Coagulation of the Bacterium Escherichia coli by Aluminum Nitrate Alan J. Rubin and George P. Hanna' College of Engineering, University of Cincinnati, Cincinnati, Ohio 45221
The coagulation of the gram-negative bacterium Escherichia coli by freshly prepared aluminum nitrate solutions was investigated. Changes in turbidity during settling were used t o determine critical p H values of coagulation and stabilization as a function of aluminum concentration. These and critical coagulation concentration data were used t o establish the entire log AI(N03)3 concentration-pH domain of stability for the coagulation of the organisms. The domain is very similar t o those described for the negative lyophobic sols, with some notable differences. The organisms are slowly coagulated by hydrogen ion at p H values less than 5.65 and restabilized by aluminum below p H 3.5. In the p H range 5.65 t o about 7.5, the critical aluminum nitrate coagulation concentration is 2.15 x 10-jM; restabilization occurs above about 2 X 10-4M. Presumably, the coagulating and restabilizing species in these regions is the A18(OH)204+ ion. Critical coagulation concentrations for the nitrates of sodium and bsrium were also determined. These studies revealed that E. coli follow the Schulze-Hardy rule as d o the lyophobic sols. A sweep zone of aluminum hydroxide precipitation and rapid settling, very similar to that described for clays, was observed. These results support the enmeshing theory. As with other sols, the coagulation of Escherichia coli is dependent upon the pH and applied concentration of hydrolyzing coagulant.
C
hemical coagulation-flocculation is a process in which particles colloidally dispersed in water are destabilized t o form aggregates of dimensions sufficiently large t o permit them t o be settled or filtered and thus be removed. Two basic mechanisms of aggregation are generally recognized. The first involves the reduction by counterions of the protective repulsive electrical forces at the particle surface. This is known commonly as coagulation. The second mechanism is called flocculation, whereby aggregation is brought about by the formation of bridges between the particles (LaMer and Healy, 1963).
Present address, Water Resources Center, The Ohio State University, Columbus, Ohio 43210 358 Environmental Science and Technology
Polyelectrolytes are postulated t o operate in the latter fashion while destabilization of colloidal sols by neutral salts results from coagulation. The mechanism of the destabilizing action of hydrolyzing electrolytes such as the aluminum and iron salts, however, is not clear (O'Melia and Stumm, 1967). For example, MatijeviE and coworkers (1964, 1966a: b) have put forth convincing evidence that such salts are true coagulants and that destabilization is brought about b> highly charged soluble hydrolysis products of these metals. Conversely, the bridging model (flocculation) has been suggested as the mechanism by which hydrolyzing metals destabilize colloids since at certain pH values they are assumed t o form long chain polymers. Tenney and Stumm (1965) and Rubin, Cassell, et al. (1966) have interpreted their coagulation-flocculation results with bacteria using this model. Packham (1963) in his studies of the aggregation of clays by aluminum sulfate interpreted the results in terms of the enmeshing of the particles "in the mass of rapidly precipitating and flocculating aluminum hydroxide." He also reported (Packham, 1965) that coagulation of dilute suspensions is almost independent of the nature of the dispersed phase. Because of these semantic inconsistencies, the term coagulation is used here without necessarily implying a mechanism. Apparently, the literature shows that the mechanism of destabilization by hydrolyzing electrolytes is a function both of the coagulant concentration and the pH of the dispersion medium. Many investigations on the coagulation of clays and other inorganic sols have been described in the literature. However, there is a scarcity of such research reported on the bacteria. The objectives of the research described in this paper were t o examine the coagulation of the gram negative bacterium Escherichia coli by freshly prepared aluminum nitrate solutions and to determine the relationship between coagulant concentration and pH for these organisms. The experimental techniques developed and successfully applied t o the study of the coagulation of silver halide sols by Teiak, MatijeviC, et a/. (1951a, b) were employed with suitable modifications. Bacterial concentration and growth stage, and therefore their size, surface area, and surface properties, were kept constant. Turbidity changes, used as a measure of coagulation, were estimated by absorbance measurements during sedimentation. Critical p H and concentration values defining limits of coagulation and stabilization extrapolated from curves of the turbidity data were used t o establish the entire log AI(NO& concentration-pH domain of stability for the coagulation of E. coli.
