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Survival of E. coli and Enterococci in sediment-water systems of lake kinneret under (feedback) controlled concentrations of hydrogen sulfide
War. Res. Vol. 22, No. 2, pp. 233-240, 1988 Printed in Great Britain. All rights reserved
0043-1354/88 $3.00 + 0.00 Copyright © 1988 Pergamon Journals Ltd
SURVIVAL OF E. COLI AND ENTEROCOCCI IN SEDIMENT-WATER SYSTEMS OF LAKE KINNERET UNDER (FEEDBACK) CONTROLLED CONCENTRATIONS OF HYDROGEN SULFIDE STEFAN PEIFFER1., TALYA BERGSTEIN-BENDAN2, T6NNIES FREVERT3 and BEN ZION CAVARI4 ~Lehrstuhl fiir Hydrologic, Universit/it Bayreuth, Postfach 10 12 51, D-8580 Bayreuth, F.R.G., 2Kinneret LimnologicalLaboratory, Tiberias, Israel, 3FreseniusConsult GmbH, D-6204 Taunusstein 4, F.R.G. and 41srael Oceanographic and Limnological Research Ltd, Haifa, Israel (Received April 1987)
Abstract--A feedback control system was applied to establish defined redox conditions in sediment-water systems: the H2S release was counterbalanced by adding of air oxygen on arbitrarily chosen pH2S setpoints, pH2S was measured by a pH2S electrode cell as the controlled variable. E. coli and Enterococci were inoculated into the system to study their survival. Applying a new kinetic approach different growth patterns were revealed, indicating that E. coli, in contrast to Enterococci, shows significant dependence on the predominating redox conditions. Enterococci may thus be a more reliable indicator of fecal contamination in cases were sulfide is present. Key words--feedback control, survival rate, E. coli, Enterococci, hydrogen sulfide, sediment-water systems, Lake Kinneret, modeling
INTRODUCTION
Lake Kinneret is the only natural fresh water body in Israel. It plays an important role in the water supply: 300-400 million m 3 are pumped annually through the National Water Carrier system, which is about onequarter of the total water requirement of the country (Serruya, 1978). The lake is also heavily used for recreation especially during the spring-summer seasons and for fishing. Lake Kinneret is a warm monomictic lake with temperatures ranging from 15 to 29°C. The lake is stratified from May to December with rapid decrease in oxygen in the hypolimnion. In late May the hypolimnion is completely depleted of oxygen and sulfide starts to appear above the sediments. Later on, sulfide can be found in all the hypolimnetic water. In mid-summer (July-August) sulfide concentrations are 5-6 mg 1-~ at the sediment-water interface and 3-4 mg 1-~ in the metalimnion (Eckert and Frevert, 1984). The large number of tourists in the lake region contributes, either directly or indirectly, to increased fecal contamination of the water. Another source of such pollution is the River Jordan bringing sewage from Upper Galilee which usually occurs during the winter flooding. For many years fecal coliforms as well as the pathogenic Enterococci (Facklam and Wilkinson, 1981) have been used as an indicator of sewage contamination in aquatic systems (Bordner and *To whom all correspondence should be addressed.
Winter, 1978). The present study was carried out to learn the effect of oxic and anoxic conditions on the indicator bacteria entering the lake and to find out their fate when reaching sulfide containing water.
SEDIMENT-WATERSYSTEM Sediments in contact with sulfate containing pore waters can be characterized by their permanent release of H2S (Wetzel, 1981). This continuous release can be understood as a flow of an electron donor into the overlying water. Various bacteria in lakes are able to use this electron donor for their energy gain (Bergstein et al., 1979; Ingvorsen and Brock, 1982; Eckert et al., i 986). This process of permanent energy flow from the sediment into the overlying water is shown schematically in Fig. 1. The electron flow can be measured in two ways: (i) by detecting the electron donor--H2S; or (ii) by detecting the change of electron activity as a result of changing concentrations of the electron donor. By adding a suitable electron acceptor, like oxygen, the H2S corresponding electron activity can be regulated on an arbitrarily chosen level using an appropriate electron activity sensor for a feedback control devicing. Corresponding control experiments were applied to investigate phosphorus and manganese release from sediments into the overlying water (Ostendorp and Frevert, 1979; Frevert, 1979, 1980). In view of H2S as most pertinent for the investigated bacteria, the electron flow was measured with a H2S sensitive electrode cell (Frevert and Galster, 1978).
233
STEFAN PEIFFER e l al.
234
WATER-S U R F A C E
E L E C ~
ELECTRON-I 4-- I
---.
-4
DONA--T-OR[-~-~L ISEDIMENT-WATER .;~P'f~ .~.~z.~_f~__f.~j tNTERFACE
MS
Fig. 1. Electron transfer from sediment into overlying water. ~
The simulation of the natural process in the sediment-water system suggests the feedback control circuit as presented in Fig. 2. pH2S is the controlled variable in the sedimentwater system, where p H 2 S = - - l o g a H 2 s . It is continuously measured and compared to a chosen setpoint. The deviation of the controlled variable from the setpoint results from the permanent microbial sulfate reduction to H2S and a consequent change of the potential at the electrode cell. This interference has to be compensated by the final control element. The rapid oxidation of reduced divalent sulfur by oxygen (O'Brien and Birkner, 1977) suggests the application of atmospheric oxygen as final control element to be injected by an air pump into the system. Two kinds of responses are possible: (i) air pump on; sulfide will be oxidized: pH2S rises during oxidation: dpH2 S
- -
dt
~-k|
leading to a pseudo-first-order reaction kinetic, which was reported for the oxidation of divalent sulfur (O'Brien and Birkner, 1977), However, the rate constant may include the "strip o u t " effect of gaseous
l'
Ii2
FS -E -.IY
FCE
C
Fig. 3. Experimental mounting. A P = a i r pump; S =sensors, 1--pH2S, 2--pe, 3--pH; MS =microsensor; C = controller; TF = transformator; CP = circulation pump; RE = recorder; SWS = sediment-water system. H2S by the injected air; (ii) air pump off; release of H2S goes on: PH2S drops by the molecular diffusion of H2S from the sediment into the water. Thus, assuming completely mixing in the overlying water, we obtain: dpH2S dt = k2f(t) corresponding to a higher-order kinetics. MATERIALS AND METHODS Experimental devicing Figure 3 shows the experimental mounting. The screw plugs of the Perspex tubes, in which the sediment cores were sampled, had threads, in which the electrode cells could be screwed, pH2S was measured with a combined glass/Ag °, Ag2S electrode cell (pH 2S electrode cell, INGOLD Ag-27585-6329; Frevert and Galster, 1978). Its potential is a linear function of pH2S. This electrode cell performed Nernstian response down to pH2S = 18.2 (Peiffer and Frevert, 1987). It was calibrated according to Peters et al. (1984). pe (=-loga,_t~n~or); Frevert, 1984) and pH were determined using commercially available Pt and glass electrode
WA
CT ST
CP x
C
Us¥ ME PF
Fig. 2. Circuit scheme of the feedback control circuit. CP = control plant; z~/z2 = interferences, z~ --- oxygen input by air pump, z2=biogenic H2S release; SE=sensor; x = controlled variable; C = controller; M = meter; w = set point; IV = index value; CE = comparator; y = final control variable; FCE = final control element.
Fig. 4. Installation of circulation tube (CT) sampling syringe (SY) and pH 2S microelectrode (ME). WA = water; SE = sediment; ST = rubber stopper; PF = parafilm wrapping.
