 1 min time curve illustrates the initial step in coagulation which is an increase in turbidity due to increase in particle size through clumping. These 1 min turbidities were the maximum observed for magnesium ion. Apparently the cells aggregated immediately upon adding the salt solution, and the coagulum remained in suspension for over 30 min without a decrease in turbidity. Essentially complete settling was observed within 10 hr which was chosen as the critical time because of the reproducibility of the steep slope of the curve. Notice that although settling continued for 24 hr that the c.c.c., determined from extrapolating the steep portions of the time curves back to the initial turbidity (relative absorbance of 1), does not vary as a function of time. Upon coagulation visible clumps were formed and after settling the coagulum was compact and dense. Experiments with magnesium nitrate were also run using E. coli ATC. The absorbances of the suspensions of these organisms also increased initially with concentrations greater than the c.c.e, but instead of settling, the turbidity remained high for over 24 hr. Apparently, the weight density of the coagulum was insufficient to allow settling. Extrapolation of the turbidity curves back to the initial, however, gave a c.c.c, value only very slightly greater than that for E. coli 198. The technique of determining c.c.c.
Coagulation of Escherichia coli by Neutral Salts
847
values by means of changes in turbidity after settling, then, is limited to those species which will indeed settle after coagulation. As shown in FIo. 2, no increase in absorbance was observed at magnesium nitrate concentrations less than the c.c.c. This was typical with all of the divalent cations studied. Conversely, the turbidity increased just before reaching the c.c.c, in all suspensions containing univalent cations. The maximum turbidity occurred at the c.c.c, and, as shown for sodium chloride in FIG. 3, the suspensions containing higher concentrations settled. Again the I0 hr curve was judged the most suitable experimentally for the critical time. It should be pointed out that the c.c.c, for univalent J I MINUTE 14 IHOUR 1.2 Z LO
~0.8
~o~ W O~
~__~,xHo2%
0.2
~
24 HOURS
0 -25
-20 LOG
~15 MOLAR
-I0 CONe.
05
FIG. 2. Effect of magnesium nitrate concentration on the turbidity of E. coli 198 suspensions as a function of time. salts is significantly greater than for multivalent ions. Thus, the organisms in such solutions are subjected to large osmotic forces. When coagulating E. coli 198 at pH 6 with low concentrations of aluminum no increase in turbidity was observed at the c.c.c. At pH 2 a much higher concentration of aluminum nitrate was required for coagulation and a very large increase in turbidity occurred upon approaching the c.c.c. (RtmIN and HANNA,1968). Of course, both very low pH and large concentrations of aluminum nitrate result in a large ionic strength. An increase in absorbance upon adding neutral salts to suspensions of gramnegative bacteria was attributed by MAGER et aL (1956) to be an osmotic phenomenon. Similar conclusions were reached by BATEMAN(1968) and LOVETT (1965) from their light scattering studies. It is evident, then, that increase in turbidity is not an adequate criterion for coagulation of biocolloids. The effects of culture age on the coagulation and subsequent settling of E. coli 198 suspensions were investigated. Magnesium nitrate was the coagulant and 3, 5, 12, 26, 48, and 72 hr cultures were used. Typical results after 10 hr of settling are shown in FIG. 4. The 5 and 48 hr curves were very similar to those shown for the 12 and 72 hr cultures, respectively. The age of the cultures influenced both the c.c.c, and the settleability of the suspensions. The older cultures, although settling at difi'erent rates,
848
A.J. RUBIN,P. L. HAYDENand G. P. HANNA,JR.
had approximately the same c.c.c. The rate o f settling of the 3-hr culture, however, was apparently less than that for the 5 and 12-hr cultures and its c.e.c, was about half. These observations reflect both the change in charge density and size o f the organisms with age. For example, electrophoretie measurements indicate that after 5 hr of growth the surface charge o f bacteria does not vary, and hence the concentration o f f MINUTE 1.4
I HOUR 1,2 = o
z
10 Q:
<[ Q8
~
Q6
0.4
24 HOURS 02
0 -I.5
1
I
I
-I,0
-05
o
LOG M O L A R
05
CONC,
FIG. 3. Effect of sodium chloride concentration on the turbidity of E. coli 198 suspensions as a function of time. ~o
o o6
~04 _1
0 -25
-2.0
-15 LOG
MOLAR
-I0
-05
CONC.
FIG. 4. Effect of culture age on the turbidity of E. coil 198 coagulated with varying concentrations of magnesium nitrate. Results after 10 hr of settling. salt required for coagulation should also remain constant. On the other hand, their size or weight density would be expected to vary continuously with age. Coagulation with neutral salts Having established the experimental procedure, studies with several salts were undertaken to determine their critical coagulation concentrations. The turbidities of 12-hr cultures redispersed in distilled water were measured after 10 hr of settling. The suspensions were maintained at a pH of about 6.