Mriter.ids rind Solririons Esclzericliiu coli. culture number 198 (Cincinnati Water Research Laboratory), were grown at 37" C. for 12 hours in batch shake cultures using 30 grams per liter of trypticase soy broth medium. Each batch was checked by turbidity measurements and related to plate counts to assure their reproducibility. Before experimentation, the organisms were centrifuged, washed, and redispersed in distilled water. This was repeated twice more, the final dispersion being made in carbonate-free distilled water. The organisms were then diluted t o a standard turbidity and were stirred at constant temperature for the duration of the experiment. Aluminum stock solutions were freshly prepared just prior to their use by dissolving reagent grade Al(NO&.9 H 2 0 in carbonate-free distilled water. Reagent grade NaOH or HNOa solutions prepared in carbonate-free distilled water were used to adjust pH. For each experiment, a 5m1. aliquot of bacteria, the exact amount of base (when used), and enough carbonate-free distilled water t o dilute t o a total volume of 8-ml. were mixed in a glass cuvette. Just after preparing these solutions, 4-ml. of aluminum nitrate solution, the exact amount of acid (when used), and,'or distilled water were added to the cuvette (time zero) and mixed vigorously for 30 seconds. E. coli concentration in the experimental dispersions were kept constant at approximately 2.5 x 108 organisms per ml. The reported AI(II1) concentrations are also those in the diluted (12-ml. total) experimental mixture. Absorbance measurements were taken at 640 mp using 19-mm. round cuvettes and a Coleman Model 14 spectrophotometer. Sargent miniature combination electrodes and a Sargent Model DR pH meter were used t o measure the pH corresponding t o each turbidity measurement.
0.6
I
1
I
I
1
1
\ I 5 MINUTES
10.2
c
\
l
i
PH
Figure 1. Coagulation of €scherichio coli as a function of pH with 2.0 X 1 0 - 3 M Al(NO& showing the slow and rapid zones of coagulation 0 15-minute settling data. 0-0 1-hour settling data. A 3-hour settling data. 121-hour settling data
Cocigiilririonr i n d Resrcihilizcirion
Most of the experiments were run by holding the aluminum nitrate concentration constant while systematically varying the pH. Each sample in a series was prepared at 2-minute intervals, and turbidity measurements were taken, typically, after 15 minutes, 1, 3, 6, and 21 hours. Typical results are shown in Figures 1, 2, and 3. From these plots the critical pH values of coagulation (pH,) and stabilization (pH,) were obtained by extrapolating the steep portions of the curves back t o the turbidity of a blank E. coli dispersion in distilled water. This technique gave very reproducible results. The pH, may be defined as a limit such that a slightly higher pH results in coagulation. The pH, is the solution pH at which stabilization (restabilization or no coagulation) is just completed. Coagulation, then, occurs in the p H range between a pH, and a pH,, while stabilization occurs in the p H range between a pH, and a pH,. Each of these extrapolated values of pH at its initial aluminum concentration gives one point along a boundary of the aluminum concentration-pH domain of stability for E. coli. This procedure was followed using bacteria dispersions without added aluminum-Le., pH adjustment with nitric acid alone-and with aluminum concentrations ranging from about 3 X 1 0 P t o 0.10M. The pH, for nitric acid without added aluminum was 5.65, and therefore E. coli would coagulate in any solution of lower pH unless the organisms were restabilized by another solution component. Above about 2 X 10-4M Al(II1) there were observed two
Figure 2. Coagulation of Escheric/zra coli as a function of pH Hith 4.0 X 10 -5MA1(NOa)ain the rapid coagulation zone SJmbols as described in Figure 1
I I 0.1
1
o!