Survival of E. coli and Enterococci ,
_
_
J
_
235
Sampling of the sediments The sediment cores were taken from maximum depth of Lake Kinneret with a Perspex sampler (Tessenow et a/., 1977; cf. Fig. 5, map). The cores were undisturbed, although slightly compressed. The sediment was sampled so that a volume of about 500 ml left as the overlying water. If necessary, it was filled up carefully with lake water from the same part of the lake. Then the cores were darkened with aluminum foil. All experiments never lasted longer than 4 days.
_
Tat
Ginosal
"rib
in-Gev
Inoculation of bacteria E. coli type 1, identified by APi 20 E and Streptococcus faecium type 1, identified by APi 20 strep, in the following referred to as Enterococci, were used for the experiments. Usually both, E. coli and Enterococci were inoculated to each core together. However, two experiments were run with separate inocula. Enumeration technique The membrane filtration technique was applied, using m-FC procedure for E. coli (APHA, 1981) with 24-h incubation at 44°C. Enterococci were counted using the method of Levin et al. (1985), which was modified by reducing the amount of indoxyl-o-glucosid from 0.75 to 0.075 g. The media were incubated after filtration at 41°C for 44~8 h.
Fig. 5. Map of Lake Kinneret (Israel). cells (INGOLD Ltd, F.R.G.), where the reference half-cell was secured by an inert electrolyte bridging, A pH2S semielectrode cell was used in order to detect and control H2S in the sediment-water interface (Fig. 4). The signals of the pH 2S electrode cell were amplified and fed into the controller, which received its current supply from a 12 V transformer. The controller was constructed following Johnston and Buhr (1983). All signals were recorded on a 2-channel recorder (LINSEIS type 7020) or read during sampling. The overlying water was continuously circulated by a circuit pump (EHEIM type I012), where the circulation speed was regulated in a way that the water was completely mixed but no disturbance of the sediment surface layers could be observed. Air was injected with an injection needle which was fixed from below in a boring of the screw plug and connected with the air pump. The air pressure was dosed with a clamp at the tube in such a way that the amplitude of the pH2S measuring signal was minimized. All tubes were made of Tygon. Since the switching range of the controller was about 48 mV, which corresponded to 1.5 pH2S units, the injection needle was brought in a close contact to the pH2S electrode cell in the overlying water. An optimum damping of pH2 S within the controlled part of the system should thus be obtained in combination with the circulation of the water body.
kaoCFU~' 7-[ --.-- pe 1
---
pH2S- Sediment
-.-*- EllghedchllCoil
"/- -I? 6- - l e IS- -18 4- -14 3-J -13 2- -t2 1 - -11
---6- Entero¢oc¢l
o-,.Io
ii
¢o ~
~o do do do
r'o
Fig. 6. Experiment No. 3.
do
do
-! -9 -2 -8 -3 -7 *4-6 -6 -IS -6 .4 3
~tth)
Bacterial sampling Four to six aliquots of different volumes and dilution steps were taken and counted. Counts between 30 and 100 per plate were accepted. Samples were taken with sterile syringes from an opening (0.1 mm) in the screw plug and from aside the experiment tube above the sediment surface (see Fig. 4). No significant difference was found between the sampling sites. Thus all further sampling was done from the overlying water. All experiments were run at 20 + I°C. To prevent pH rising by CO2 release, one pH 7 buffer tablet (WTW pH calibration buffer tablets for 100ml buffer solution) was added to the overlying waters. The corresponding phosphate concentration increase could be neglected in view of the phosphorus concentrations of Lake Kinneret sediments (Serruya, 1978).
RESULTS Figures 7 a n d 9 show typical feedback control diagrams. It can be seen t h a t the frequency o f the p u m p ' s switching is a direct function of H2S production in the sediment: the closer the setpoint is situated as c o m p a r e d to the H2S c o n c e n t r a t i o n s which are naturally f o u n d in Lake K i n n e r e t s e d i m e n t - w a t e r systems (pH2S -~ 4.3 at p H 7), the more intensive is the release after switching the p u m p off (Fig. 9). W h e n controlling pH2S o n intermediate values (Fig. 7), H2S is released m u c h slower. This indicates t h a t the redox conditions (pc ~-0, Fig. 7; pe ~- - 4 , Fig. 9) will not allow such a n intensive H2S p r o d u c t i o n as has been f o u n d with experimental conditions given in Fig. 9. Both experiments show t h a t various pH~S setpoints could be m a i n t a i n e d for a certain period of time. Different redox conditions were thus established. Typical bacterial results are given in Figs 6-10. W h e n E. coli a n d Enterococci were inoculated into an aerated core, a decrease of a b o u t 1 log o f E. coli a n d less t h a n 1/2 log of Enterococci cells after 25 h of
236
STEFANPE~FFERet al. pHaS "17 .19
~42S
I"le
pH2S - Water
pH=S - ~
.1E .14 "13 '12 .tt "tO '0 .8 'I .9
1'o
/o
~
~)
r~
~
7'o
ah
,6 ,4
I: ~b ~oo..)
log C FU, N~ ?• .--*- F J c h e d c ~ CoW S-,.-,,,- En~tococci
3
log cFU'~I 7-( .p~
2S 47 ~
i ~ S - Sediment
.~e
5-
.13 .12 •11
.10 3"
"9
"9 2-
tlO
0
20
30
410
50
~
TIO
~)
~) I= (h)
Fig. 7. Experiment No. 4. Top--feedback control diagram; setpoint--pH2S,~tc, = 9.7 (straight line).
incubation could be observed. After 40 h the cell number of E. coil decreased by 2 log and of Enterococci by less than 2 log. Both reached the same number after 60 h (Fig. 6). Under anaerobic conditions (pH2S 9-10) a slight increase of E. coli cell numbers was noticed after 15 h, followed by a slight decrease to reach a constant number of both E. cell and Enterococci (Fig. 7). When initial pH2S was about 7 and bacterial number was about 103ml -~ both bacteria increased by 1/2 log after 5 h and then decreased by about l log. pH2S rising to 9 (sulfide decreasing) after 25 h was followed by a recovery of E. coli but not Enterococci, where further decrease was observed (Fig. 8). No recovery and a sharper decrease of cell numbers was observed when a higher number of E. coil and Enterococci were inoculated ~cFu#
p829
7-
~.i ---
6"
-4•.-o- E n t e r o c o c c i
5"
4"
pe pH2S - Sedlms~t PH2S - W a t e r Escherichia CoW
.~.
7. -'17 6- -16
01
,b
o
z6
/o
,b
sb
3
6b
/0
~
,oo,(.)
Fig. 9. Experiment No. 6. Top--feedback control diagram; setpoint--pH2S,,tc, = 6.3 (straight line). ( > 105 C F U m l - ' ) under the same conditions (Fig. 9). However, separate inoculation of high numbers of bacteria lead to a reduced decrease ofEnterococci (cf. Table 1, experiment 7 and Table 2, experiment 8, respectively). A combination of low pH2S of about 4.5 and relatively low number of cells (103-104CFUml -~) gave an increase of more than 1 log in E. coil cells and a constant number of EnterococcL As the redox conditions seemed to influence the mortality rate of E. coli more than Enterococci, statistical analysis were performed. However, no kinetical coefficients can be obtained assuming firstorder die-off, since growth was observed in some experiments before dying. This lack was solved by applying a new kinetic approach. logCFUmi' i
p~2S
~
---~
5. -15 4- -14 3- -13 2. L12
pH2S - Water PH2S " Sediment Escherichia Coli
-i}' -16 -15
En|efo
-14 -12 -I1
~
I. -11 o, "10
3~
~
-I" - 9
-2. ~-a 2"
-3. ~-7 -4" P6 -5' ~-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
o
ib
ab
ab
,6
,~
eb
7'o
Fig. 8. Experiment No. 5.
ab
b3
lO0 I(n)
0
10
~1
30
40
50
60
70
Fig. 10. Experiment No. 10.