Coagulation of Escherichia coli by Neutral Salts
849
The first studies in this series were performed using NaCI, Na2SO4, NaNO3, and LiNO3. Since bacteria and all other known biocolloids are negatively charged, the effect of the anion associated with the coagulating cation should be very slight at best. For example, it has been demonstrated with negative silver halide sols that the sulfate coagulates at slightly higher concentrations than the nitrate of the same counterion (TE~'AK et al., 1955). The results with E. coli 198, as shown in FIG. 5, would indicate
I0 LITHIUM NITRATE NITRATE
o ~O6
,~
o4
.d SODIUM SULFATE
O2
O -15
-LO
-05 0 1.0(3 MOLAR CONC.
05
Fio. 5. Coagulation of E. coli 198 with sodium nitrate, lithium nitrate, and sodium sulfate. Results after 10 hr of settling. ~_018 x
Mg *+
~5
§,4 o
i0
U
6
d ~
2
I
85
9,0 HYDRATED
95 RADfUS
[
i
I00
105
IT0
IN A N G S T R O M S
Fro. 6. Relationship between critical coagulation concentration and the hydrated radii of divalent counterions. Ten hour settling data for E coli 198.
the opposite effect. The difference, however, between the critical values, 0.32 N and 0.34 N for sodium sulfate and sodium nitrate, respectively, is not significantly different. The results with lithium nitrate were similar to those for sodium chloride (FIG. 3) having c.c.c, values of 0.45 and 0.42 N, respectively. Comparing NaNO 3 and LiNO3, the results are as expected since sodium ion has the larger crystallographic radius and therefore should be a more effective coagulant than lithium ion (TE£AK et al., 1955). To examine the effect of the counterion size more closely, several experiments were run with the nitrates of barium, calcium, magnesium, and strontium. The age of these salt solutions did not appear to affect their c.c.c, values. The results are summarized in FIG. 6 showing the critical coagulation concentrations as a linear function of the hydrated radius of the coagulating cation. Since the hydrated radii of simple ions is
850
A.J. RUBIN, P. L. HAYDENand G. P. HANNA, JR.
inversely p r o p o r t i o n a l to their crystallographic (naked) radii it is a p p a r e n t that the c o a g u l a t i n g power of the c o u n t e r i o n is p r o p o r t i o n a l to its crystallographic radius. The experiments with b a r i u m , m a g n e s i u m , a n d calcium were repeated a n d the results r e p r o d u c e d within a few per cent. The c.c.c, for b a r i u m was also determined at p H 6.0 using s o d i u m acetate-acetic acid buffer a n d again the results were in excellent agreement. U s i n g the p H 6 acetate buffer the critical c o a g u l a t i o n c o n c e n t r a t i o n s were deter= m i n e d for the salts Zn(NO3)2, AI(NO3)3, Al2(SO4)a, a n d La(NO3)3. The results, a n d TABLE 1. TYPICAL VALUES OF MILLIMOLAR CRITICAL COAGULATION CONCENTRATIONS FOR NEGATIVE SOLS
Electrolyte
Gold*
Silver iodide*
Silver bromidet
E. coli
Univalent cations LiNOa NaCI NaNO3 KNO3 1/2 K2SO, 1/2 Na2SO, Mean
-24 -25 23 -24
165 -140 136 --142
------180
450 415 340 --320 381
Divalent cations Mg(NO3)2 Ca(NO3)2 Sr(NOa)2 Ba(NO3)2 Zn(NO3)2 Mean
---0.35 -0.38
2.60 2.40 -2.26 2.50 2.43
-----2.65
Trivalent cations AI(NOa)3 1/2 A12(SO4)a La(NOa)3 Mean
---0.006
0.067 -0.069 0.068
---0.0367
0.0218 0.0190 0.026
Tetravalent cations Th(NOa), A18(OH)2o 4+
0.0009 __
0.013 __
-0.0010
-0.00272
15 9.5 9.0 7.0 7.0 9.4
Data from: * KRUYT, 1952, and t MATU~VI6et al., 1959. those for the n e u t r a l salts, are s u m m a r i z e d i n Table 1. These metals hydrolyze even in solution o f low p H f o r m i n g m o n o n u c l e a r a n d p o l y n u c l e a r species whose charge depends u p o n their c o n c e n t r a t i o n a n d the p H of the solution. Thus at p H 6 zinc would n o t be expected to be 2 + a n d a l u m i n u m a n d l a n t h a n u m would n o t be 3 + . F o r a l u m i n u m nitrate the c.c.c, was 2.15 x 10 -5 M. O f the various soluble polymeric h y d r o x o species that have been proposed for a l u m i n u m , the most reasonable at p H 6 appears to be the octamer, AIs(OH)2o 4+, suggested by Matijevi6 a n d co-workers (e.g., MATIJEVI6 et aL, 1966). A s s u m i n g this species, its c.c.c, then is one-eighth of the applied dose or 2.7 x 10 - 6 M. This value a n d others for several negative sols are also listed for c o m p a r i s o n in TABLE 1.