4 I
1 3
I
I PH
1 7
I 9
Figure 3. Coagulation of €scherichia coli as a function of pH Hith 2.0 X 10-51MAI(N03)3 at the bottom of the rapid coagulation zone Symbols as described in Figure 1
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discrete pH ranges of coagulation. Settling in the low p H region was very slow and the critical values were taken at 18 to 24 hours (Figure 1). In this region the coagulation is due t o either hydrogen ion or soluble aluminum species. I n the higher pH coagulation region, settling was essentially complete within 1 hour, which was chosen as a critical time. Notice in Figure 1 that in this region, at 2.0 X 10-3MAl(III), the 15-minute removal is also very high. This is the p H range of aluminum hydroxide precipitation and is the socalled “sweep zone” of rapid settling. Here pH, is virtually independent of time. At this aluminum concentration there are three p H ranges of stability for E. coli. Matijevik, Janauer, et ul. (1964) have shown with silver halide sols that positive polymeric aluminum species are specifically adsorbed at pH’s just below neutrality and that restabilization due t o charge reversal occurs in this region. The stability region at high p H is due t o the formation of aluminate ion, conforming t o the equilibrium:
+
AI(OH)~(S) H20
=
AI(0H)d-
+ Hf
Figure 4. Coagulation of Esclrerichin coli as a function of Al(NO& concentration at pH 2.0 (nitric acid) and pH 6.0 (acetate
buffer) A I-hour settling data. 0 6-hour settling data. 3 12-hour settling data
(1)
The low p H region of stability will be discussed later. At somewhat lower concentrations, for example, a t 4.0 X 10-jM AI(II1) as shown in Figure 2, the slow and rapid coagulation regions merge and the central stability region does not form. The concentration of aluminum at which this occurs would depend upon the concentration-i.e., surface area-of the dispersed phase since reversal of charge is a surface adsorption phenomenon. Note also in Figure 2 that the low p H region of stability is narrowing, and that in general the rate of settling is less than at higher aluminum concentrations. At still lower aluminum concentrations, 2.0 X 10-jM as shown in Figure 3, the sweep zone no longer appears, and the low p H stability region is only partially formed. At this concentration the settling behavior is not much different than would occur if the aluminum were not present and the pH was adjusted with nitric acid alone. Application of the Scliulze-Hard-v Rule
Several experimental series were also run in which the pH was held constant while the salt concentration was systematically varied. From these plots [as shown in Figure 4, for AI(NO3), at p H 2.0 and 6.01 the critical coagulation concentrations (c.c.c.) were determined in a similar fashion as described above for the critical pH values. For aluminum, the pairs, fixed pH-c.c.c., and fixed concentration-pH,, give essentially the same results, these being points along a boundary of the stability domain. The C.C.C.values for N a N 0 3 and Ba(NO&, without pH adjustment, were also determined using solutions prepared from the reagent grade salts and carbonate-free distilled water. Restabilization, which occurs at aluminum concentrations greater than the c.c.c., was not observed for these salts. The critical concentrations for N a N 0 3 and Ba(NO& were 3.4 X 10-l and 7.0 X lOPM, respectively. 360 Environmental Science and Technology
1
For aluminum various soluble polymeric hydroxo species have been proposed. These include A1ti(OH)lj3+and AIz(OH)?4- (cited by Stumm and Morgan, 1962), Ali(OH)li4+and A113(OH)315+(Biedermann, 1964), and Als(OH)204’ (MatijeviC et al., 1961, 1964, 1966b). From coagulation data there is extensive evidence that in the p H range 5 t o 7 the principal coagulating ion in dilute solutions is a four-plus charged specie. Assuming the octameric structure proposed by MatijeviC, the authors results at pH 6.0 indicate a C.C.C. of 2.7 X 10-6M-i.e., one-eighth of 2.15 X 10-jM for the applied aluminum dose. Note that assuming the A1i(OH)n4+ structure would not significantly change the logarithm of the C.C.C.