80
90
-4 3
|0~ I(h)
Survival of E. coli and Enterococci
237
Table 1. Comparison of kinetic coefficients k, as obtained by application of Chick's law and the t -model and correlation coefficients r between experimental and predicted data for E. coli Type of model
n
k (h I)
C*
4
-0.1862 (-)0.2275 -0.1306 (-)0.1489 -0.0923 (-)0.1019 -0.0545 (-)0.0606 -0.0469 (-)0.0491 -0.0111 0.0127 -0.0206 0.0449 -0.0089 0.0596 -0.0048 0.1308
t~"
4
c
6
c
5
C
6
c
4
C
6
c
5
c
5
C
tm,~ (h)
a
0
1
0
1
0
1
0
I
0
l
4
1
15
5
19
10
44
45
r 0.927 0.866 0.935 0.897 0.999 0.995 0.970 0.960 0.905 0.875 0.574 0.562 0.642 0.860 0.292 0.839 -0.061 0.961
P(~<0.05)
Experiment No.
+ -+ +
7~
+
3
6
+ + + + + ---+ -+ -+
1 2 5 4 9 10
*Chick's law. tt-model. ~:Experiment run only with E. coli. Significance higher than the 95% level for r is marked by +.
Table 2. As Table 1 but for Enterococci Type of model
n
k (h- i)
C
6
-0.1032 -0.2398 -0.1761 -0.2018 -0.0610 -0.1042 -0.0971 -0.1040 -0.0458 -0.0589 -0.0222 0.0280 -0.0219 -0.0234 -0.0192 -0.0203 -0.0724 0.1314
4
c
5
c
6
c
4
c
5
C
5
C
6
c
4
C l
tin,~ (h)
a
0
I
0
I
0
1
0
1
0
I
1
2
0
1
0
1
2.5
4
r
P( ~<0.05)
Experiment No.
0.798 0.866 0.988 0.962 0.915 0.946 0.993 0.987 0.894 0.930 0.922 0.950 0.902 0.880 0.885 0.871 0.983 0.994
+ + + + + + + + + + + + + + + + + +
3 6 1 2 5 9 10 4 8*
*Experiment run only with Enterococci.
STATISTICAL ANALYSIS
is
a
Theoretical consideration C h i c k ' s law o f disinfection (Chick, 1908), a first-order r e a c t i o n kinetic, is u s u a l l y applied for a m a t h e m a t i c a l d e s c r i p t i o n o f bacterial die-off: dc --
=
-kc.
dt
(1)
H o w e v e r , r e p o r t s are existing in literature, w h e r e the die-off is p r e c e d e d by a significant a f t e r - g r o w t h o f cells in field studies ( E v a n s , 1968) as well as in l a b o r a t o r y studies ( C a n a l e et al., 1973; M a n c i n i , 1980; S a v a g e a n d H a n e s , 1971). Since, this p h e n o m e n o n w a s o b s e r v e d in this s t u d y , m o s t o b v i o u s in e x p e r i m e n t 10, e q u a t i o n (1) w a s varied in the following way: T h e function: dc
general
expression
of
Chick's
law,
where
f ( t ) = c o n s t a n t . Setting:
-- = k'c = kf(t)c dt
(2)
k'=kf(t)=k
tmax-t-, t+a
(3)
tn~x = time o f m a x i m u m cell density, a =parameter c o n t r o l l i n g the s k e w n e s s a s s y m e t r y o f the curve (a > 1),
or
a f u n c t i o n w i t h the f o l l o w i n g characteristics will be found:
(i) it c h a n g e s its sign f o r t = tm~x ( g r o w t h - d y i n g ) , (ii) k is a l w a y s negative w i t h tr~x = 0, (iii) k c o n v e r g e s vs - k w i t h large t (lim k f ( t ) =
-k).
t~o~
238
ST~AN PEIFFERet al.
Table 3. Data for computing the Varimax Rotated Principal Component Analysis Inoculum Experiment No. k (h- ~) (cellsml- i) pc, E. coil 7 -0.2275 136.20 -4.1 6 --0.1489 119.00 -4.0 3 -0.1019 25.80 4.2 I -0.0606 12.63 4.3 2 -0.0491 100.00 2.1 5 0.0127 4.15 0.1 4 0.0449 1,91 -0,2 9 0.0596 4.04 --4,0 10 0.1308 11.80 -3,9 Enterococci 3 --0.2398 48.50 4,2 6 -0.2018 4610.00 -4.0 I -0.1042 45.00 4.3 2 -0.1040 93.00 2.1 5 --0.0589 68 0.1 9 0.0280 7.9 -4.0 10 -0.0234 1.85 -3.9 4 -0.0203 43.20 -0.2 8 0.1314 362.50 -3.1 *Mean value.
The complete growth function can be written:
dc
--
dt
= k
/max -- t c t +a
(4)
and its integrated form (Bronstein and Semendjajev, 1979): c = b exp[k(tma x + a ) l n ( t + a) - t],
(5)
where b is a parameter. N u m e r i c a l solution
Nonlinear regression analysis was computed using the least-squares method on a VAX-1 1/780 computer system for both models, the first-order reaction and the time dependent growth function, in the following called t-model. The parameter a was determined iteratively by optimizing the correlation coefficient r between experimental and computed data. The iteration was stopped at step i + 1 if: (i) Iri+ ~- ril was smaller than 0.01; or (ii) Iri+l < ril. After each iteration step, a was enhanced by 1.
statistical significance with the t-model. Taking into account that with tmx---0 (die-off) the parameter a will be 1 (see Tables 1 and 2) and by that the function will converge vs - k in equation (4), thus corresponding to a first-order reaction rate. This is indicated by the minus signs in brackets. In case of E n t e r o c o c c i a similar sequence cannot be obseved. This can be explained by the fact that a tmax very close to 0 is corresponding to a very slight increase of cell density thus indicating rather a statistical deviation that a growth. S t a t i s t i c a l results
A Varimax Rotated Principal C o m p o n e n t Analysis (Oberla, 1971; V R P C A ) was computed with the kinetic coefficient of the t-model k, inoculum and pe-value as variables and the data of the 9 experiments (Table 3). Inoculum was taken as a variable to check whether its impact on survival is significant. This method coordinates data in selecting variables due to their degree of intercorrelation and in combining a multitude o f variables to new parameters (principal components). The resulting " l o a d i n g " matrices tabulate significant correlation coefficients between the principal components and the original variables; each column represents a principal component due to the intercorrelated variables (rows). Hence this method gives hints for intercorrelations between variables loading one principal component. Table 4 shows the loading matrices for E. coli and E n t e r o c o c c i with correlation coefficients significant on the 95% level. Neither inoculum nor redox potential influenced the growth of Enterococci. The same picture can be seen with E. coli. However, when repeating the V R P C A reduced by experiments with high inoculum (experiments 6 and 7 with E. coli and 6 and 8 with E n t e r o c o c c i ) high correlation between redox potential and kinetic coefficient was revealed (Table 5). Consequently a correlation coefficient between pe-value and k was calculated to be 0.963 (P = 0.001).