Coagulation of Escherichia coil by Neutral Salts
851
Coagulation is effected by ions of opposite charge to that of the sol, that is, by counterions. This is the Schulze-Hardy rule, which further states that the coagulating power of a counterion increases greatly with its charge. FIGURE7 shows that a plot of the logarithm of the critical coagulation concentrations of the counterions against their charge yields a straight line giving a quantitative expression of the rule. Thus, the
z 0 u
-2
,< 0 u
I-= u a:
0 -4
B -5
-6 I
2
3
COUNTERION CHARGE
FIG. 7. Relation between critical coagulation concentration and the charge of the coagulating cations. Ten hour settling data for E. coli 198 using average values from TABLE1.
coagulation of E. coli 198 follows the Schulze-Hardy rule as for the negative hydrophic sols. It is interesting that the slopes of the lines for E. coli 198 and silver bromide are similar and that the difference between the curves is well within an order of magniture in c.c.c. CONCLUSION
A method for the systematic study of the aggregation of settleable bacterial suspensions has been described. The results are reproducible provided a pure and viable culture is maintained. Factors affecting settleability include culture age, solution pH, and the type and concentrations of ions present. It was demonstrated that E. coli 198 are readily coagulated in salt solutions. The critical coagulation concentrations for a series of salts was determined on 12 hr cultures after a 10-hr settling period. The amount of neutral salt required to coagulate the bacterial suspensions varied both with the charge and ionic size of the counterion as predicted by the Schulze-Hardy rule and the lyotropic series of ions. Graphical representations of these rules correlated very well with published data on hydrophobic colloids. The organisms were coagulated by hydrogen ion at concentrations considerably below that of other univalent cations. Apparently, since coagulation by this ion does not follow the Schulze-Hardy rule, it is preferentially adsorbed by the organisms (see McCALLA, 194 I).
852
A.J. RUBIN, P. L. HAYDENand G. P. HANNA,JR.
Acknowledgement---The work described in this paper was conducted at, and supported in part by the College of Engineering, University of Cincinnati, Cinicinati, Ohio. REFERENCES BATEMANJ. B. (1968) Osmotic responses and light scattering of bacteria. Natn. colloidSymp. Preprints 42, 178-194. HARDENV. P. and HARRISJ. C. (1953) The isoelectric point of bacterial cells. J. Bact. 65, 198-202. HODOEH. M. and M~TCALrS. N., Jr. (1958) Flocculation of bacteria by hydrophilic colloids. J. Bact. 75, 258-264. KRUYTH. R. (1952) Colloid Science Vol. 1, Elsevier, New York. LAMANNAC. and MALLETTEM. F. (1965) Basic Bacteriology and its Biological and Chemical Background 3rd Edn. Williams & Wilkins, Baltimore. LOVETTS. (1965) Rapid changes in bacteria following introduction into hypertonic media. Proc. Soc. exp. biol. Med. 120, 567-569. MAGER J., KUCZYNSKIM., SCHATZBERGG. and AvI-DoR Y. (1956) Turbidity changes in bacterial suspensions in relation to osmotic pressure. J. gen. Microbiol. 14, 69-75. MATLIEVI~ E., BROADHURSTD. and KERKER M.(1959) On coagulation effects of highly charged counterions. J. phys. Chem. 63, 1552-1557. MATIJEVI(~E., JANAUERG. E. and KERKER M. (1964) Reversal of charge of lyophobic colloids by hydrolyzed metal ions--I. Aluminum nitrate. J. colloid Sci. 19, 333-346. MCCALLAT. M. (1941) The adsorption of H + by bacteria as measured by the glass electrode. J. Bact. 41, 775-784. MOYERL. S. (1936) Changes in the electrokinetic potential of bacteria at various phases of the culture Cycle. J. Bact. 32, 433-464. RUBIN A. J. and HANNA G. P. (1968) Coagulation of the bacterium Escherichia coli by aluminum nitrate. Environ. Sci. TechnoL 2, 358-362. TE~'.AKB., MATUEVI6E. and SCHULZK. F. (1955) Coagulation of hydrophobic sols in statu nascendi-IIL The influence of the ionic size and valency of the counterion. J. phys. Chem. 59, 769-773. WELCHER F. J. (1958) The Analytical Uses of Ethylenediaminetetraacetic Acid Van Nostrand, New York.