The Schulze-Hardy rule states that the coagulating power of a counterion increases greatly with its charge. Teiak, MatijeviC, et al. (1955) have shown with silver halide sols that a plot of log C.C.C. against the counterion charge yields a straight line giving a quantitative expression of the rule. Plotting the log of the critical concentrations for Na+, Ba?+, and Alg(OH)co4+ against their charge gives a straight line demonstrating the applicability of the Schulze-Hardy rule t o the coagulation of E. coli. Figure 5 shows that the plot for E. coli falls between those for the negative lyophobic sols, silver bromide (MatijeviC, Broadhurst, et ul., 1959) and latex (MatijeviC, 1967). The difference between the AgBr sol and E. coli curves is much less than an order of magnitude in C.C.C. E. coli also follow the Schulze-Hardy rule for coagulation by LiT, Ca?+, Mg2+,Zn2&,and Sr?+(Hayden, 1967). Domain of Stabilityfor E . coli
The critical coagulation and stabilization values from Figures 1, 2, 3, and 4 and similar data were plotted logarithm of the initial AI(NO& concentration against the solution p H
(Figure 6). Critical values obtained after 1 hour's settling are circles which form the boundaries for the rapid settling zones; 18- to 24-hour critical values for the turbidity-pH data are plotted a s squares forming the boundaries for the slow coagulation regions. Diamonds are critical coagulation concentration values taken after 12 hours of settling. Blackened symbols indicate stabilization data, and open symbols the critical coagulation values. Figure 6 defines the entire log A1(NOJ), concentration-pH domain of stability for this strain of Eschrrichiri coli at its specified concentration and harvest and growth conditions. At the bottom of the domain the vertical line passing through pH 5.65 is the pH, for the organism without added aluminum. Thus. as mentioned before, the region t o its left is slow coagulation by the H' ion. The vertical line meets a horizontal line at -4.67 logs, which in turn is joined by a diagonal line. These form the boundaries of a region of no coagulation. The upper boundaries of this stability region are similar to the results with silver halide sols (MatijeviC et al., 1961. 1964). In this region, below the diagonal line, the soluble Al(OH),-- ion is the predominate specie. As predicted in Equation 1 the slope of the line is fl. From the data, using the method of least squares, the calculated slope and pK are 1.02 and 12.3, respectively. A literature value for the latter is 12.71 (Stumm and Morgan, 1962). Thus, this part ofthe boundar) i s independent of the nature and concentration
of the dispersed phase. The horizontal portion of the boundary, however, is dependent upon the nature of the dispersed phase and may be predicted from results such as those shown in Figures 4 and 5 . Above the horizontal boundary of the region of no coagulation is a zone of rapid coagulation which bridges the slow coagulation region and the sweep zone. Coagulation in the rapid coagulation zone is shown in Figure 2. According to MatijeviC the primary aluminum species in this zone and the restabilization zone just above is the four-plus charged octamer. At the boundary, then, between this restabilization zone and the sweep zone:
+ 4H20 = SAI(OH)j(s) + 4HS.
A&(OH)?o.'+
I
I
-51
-6' I
(2)
For this reaction, at equilibrium and if the octamer were the predominate species, the predicted slope of the boundary is -4. The slope calculated from the data by the method of least squares is - 3.5. Therefore, direct conclusions regarding the principal coagulating and restabilizing aluminum species in these regions around the sweep zone cannot be drawn from the present data. The results, however, are entirely consistent with other studies. The stability region is very similar to those described for silver halide sols, except that it terminates on the bottom at a higher AI(II1) concentration. As indicated earlier, this would depend upon the surface area of the dispersed phase.
I
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3
SLOW COAGULATION ZONE
1'
Nitrates o f Na +.Ba2+,and AI,(OH)?o'' for E. coli
COAGULATION
i
4
1
COUNTERION CHARGE
Figure 5. Relationship for negative sols between the critical coagulation concentration and the charge of the coagulating cations
NO
PH Figure 6. Log AI(NO& concentration-pH domain of stability for Eschericlzin coli 0,O 1-hour and O ,. 18- to 24-hour turbiditj-pH settling data. 0 12-hour turbiditj -concentration settling data. O,C, 0 coagulation values. 0. stabilization values
Volume 2, Number 5, May 1968 361