Validation
Comparison of the two models (Tables 1 and 2) reveals a better fit of the experimental data by the t-model, especially in case of experiment 10. An arrangement of the experiments according to their kinetic coefficient k, provides the same sequence with both models for E. coli, but with a more frequent Table 4. Loading matrices as obtained by VRPCA with all data for E. coli and Enterococci with correlation coefficients, the 95% significance level Principal components Variable 1 2 3 E. coli k 0.9316 lnoculum 0.7869 pe 0.9928 k 0.9993 Enterococci lnoculum 0.9786 pe -0.9826
DISCUSSION The feedback control, applied to the sedimentwater system, avoided a number of disadvantages of in vitro studies, discussed in literature (Wuhrmann, 1972; Hendricks, 1974): chemical parameter of the overlying waters like pH, alkalinity, salinity or Table 5. Loading matrix with data redued by the experiments with high inoculum (6, 7 E. coli and 6, 8 Enterococci) (P ~<0.05) Principal components Variable I 2 3 k -0.9527 E. coli 0.9806 Inoculum pe 0.9730 k 0.9923 Enterococci Inoculum 0.9850 pe 0.9950
Survival of E. coli and Enterococci
239
Bronstein I. N. und Semendjajev K. A. (1979) Taschenbuch der Mathematik, p. 13. Verlag H. Deutsch, Ziirich. Canale R. P., Patterson R. L., Gannon J. J. and Powers W. F. (1973) Water quality models for total coliforms. J. Wat. Pollut. Control. Fed. 45, 325-326. Chick H. (1908) Investigation of the law of disinfection. J. Hyg. 8, 92-158. Eckert W. and Frevert T. (1984) In situ monitoring of hydrogen sulfide in water and sediment of Lake Kinneret (Israel). In 4th Symposium on Ion-Selective Electrodes, Mhtrafiired (Hungary) (Edited by Pungor E.), Hungarian Academy of Sciences. Eckert W., Frevert T., Bergstein-Ben Dan T. and Cavari B. Z. (1986) Competitive development of Thiocapsa roseopersicina and Chlorobium phaeobacteroides in Lake Kinneret. Can. J. Microbiol. 32, 917-921. Evans F. L. (1968) Treatment of urban stormwater runoff. .L Wat. Pollut. Control Fed. 40, R162-RI67. Facklam R. F. and Wilkinson H. W. (1981) The family Streptococcoceae (medical aspects). In The Prokaryotes, A Handbook on Habitats. Isolation and Identification of Bacteria (Edited by Starr M. P., Stolp H., Triiper H. C., Balows A. and Schlegel H. G.), pp. 1572-1597. Frevert T. (1979) The pe redox concept in natural sediment-water systems; its role in controlling phosphorus release from lake sediments. Arch. Hydrobiol. (Suppl.) 55, 278-297. Frevert T. (1980) Dissolved oxygen dependent phosphorus release from profundal sediments of Lake Constance (Obersee). Hydrobiologia 74, 17-28. Frevert T. (1984) Can the redox conditions in natural waters be predicted by a single parameter? Schweiz. Z. Hydrol. 46, 269-290. Frevert T. and Galster H. (1978) Schnelle und einfache Methode zur in-situ-Bestimmung von Schwefelwasserstoff in Gew~sern und Sedimenten. Schweiz. Z. Hydrol. 40, 199-208. Hendricks C. W. (1974) Sorption of heterotrophic and enteric bacteria to glass surfaces in the continuous culture of river water. Appl. Microbiol. 28, 572-582. Ingvorsen K. and Brock T. D. (1982) Electron flow via sulfate reduction and methanogenesis in the anaerobic hypolimnion of Lake Mendota. Limnol. Oceanogr. 27, 559-564. Johnston N. R. and Buhr H. O. (1983) A simple scheme for controlling dissolved oxygen and measuring oxygen utilisation in lab-scale activated sludge units. Wat. SA 8, 23-25. Levin M. A., Fischer D. R. and Cabelli V. J. (1975) Membrane filter technique for enumeration of Enterococci in marine waters. Appl. Microbiol. 30, 66-71. Mancini J. L. (1980) Numerical estimates of coliform mortality rates under various conditions. J. Wat. Pollut. Control Fed. 50, 2477-2484. Acknowledgements--This study was granted by the Bay- Milne D. P., Curran J. C. and Wilson L. (1986) Effects of erisches Staatsministerium fiir Wissenschaft und Kunst, sedimentation on removal of faecal coliform bacteria which is gratefully acknowledged. The authors further wish from effluents in estuarine waters. Wat. Res. 20, to thank Drs A. Mattes and Y. Forer from the Israelian 1493-1496. Health Authority for the identification of the bacteria. O'Brien D. J. and Birkner F. B. (1977) Kinetics of oxidation of reduced sulphur species in aqueous solution. Envir. Sci. Technol. 11, 1114-1120. Ostendorp W. and Frevert T. (1979) Untersuchungen zur REFERENCES Manganfreisetzung und zum Mangangehalt der SedimenAPHA (1981) Standard Methods for the Examination toberschicht im Bodensee. Arch. Hydrobiol. (Suppl.) 55, of Water and Wastewater. American Public Health 255-277. Association, Washington, D.C. Peiffer S. and Frevert T. (1987) Potentiometric deterBergstein T., Henis Y. and Cavari B. Z. (1979) Investimination of heavy metal sulfide solubilities by use of the gations on the photosynthetic sulfur bacterium ChloroPH2S (glass/Ag°, Ag2S) Electrode cell. The Analyst 112, bium phaeobacteroides causing seasonal blooms in Lake 951-954. Kinneret. Can. J. Microbiol. 25, 999-1007. Peters K., Huber G., Netsch S. and Frevert T. Bordner R. and Winter J. (Eds) (1978) Microbiological (1984) Methode zur direktpotentiometrischen in-situMethods for Monitoring and Environment. EPA Registrierung yon Schwefelwasserstoff in einer Kl~ir600/8-78-017, U.S. Environmental Protection Agency. anlage. Wass. Abwass. 125, 386-390.
nutrients are buffered by the sediment. The biocenosis in the sediment and at the sediment surface is the same as under natural conditions. Variation of the redox-chemical conditions was thus performed with a minimum of disturbance of the environmental conditions and with a satisfying precision. The precision, however, could be increased by application of more sophisticated equipment, which was not at the author's disposal for this study. E. coli showed a higher response to aerobiosisanaerobiosis than Enterococci. Enterococci were more stable in most of the experimental conditions. However, care should be taken, when investigating two different bacteria in one experiment at the same time. As was shown, significant differences in the mortality rate were only found in experiments with reduced inoculum. The inoculum should not be higher than the total bacterial count usually found ( I & - 1 0 5 C F U m l -~) in sediment-water systems of Lake Kinneret). We may therefore propose that Enterococci will be a more faithful indicator for fecal contamination in environments of frequently changing redox conditions, i.e. environments of changing water levels. Those environments in combination with sulfate reduction as the predominant anaerobic metabolism in sediments will mainly exist in estuaries (Presley and Trefry, 1980), where also high loads of fecal polluted waters can be expected (Milne et al., 1986). Die-off functions for modeling bacterial pollution (Milne et al., 1986) should thus be extended by a hydrochemical parameter. As the Israeli water authorities intend to further decrease the water level of Lake Kinneret, the nearshore sediments will change from coarse to silty material. Hence environmental conditions in the area of potential fecal pollution at Lake Kinneret sediments will become similar to the simulated ones. Our results, therefore, strongly suggest that the survival of other fecal contaminants, e.g. viruses, in sedimentwater systems of Lake Kinneret should be investigated under the view presented in this study.
240
STEFANPEIFFERet al.
Presley B. J. and Trefry J. H. (1980) Sediment-water interaction and the geochemistry of interstitial waters. In Chemistry and Biogeochemistry of Estuaries (Edited by Olausson E. and Cato I.), Chap. 5. Wiley, New York. Savage H. P. and Hanes N. B. (1971) Toxicity of seawater to coliform bacteria. J. Wat. Pollut. Control Fed. 43, 854-858. Serruya C. (1978) Lake Kinneret. Junk, The Hague. Tessenow U., Frevert T., Hofg/irtner W. and Moser A. (1977) Ein simultan schliel3ender Serienwassersch6pfer
fiir Sedimentkontaktwasser mit fotoelektrischer S¢lbstausl6sung und fakultativem Sedimentstecher. Arch. Hydrobiol. (Suppl.) 48, 438-452. Uberla K. (1971) Faktorenanalyse. Springer, Heidelberg. Wetzel R. G. (1981) Limnology. 2nd edition. Saunders, Philadelphia. Wuhrmann F. (1972) Stream purification. In Water Pollution Microbiology (Edited by Mitchell R.), Vol. 1. Wiley-lnterscience, New York.
0043-1354/88 $3.00 + 0.00 Copyright © 1988 Pergamon Journals Ltd
SURVIVAL OF E. COLI AND ENTEROCOCCI IN SEDIMENT-WATER SYSTEMS OF LAKE KINNERET UNDER (FEEDBACK) CONTROLLED CONCENTRATIONS OF HYDROGEN SULFIDE STEFAN PEIFFER1., TALYA BERGSTEIN-BENDAN2, T6NNIES FREVERT3 and BEN ZION CAVARI4 ~Lehrstuhl fiir Hydrologic, Universit/it Bayreuth, Postfach 10 12 51, D-8580 Bayreuth, F.R.G., 2Kinneret LimnologicalLaboratory, Tiberias, Israel, 3FreseniusConsult GmbH, D-6204 Taunusstein 4, F.R.G. and 41srael Oceanographic and Limnological Research Ltd, Haifa, Israel (Received April 1987)
Abstract--A feedback control system was applied to establish defined redox conditions in sediment-water systems: the H2S release was counterbalanced by adding of air oxygen on arbitrarily chosen pH2S setpoints, pH2S was measured by a pH2S electrode cell as the controlled variable. E. coli and Enterococci were inoculated into the system to study their survival. Applying a new kinetic approach different growth patterns were revealed, indicating that E. coli, in contrast to Enterococci, shows significant dependence on the predominating redox conditions. Enterococci may thus be a more reliable indicator of fecal contamination in cases were sulfide is present. Key words--feedback control, survival rate, E. coli, Enterococci, hydrogen sulfide, sediment-water systems, Lake Kinneret, modeling
INTRODUCTION
Lake Kinneret is the only natural fresh water body in Israel. It plays an important role in the water supply: 300-400 million m 3 are pumped annually through the National Water Carrier system, which is about onequarter of the total water requirement of the country (Serruya, 1978). The lake is also heavily used for recreation especially during the spring-summer seasons and for fishing. Lake Kinneret is a warm monomictic lake with temperatures ranging from 15 to 29°C. The lake is stratified from May to December with rapid decrease in oxygen in the hypolimnion. In late May the hypolimnion is completely depleted of oxygen and sulfide starts to appear above the sediments. Later on, sulfide can be found in all the hypolimnetic water. In mid-summer (July-August) sulfide concentrations are 5-6 mg 1-~ at the sediment-water interface and 3-4 mg 1-~ in the metalimnion (Eckert and Frevert, 1984). The large number of tourists in the lake region contributes, either directly or indirectly, to increased fecal contamination of the water. Another source of such pollution is the River Jordan bringing sewage from Upper Galilee which usually occurs during the winter flooding. For many years fecal coliforms as well as the pathogenic Enterococci (Facklam and Wilkinson, 1981) have been used as an indicator of sewage contamination in aquatic systems (Bordner and *To whom all correspondence should be addressed.
Winter, 1978). The present study was carried out to learn the effect of oxic and anoxic conditions on the indicator bacteria entering the lake and to find out their fate when reaching sulfide containing water.
SEDIMENT-WATERSYSTEM Sediments in contact with sulfate containing pore waters can be characterized by their permanent release of H2S (Wetzel, 1981). This continuous release can be understood as a flow of an electron donor into the overlying water. Various bacteria in lakes are able to use this electron donor for their energy gain (Bergstein et al., 1979; Ingvorsen and Brock, 1982; Eckert et al., i 986). This process of permanent energy flow from the sediment into the overlying water is shown schematically in Fig. 1. The electron flow can be measured in two ways: (i) by detecting the electron donor--H2S; or (ii) by detecting the change of electron activity as a result of changing concentrations of the electron donor. By adding a suitable electron acceptor, like oxygen, the H2S corresponding electron activity can be regulated on an arbitrarily chosen level using an appropriate electron activity sensor for a feedback control devicing. Corresponding control experiments were applied to investigate phosphorus and manganese release from sediments into the overlying water (Ostendorp and Frevert, 1979; Frevert, 1979, 1980). In view of H2S as most pertinent for the investigated bacteria, the electron flow was measured with a H2S sensitive electrode cell (Frevert and Galster, 1978).
233
STEFAN PEIFFER e l al.
234
WATER-S U R F A C E
E L E C ~
ELECTRON-I 4-- I
---.
-4
DONA--T-OR[-~-~L ISEDIMENT-WATER .;~P'f~ .~.~z.~_f~__f.~j tNTERFACE
MS
Fig. 1. Electron transfer from sediment into overlying water. ~
The simulation of the natural process in the sediment-water system suggests the feedback control circuit as presented in Fig. 2. pH2S is the controlled variable in the sedimentwater system, where p H 2 S = - - l o g a H 2 s . It is continuously measured and compared to a chosen setpoint. The deviation of the controlled variable from the setpoint results from the permanent microbial sulfate reduction to H2S and a consequent change of the potential at the electrode cell. This interference has to be compensated by the final control element. The rapid oxidation of reduced divalent sulfur by oxygen (O'Brien and Birkner, 1977) suggests the application of atmospheric oxygen as final control element to be injected by an air pump into the system. Two kinds of responses are possible: (i) air pump on; sulfide will be oxidized: pH2S rises during oxidation: dpH2 S
- -
dt
~-k|
leading to a pseudo-first-order reaction kinetic, which was reported for the oxidation of divalent sulfur (O'Brien and Birkner, 1977), However, the rate constant may include the "strip o u t " effect of gaseous
l'
Ii2
FS -E -.IY
FCE
C
Fig. 3. Experimental mounting. A P = a i r pump; S =sensors, 1--pH2S, 2--pe, 3--pH; MS =microsensor; C = controller; TF = transformator; CP = circulation pump; RE = recorder; SWS = sediment-water system. H2S by the injected air; (ii) air pump off; release of H2S goes on: PH2S drops by the molecular diffusion of H2S from the sediment into the water. Thus, assuming completely mixing in the overlying water, we obtain: dpH2S dt = k2f(t) corresponding to a higher-order kinetics. MATERIALS AND METHODS Experimental devicing Figure 3 shows the experimental mounting. The screw plugs of the Perspex tubes, in which the sediment cores were sampled, had threads, in which the electrode cells could be screwed, pH2S was measured with a combined glass/Ag °, Ag2S electrode cell (pH 2S electrode cell, INGOLD Ag-27585-6329; Frevert and Galster, 1978). Its potential is a linear function of pH2S. This electrode cell performed Nernstian response down to pH2S = 18.2 (Peiffer and Frevert, 1987). It was calibrated according to Peters et al. (1984). pe (=-loga,_t~n~or); Frevert, 1984) and pH were determined using commercially available Pt and glass electrode
WA
CT ST
CP x
C
Us¥ ME PF
Fig. 2. Circuit scheme of the feedback control circuit. CP = control plant; z~/z2 = interferences, z~ --- oxygen input by air pump, z2=biogenic H2S release; SE=sensor; x = controlled variable; C = controller; M = meter; w = set point; IV = index value; CE = comparator; y = final control variable; FCE = final control element.