To the extreme left at low p H is another stability region, being presumably a zone of restabilization since the organisms would be expected t o be coagulated by hydrogen ion. The nature of this region is ambiguous and has not been described for silver halide sols or clays. Coagulation results indicate that the primary coagulating species in solutions less than pH 4 is the unhydrolyzed AI3+ ion (MatijeviC, Janauer, et ul., 1964). However, similar studies have also shown that unhydrolyzed metal ions are ineffective for charge reversal. It is possible that very small concentrations of hydrolyzed species in equilibrium with AI3+ a t very low p H may be responsible for the restabilization. Conclusion
As with other sols, the coagulation of Escherichia coli is strongly dependent upon the p H and applied concentration of hydrolyzing coagulant. These organisms obey the SchulzeHardy rule for coagulation by univalent and divalent neutral salts, and presumably a polymeric species such as A18(OH)2a4f. They are slowly coagulated by hydrogen ion at concentrations considerably below that of other univalent cations. The entire log aluminum nitrate concentration-pH domain of stability for the coagulation of this strain of E. coli at a single concentration of the organisms was determined as indicated by settling rates. The stability domain is very similar t o those described for silver halide sols except for the boundary at pH 5.65 and the region of restabilization at low pH. Three regions of different settling rates were observed. The rate of settling in the rapid coagulation zone was greater than in the slow zone but considerably lower than in the sweep zone. Coagulation in the rapid zone is best explained by assuming the predominate aluminum species t o have an octameric or similar structure and to be four-plus charged. The sweep zone of aluminum hydroxide precipitation and rapid settling for E. coli was completely defined and occurs in similar concentration and p H ranges previously described for clays (Packham, 1963, 1965). The data support the contention of Packham that the dispersed particles are being enmeshed and carried down by precipitating gelatinous aluminum hydroxide (aluminum “floc”). Of course, as Packham (1963) has pointed out, physicochemical forces of attraction between the sol and the precipate may be important. Ives (1959) also observed that algae attract floc particles to themselves. Some workers have found that the nature of the dispersed phase has little if any effect on coagulation. Because of the
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En\irontnetital Science and Technologg
similarity of the results for E. coli with silver halide sols in the rapid coagulation zone and clays in the sweep zone, it appears that this view is essentially correct with certain limitations. Certainly, restabilization will depend both upon the nature and concentration of the dispersed phase. Coagulation by hydrogen ion also appears t o be dependent upon the nature of the sol, and may be dependent upon its concentration. Acknowledgment
The authors are indebted t o Egon Matijevii? of Clarkson College for his interest in this work and valuable discussions of it. The authors also gratefully acknowledge the assistance of Phillip Hayden with some of the experiments. Lirernture Cited
Biedermann, G.,Scensk. Kern. Tidskr. 76, 362 (1964). Hayden, P. L., M.S. thesis and unpublished data, University of Cincinnati, Cincinnati, Ohio, 1967. Ives, K. J., J . Biochem. Microbiol. Tech. Eng. 1, 37 (1959). LaMer, V. K., Healy, T. W., Rec. Pure Appl. Clieni. (Aiistralia) 13, 112 (1963). MatijeviC, E., Clarkson College, Potsdam, N. Y . , private communication, 1967. Matiievif. E.. Broadhurst, D., Kerker, M.. J . P/i\..s. Clieni. 63, 1552 (1959). MatijeviC, E., Janauer, G. E., J . Colloid lnrertace Sci. 21, 197 (1966a). MatijeiiC, E.; Janauer, G. E.. Kerker, M.,J . Co//oid Sci. 19, 333 (1964). MatiieviC. E.. Mathai, K. G.. Ottewill, R. H., Kerker, M., J . Pliys. Cliem. 65, 826 (1961). Matiievii.. E.. Strvker. L. J.. J . Colloid Inlertme Sci. 22, 68”(1966b).’ ’ O’Melia, C. R., Stumm, W., J . Colloid In~erfiiceSci. 23, 437 (1967). Packham, R’. F., J. Colloid Sci. 20, 81 (1965). Packham, R. F., Proc. Soc. Wuter Trear. E.~rcni.12, 15 (1963). Rubin, A. J., Cassell, E. A., Henderson, O., Johnson. J. D., Lamb, J. C., Biorechnol. Bioeng. 8, 135 (1966). Stumm, W., Morgan, J. J., J . Ani. Wmrr ItivXs .4ssoc. 54, 971 (1962). Tenney, M. W., Stumm, W., J . Wciter Pollirrion Control Fed. 37, 1370 (1965). Teiak, B., Matijevic, E., Schulz, K. F., J . P/IJ.S.Cliein. 55, 1557 (1951a). Teiak. B.. MatiieviC. E.. Schulz. K. F.. J . Phi.s. Cheriz. 55, 1567 (1951b). Teiak, B., Matijevii, E.. Schulz, K . F., J . P/ij.,. Clieriz. 59, 769 (1955). Receiredfbs reriew J ~ l 17, y 1967. Accepted .!fitrr~c~li21, 1968. G.P.H. as slipported bj, n N.S.F. felloitdiip.