Fig. 4. Installation of circulation tube (CT) sampling syringe (SY) and pH 2S microelectrode (ME). WA = water; SE = sediment; ST = rubber stopper; PF = parafilm wrapping.
Survival of E. coli and Enterococci ,
_
_
J
_
235
Sampling of the sediments The sediment cores were taken from maximum depth of Lake Kinneret with a Perspex sampler (Tessenow et a/., 1977; cf. Fig. 5, map). The cores were undisturbed, although slightly compressed. The sediment was sampled so that a volume of about 500 ml left as the overlying water. If necessary, it was filled up carefully with lake water from the same part of the lake. Then the cores were darkened with aluminum foil. All experiments never lasted longer than 4 days.
_
Tat
Ginosal
"rib
in-Gev
Inoculation of bacteria E. coli type 1, identified by APi 20 E and Streptococcus faecium type 1, identified by APi 20 strep, in the following referred to as Enterococci, were used for the experiments. Usually both, E. coli and Enterococci were inoculated to each core together. However, two experiments were run with separate inocula. Enumeration technique The membrane filtration technique was applied, using m-FC procedure for E. coli (APHA, 1981) with 24-h incubation at 44°C. Enterococci were counted using the method of Levin et al. (1985), which was modified by reducing the amount of indoxyl-o-glucosid from 0.75 to 0.075 g. The media were incubated after filtration at 41°C for 44~8 h.
Fig. 5. Map of Lake Kinneret (Israel). cells (INGOLD Ltd, F.R.G.), where the reference half-cell was secured by an inert electrolyte bridging, A pH2S semielectrode cell was used in order to detect and control H2S in the sediment-water interface (Fig. 4). The signals of the pH 2S electrode cell were amplified and fed into the controller, which received its current supply from a 12 V transformer. The controller was constructed following Johnston and Buhr (1983). All signals were recorded on a 2-channel recorder (LINSEIS type 7020) or read during sampling. The overlying water was continuously circulated by a circuit pump (EHEIM type I012), where the circulation speed was regulated in a way that the water was completely mixed but no disturbance of the sediment surface layers could be observed. Air was injected with an injection needle which was fixed from below in a boring of the screw plug and connected with the air pump. The air pressure was dosed with a clamp at the tube in such a way that the amplitude of the pH2S measuring signal was minimized. All tubes were made of Tygon. Since the switching range of the controller was about 48 mV, which corresponded to 1.5 pH2S units, the injection needle was brought in a close contact to the pH2S electrode cell in the overlying water. An optimum damping of pH2 S within the controlled part of the system should thus be obtained in combination with the circulation of the water body.
kaoCFU~' 7-[ --.-- pe 1
---
pH2S- Sediment
-.-*- EllghedchllCoil
"/- -I? 6- - l e IS- -18 4- -14 3-J -13 2- -t2 1 - -11
---6- Entero¢oc¢l
o-,.Io
ii
¢o ~
~o do do do
r'o
Fig. 6. Experiment No. 3.
do
do
-! -9 -2 -8 -3 -7 *4-6 -6 -IS -6 .4 3
~tth)
Bacterial sampling Four to six aliquots of different volumes and dilution steps were taken and counted. Counts between 30 and 100 per plate were accepted. Samples were taken with sterile syringes from an opening (0.1 mm) in the screw plug and from aside the experiment tube above the sediment surface (see Fig. 4). No significant difference was found between the sampling sites. Thus all further sampling was done from the overlying water. All experiments were run at 20 + I°C. To prevent pH rising by CO2 release, one pH 7 buffer tablet (WTW pH calibration buffer tablets for 100ml buffer solution) was added to the overlying waters. The corresponding phosphate concentration increase could be neglected in view of the phosphorus concentrations of Lake Kinneret sediments (Serruya, 1978).
RESULTS Figures 7 a n d 9 show typical feedback control diagrams. It can be seen t h a t the frequency o f the p u m p ' s switching is a direct function of H2S production in the sediment: the closer the setpoint is situated as c o m p a r e d to the H2S c o n c e n t r a t i o n s which are naturally f o u n d in Lake K i n n e r e t s e d i m e n t - w a t e r systems (pH2S -~ 4.3 at p H 7), the more intensive is the release after switching the p u m p off (Fig. 9). W h e n controlling pH2S o n intermediate values (Fig. 7), H2S is released m u c h slower. This indicates t h a t the redox conditions (pc ~-0, Fig. 7; pe ~- - 4 , Fig. 9) will not allow such a n intensive H2S p r o d u c t i o n as has been f o u n d with experimental conditions given in Fig. 9. Both experiments show t h a t various pH~S setpoints could be m a i n t a i n e d for a certain period of time. Different redox conditions were thus established. Typical bacterial results are given in Figs 6-10. W h e n E. coli a n d Enterococci were inoculated into an aerated core, a decrease of a b o u t 1 log o f E. coli a n d less t h a n 1/2 log of Enterococci cells after 25 h of
236
STEFANPE~FFERet al. pHaS "17 .19
~42S
I"le
pH2S - Water
pH=S - ~
.1E .14 "13 '12 .tt "tO '0 .8 'I .9
1'o
/o
~
~)
r~
~
7'o
ah
,6 ,4
I: ~b ~oo..)
log C FU, N~ ?• .--*- F J c h e d c ~ CoW S-,.-,,,- En~tococci
3
log cFU'~I 7-( .p~
2S 47 ~
i ~ S - Sediment
.~e
5-
.13 .12 •11
.10 3"
"9
"9 2-
tlO
0
20
30
410
50
~
TIO
~)
~) I= (h)
Fig. 7. Experiment No. 4. Top--feedback control diagram; setpoint--pH2S,~tc, = 9.7 (straight line).
incubation could be observed. After 40 h the cell number of E. coil decreased by 2 log and of Enterococci by less than 2 log. Both reached the same number after 60 h (Fig. 6). Under anaerobic conditions (pH2S 9-10) a slight increase of E. coli cell numbers was noticed after 15 h, followed by a slight decrease to reach a constant number of both E. cell and Enterococci (Fig. 7). When initial pH2S was about 7 and bacterial number was about 103ml -~ both bacteria increased by 1/2 log after 5 h and then decreased by about l log. pH2S rising to 9 (sulfide decreasing) after 25 h was followed by a recovery of E. coli but not Enterococci, where further decrease was observed (Fig. 8). No recovery and a sharper decrease of cell numbers was observed when a higher number of E. coil and Enterococci were inoculated ~cFu#
p829
7-
~.i ---
6"
-4•.-o- E n t e r o c o c c i
5"
4"
pe pH2S - Sedlms~t PH2S - W a t e r Escherichia CoW
.~.
7. -'17 6- -16
01
,b
o
z6
/o
,b
sb
3
6b
/0
~
,oo,(.)
Fig. 9. Experiment No. 6. Top--feedback control diagram; setpoint--pH2S,,tc, = 6.3 (straight line). ( > 105 C F U m l - ' ) under the same conditions (Fig. 9). However, separate inoculation of high numbers of bacteria lead to a reduced decrease ofEnterococci (cf. Table 1, experiment 7 and Table 2, experiment 8, respectively). A combination of low pH2S of about 4.5 and relatively low number of cells (103-104CFUml -~) gave an increase of more than 1 log in E. coil cells and a constant number of EnterococcL As the redox conditions seemed to influence the mortality rate of E. coli more than Enterococci, statistical analysis were performed. However, no kinetical coefficients can be obtained assuming firstorder die-off, since growth was observed in some experiments before dying. This lack was solved by applying a new kinetic approach. logCFUmi' i
p~2S
~
---~
5. -15 4- -14 3- -13 2. L12
pH2S - Water PH2S " Sediment Escherichia Coli
-i}' -16 -15
En|efo
-14 -12 -I1
~
I. -11 o, "10
3~
~
-I" - 9
-2. ~-a 2"
-3. ~-7 -4" P6 -5' ~-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
o
ib
ab
ab
,6
,~
eb
7'o
Fig. 8. Experiment No. 5.
ab
b3
lO0 I(n)
0
10
~1
30
40
50
60
70
Fig. 10. Experiment No. 10.
80
90
-4 3
|0~ I(h)
Survival of E. coli and Enterococci
237
Table 1. Comparison of kinetic coefficients k, as obtained by application of Chick's law and the t -model and correlation coefficients r between experimental and predicted data for E. coli Type of model
n
k (h I)
C*
4
-0.1862 (-)0.2275 -0.1306 (-)0.1489 -0.0923 (-)0.1019 -0.0545 (-)0.0606 -0.0469 (-)0.0491 -0.0111 0.0127 -0.0206 0.0449 -0.0089 0.0596 -0.0048 0.1308
t~"
4
c
6
c
5
C
6
c
4
C
6
c
5
c
5
C
tm,~ (h)
a
0
1
0
1
0
1
0
I
0
l
4
1
15
5
19
10
44
45
r 0.927 0.866 0.935 0.897 0.999 0.995 0.970 0.960 0.905 0.875 0.574 0.562 0.642 0.860 0.292 0.839 -0.061 0.961
P(~<0.05)
Experiment No.
+ -+ +
7~
+
3
6
+ + + + + ---+ -+ -+
1 2 5 4 9 10
*Chick's law. tt-model. ~:Experiment run only with E. coli. Significance higher than the 95% level for r is marked by +.
Table 2. As Table 1 but for Enterococci Type of model
n
k (h- i)
C
6
-0.1032 -0.2398 -0.1761 -0.2018 -0.0610 -0.1042 -0.0971 -0.1040 -0.0458 -0.0589 -0.0222 0.0280 -0.0219 -0.0234 -0.0192 -0.0203 -0.0724 0.1314
4
c
5
c
6
c
4
c
5
C
5
C
6
c
4
C l
tin,~ (h)
a
0
I
0
I
0
1
0
1
0
I
1
2
0
1
0
1
2.5
4
r
P( ~<0.05)
Experiment No.
0.798 0.866 0.988 0.962 0.915 0.946 0.993 0.987 0.894 0.930 0.922 0.950 0.902 0.880 0.885 0.871 0.983 0.994
+ + + + + + + + + + + + + + + + + +
3 6 1 2 5 9 10 4 8*
*Experiment run only with Enterococci.
STATISTICAL ANALYSIS
is
a
Theoretical consideration C h i c k ' s law o f disinfection (Chick, 1908), a first-order r e a c t i o n kinetic, is u s u a l l y applied for a m a t h e m a t i c a l d e s c r i p t i o n o f bacterial die-off: dc --
=
-kc.
dt
(1)
H o w e v e r , r e p o r t s are existing in literature, w h e r e the die-off is p r e c e d e d by a significant a f t e r - g r o w t h o f cells in field studies ( E v a n s , 1968) as well as in l a b o r a t o r y studies ( C a n a l e et al., 1973; M a n c i n i , 1980; S a v a g e a n d H a n e s , 1971). Since, this p h e n o m e n o n w a s o b s e r v e d in this s t u d y , m o s t o b v i o u s in e x p e r i m e n t 10, e q u a t i o n (1) w a s varied in the following way: T h e function: dc
general
expression
of
Chick's
law,
where
f ( t ) = c o n s t a n t . Setting:
-- = k'c = kf(t)c dt
(2)
k'=kf(t)=k
tmax-t-, t+a
(3)
tn~x = time o f m a x i m u m cell density, a =parameter c o n t r o l l i n g the s k e w n e s s a s s y m e t r y o f the curve (a > 1),
or
a f u n c t i o n w i t h the f o l l o w i n g characteristics will be found:
(i) it c h a n g e s its sign f o r t = tm~x ( g r o w t h - d y i n g ) , (ii) k is a l w a y s negative w i t h tr~x = 0, (iii) k c o n v e r g e s vs - k w i t h large t (lim k f ( t ) =
-k).
t~o~
238
ST~AN PEIFFERet al.
Table 3. Data for computing the Varimax Rotated Principal Component Analysis Inoculum Experiment No. k (h- ~) (cellsml- i) pc, E. coil 7 -0.2275 136.20 -4.1 6 --0.1489 119.00 -4.0 3 -0.1019 25.80 4.2 I -0.0606 12.63 4.3 2 -0.0491 100.00 2.1 5 0.0127 4.15 0.1 4 0.0449 1,91 -0,2 9 0.0596 4.04 --4,0 10 0.1308 11.80 -3,9 Enterococci 3 --0.2398 48.50 4,2 6 -0.2018 4610.00 -4.0 I -0.1042 45.00 4.3 2 -0.1040 93.00 2.1 5 --0.0589 68 0.1 9 0.0280 7.9 -4.0 10 -0.0234 1.85 -3.9 4 -0.0203 43.20 -0.2 8 0.1314 362.50 -3.1 *Mean value.
The complete growth function can be written:
dc
--
dt
= k
/max -- t c t +a
(4)
and its integrated form (Bronstein and Semendjajev, 1979): c = b exp[k(tma x + a ) l n ( t + a) - t],
(5)
where b is a parameter. N u m e r i c a l solution
Nonlinear regression analysis was computed using the least-squares method on a VAX-1 1/780 computer system for both models, the first-order reaction and the time dependent growth function, in the following called t-model. The parameter a was determined iteratively by optimizing the correlation coefficient r between experimental and computed data. The iteration was stopped at step i + 1 if: (i) Iri+ ~- ril was smaller than 0.01; or (ii) Iri+l < ril. After each iteration step, a was enhanced by 1.
statistical significance with the t-model. Taking into account that with tmx---0 (die-off) the parameter a will be 1 (see Tables 1 and 2) and by that the function will converge vs - k in equation (4), thus corresponding to a first-order reaction rate. This is indicated by the minus signs in brackets. In case of E n t e r o c o c c i a similar sequence cannot be obseved. This can be explained by the fact that a tmax very close to 0 is corresponding to a very slight increase of cell density thus indicating rather a statistical deviation that a growth. S t a t i s t i c a l results
A Varimax Rotated Principal C o m p o n e n t Analysis (Oberla, 1971; V R P C A ) was computed with the kinetic coefficient of the t-model k, inoculum and pe-value as variables and the data of the 9 experiments (Table 3). Inoculum was taken as a variable to check whether its impact on survival is significant. This method coordinates data in selecting variables due to their degree of intercorrelation and in combining a multitude o f variables to new parameters (principal components). The resulting " l o a d i n g " matrices tabulate significant correlation coefficients between the principal components and the original variables; each column represents a principal component due to the intercorrelated variables (rows). Hence this method gives hints for intercorrelations between variables loading one principal component. Table 4 shows the loading matrices for E. coli and E n t e r o c o c c i with correlation coefficients significant on the 95% level. Neither inoculum nor redox potential influenced the growth of Enterococci. The same picture can be seen with E. coli. However, when repeating the V R P C A reduced by experiments with high inoculum (experiments 6 and 7 with E. coli and 6 and 8 with E n t e r o c o c c i ) high correlation between redox potential and kinetic coefficient was revealed (Table 5). Consequently a correlation coefficient between pe-value and k was calculated to be 0.963 (P = 0.001).
Validation
Comparison of the two models (Tables 1 and 2) reveals a better fit of the experimental data by the t-model, especially in case of experiment 10. An arrangement of the experiments according to their kinetic coefficient k, provides the same sequence with both models for E. coli, but with a more frequent Table 4. Loading matrices as obtained by VRPCA with all data for E. coli and Enterococci with correlation coefficients, the 95% significance level Principal components Variable 1 2 3 E. coli k 0.9316 lnoculum 0.7869 pe 0.9928 k 0.9993 Enterococci lnoculum 0.9786 pe -0.9826
DISCUSSION The feedback control, applied to the sedimentwater system, avoided a number of disadvantages of in vitro studies, discussed in literature (Wuhrmann, 1972; Hendricks, 1974): chemical parameter of the overlying waters like pH, alkalinity, salinity or Table 5. Loading matrix with data redued by the experiments with high inoculum (6, 7 E. coli and 6, 8 Enterococci) (P ~<0.05) Principal components Variable I 2 3 k -0.9527 E. coli 0.9806 Inoculum pe 0.9730 k 0.9923 Enterococci Inoculum 0.9850 pe 0.9950
Survival of E. coli and Enterococci
239
Bronstein I. N. und Semendjajev K. A. (1979) Taschenbuch der Mathematik, p. 13. Verlag H. Deutsch, Ziirich. Canale R. P., Patterson R. L., Gannon J. J. and Powers W. F. (1973) Water quality models for total coliforms. J. Wat. Pollut. Control. Fed. 45, 325-326. Chick H. (1908) Investigation of the law of disinfection. J. Hyg. 8, 92-158. Eckert W. and Frevert T. (1984) In situ monitoring of hydrogen sulfide in water and sediment of Lake Kinneret (Israel). In 4th Symposium on Ion-Selective Electrodes, Mhtrafiired (Hungary) (Edited by Pungor E.), Hungarian Academy of Sciences. Eckert W., Frevert T., Bergstein-Ben Dan T. and Cavari B. Z. (1986) Competitive development of Thiocapsa roseopersicina and Chlorobium phaeobacteroides in Lake Kinneret. Can. J. Microbiol. 32, 917-921. Evans F. L. (1968) Treatment of urban stormwater runoff. .L Wat. Pollut. Control Fed. 40, R162-RI67. Facklam R. F. and Wilkinson H. W. (1981) The family Streptococcoceae (medical aspects). In The Prokaryotes, A Handbook on Habitats. Isolation and Identification of Bacteria (Edited by Starr M. P., Stolp H., Triiper H. C., Balows A. and Schlegel H. G.), pp. 1572-1597. Frevert T. (1979) The pe redox concept in natural sediment-water systems; its role in controlling phosphorus release from lake sediments. Arch. Hydrobiol. (Suppl.) 55, 278-297. Frevert T. (1980) Dissolved oxygen dependent phosphorus release from profundal sediments of Lake Constance (Obersee). Hydrobiologia 74, 17-28. Frevert T. (1984) Can the redox conditions in natural waters be predicted by a single parameter? Schweiz. Z. Hydrol. 46, 269-290. Frevert T. and Galster H. (1978) Schnelle und einfache Methode zur in-situ-Bestimmung von Schwefelwasserstoff in Gew~sern und Sedimenten. Schweiz. Z. Hydrol. 40, 199-208. Hendricks C. W. (1974) Sorption of heterotrophic and enteric bacteria to glass surfaces in the continuous culture of river water. Appl. Microbiol. 28, 572-582. Ingvorsen K. and Brock T. D. (1982) Electron flow via sulfate reduction and methanogenesis in the anaerobic hypolimnion of Lake Mendota. Limnol. Oceanogr. 27, 559-564. Johnston N. R. and Buhr H. O. (1983) A simple scheme for controlling dissolved oxygen and measuring oxygen utilisation in lab-scale activated sludge units. Wat. SA 8, 23-25. Levin M. A., Fischer D. R. and Cabelli V. J. (1975) Membrane filter technique for enumeration of Enterococci in marine waters. Appl. Microbiol. 30, 66-71. Mancini J. L. (1980) Numerical estimates of coliform mortality rates under various conditions. J. Wat. Pollut. Control Fed. 50, 2477-2484. Acknowledgements--This study was granted by the Bay- Milne D. P., Curran J. C. and Wilson L. (1986) Effects of erisches Staatsministerium fiir Wissenschaft und Kunst, sedimentation on removal of faecal coliform bacteria which is gratefully acknowledged. The authors further wish from effluents in estuarine waters. Wat. Res. 20, to thank Drs A. Mattes and Y. Forer from the Israelian 1493-1496. Health Authority for the identification of the bacteria. O'Brien D. J. and Birkner F. B. (1977) Kinetics of oxidation of reduced sulphur species in aqueous solution. Envir. Sci. Technol. 11, 1114-1120. Ostendorp W. and Frevert T. (1979) Untersuchungen zur REFERENCES Manganfreisetzung und zum Mangangehalt der SedimenAPHA (1981) Standard Methods for the Examination toberschicht im Bodensee. Arch. Hydrobiol. (Suppl.) 55, of Water and Wastewater. American Public Health 255-277. Association, Washington, D.C. Peiffer S. and Frevert T. (1987) Potentiometric deterBergstein T., Henis Y. and Cavari B. Z. (1979) Investimination of heavy metal sulfide solubilities by use of the gations on the photosynthetic sulfur bacterium ChloroPH2S (glass/Ag°, Ag2S) Electrode cell. The Analyst 112, bium phaeobacteroides causing seasonal blooms in Lake 951-954. Kinneret. Can. J. Microbiol. 25, 999-1007. Peters K., Huber G., Netsch S. and Frevert T. Bordner R. and Winter J. (Eds) (1978) Microbiological (1984) Methode zur direktpotentiometrischen in-situMethods for Monitoring and Environment. EPA Registrierung yon Schwefelwasserstoff in einer Kl~ir600/8-78-017, U.S. Environmental Protection Agency. anlage. Wass. Abwass. 125, 386-390.
nutrients are buffered by the sediment. The biocenosis in the sediment and at the sediment surface is the same as under natural conditions. Variation of the redox-chemical conditions was thus performed with a minimum of disturbance of the environmental conditions and with a satisfying precision. The precision, however, could be increased by application of more sophisticated equipment, which was not at the author's disposal for this study. E. coli showed a higher response to aerobiosisanaerobiosis than Enterococci. Enterococci were more stable in most of the experimental conditions. However, care should be taken, when investigating two different bacteria in one experiment at the same time. As was shown, significant differences in the mortality rate were only found in experiments with reduced inoculum. The inoculum should not be higher than the total bacterial count usually found ( I & - 1 0 5 C F U m l -~) in sediment-water systems of Lake Kinneret). We may therefore propose that Enterococci will be a more faithful indicator for fecal contamination in environments of frequently changing redox conditions, i.e. environments of changing water levels. Those environments in combination with sulfate reduction as the predominant anaerobic metabolism in sediments will mainly exist in estuaries (Presley and Trefry, 1980), where also high loads of fecal polluted waters can be expected (Milne et al., 1986). Die-off functions for modeling bacterial pollution (Milne et al., 1986) should thus be extended by a hydrochemical parameter. As the Israeli water authorities intend to further decrease the water level of Lake Kinneret, the nearshore sediments will change from coarse to silty material. Hence environmental conditions in the area of potential fecal pollution at Lake Kinneret sediments will become similar to the simulated ones. Our results, therefore, strongly suggest that the survival of other fecal contaminants, e.g. viruses, in sedimentwater systems of Lake Kinneret should be investigated under the view presented in this study.
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