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V,:iter Re~cJrch Vol. I1. pp 72'" to "29 Pergamon Pres~ t ~' " Pr:nted in Great Britain.

MECHANISM OF DISINFECTION: EFFECT OF CHLORINE


ON CELL MEMBRANE FUNCTIONS
C. VENKOBACH.M~.. LEELA IYENGAR and A. V S. PRABHAKARA RAO
Environmental Engineering Laboratory. Department of Civil Engineering.
Indian Institute of Technology. Kanpur 208016. India
{Receited 12 Jul y 19761
Abstract- - To elucidate the mechanism of disinfection b? chlorine, its effects on some ~ital properties
associated with cell membrane of Escherchi~ coli were studied. There was no significant change in
Zeta potential at bactericidal doses of chlorine. Treatment with chlorine induced the leakage of macro-
molecules from the cells indicating the permeability changes of the membrane. Proteins and RNA
were detected in the supernatant when the cells were treated with chlorine dose of 1.5 mg I - t (100 lLg
CI mg- ~ N). The presence of DNA was observed only at high chlorine doses. The studies on oxidative
phosphorylation of cell free extract indicated the complete cessation of phosphate uptake at a dose
of 0.4 mg I - t (30t~g CI mg- t N). There was about 700 decrease in the oxygen uptake corresponding
to chlorine dose of 0.8 mg I- t.
I NTRODUCTI ON
Removal of pat hogeni c bac t eri a and ent eri c viruses
f rom wat er is of par amou nt i mport anc e because of
i ncreasi ng pol l ut i on of surf ace waters. Whi l e treat-
ment processes ot her t han disinf ection i mprove the
physical and chemi cal charact eri st i cs of raw water,
generally it is disinf ection t hat ensures systemic mi-
crobi al dest ruc t i on of f ering effective def ence against
t ransmi ssi on of wat er bor ne diseases. Disinf ection by
chl ori ne has been extensively used for t he last six
decades. As a result, c onsi derabl e amou nt of inf orma-
tion is avai l abl e on the physical chemi st ry of chl ori ne
in aqueous sol ut i ons as well as' t h e kinetics of its bac-
tericidal act i on ( Fai r et al., 1948; White, 1972). How-
ever the mode of act i on of chl ori ne and its com-
pounds on mi crobi al life is not yet fully underst ood.
Green and St u mpf (1946) f ound t hat glucose oxi-
dat i on by bact eri a was i nhi bi t ed by trace amou nt s
of chl ori ne while Knox et al, (1948) report ed chl ori ne
in bact eri ci dal doses i nhi bi t ed the sulf hydryl enzymes.
El ect ron mi croscopi c studies by Br i ngmann (1953)
showed no changes occurred to cell wall where as
in a conf licting report Fri berg (1957) using p32
labelled bact eri a observed the leakage of cellular com-
ponent s after chl ori ne t reat ment .
Thus, t here are conf licting report s regardi ng the
mode of act i on of chl ori ne on bact eri a in general and
on bacterial memb r ane in particular. This pr ompt ed
to investigate the effect of chl ori ne on the vital poly-
f unctions of cell memb r ane of E. coll.
MATERI ALS AND METHODS
Materials
Adenosine monophosphate, sodium succinate, ribose,
calf thymus DNA and serum albumin were purchased from
Sigma Chemicals Ltd. All other chemicals used were of
analytical grade.
Methods
Escl,erichia coil BB obtained from Environmental
Engineering Laboratory, Department of Civil Engineering,
University of Illinois. Urbana. U.S.A.. was grown on syn-
thetic medium containing bacto-tryptone 10g: yeast
extract 5 ~; sodium chloride 10~ and glucose 1 ,, l - t of
distilled water. The medium was adjusted to pH 7.0. 200 ml
portions were distributed in 500 ml Fernbech flasks and
were sterilised (Standard Methods, 1971). They were inocu-
lated with E. coil cells and incubated at 30 + TC. During
incubation the flasks were agitated in a reciprocating
shaker for 12 h. The cells thus obtained were harvested
by centrifugation, washed thrice with 0.01 M phosphate
buffer (pH 7.0) and resuspended in the same buffer for
exposure to disinfectant.
Preparation of cell free extract: all operations were car-
ried out between 0 - 4 C. A thick suspension of bacterial
ceils in chlorine demand free water was subjected to soni-
cation (Vibronics, Bombay, India) at 100 mA for 12 min.
The extract was centrifuged at 6000g for 20 rain to remove
cell debris. The supernatant obtained was used for oxida-
tive phosphorylation experiment.
Chlorine demand free water was prepared according to
Greening (1971). All reagents and buffers used in chlorinat-
ing experiments were prepared using chlorine demand free
water. Chlorine solution was prepared by dissolving the
chlorine gas, liberated by the action of concentrated HCI
on potassium permanganate in dilute NaOH. The stock
chlorine solution was stored in amber coloured bottles at
5 :C. Chlorine determinations were made using a DPD-sul-
phate indicator according to Standard Methods (1971).
Exposure of bacterial cells was done using the following
procedure. E. coil suspension of known protein concen-
tration were exposed to different chlorine doses for 15 rain
at 20~C. Samples were dechlorinated by 0.1 N sodium thio-
sulphate and then used for the assay of survivors. Standard
plate count method using nutrient agar was used for enu-
merating the survivors in accordance with Standard
Methods (19711. The cell free extracts were also subjected
to disinfectant in the similar way.
Electrophoretic mobility of bacterial cells of known
number was measured using a zetameter. Zeta potential
values reported were calculated from the average electro-
phoretic mobility of at least 10 bacterial cells tracked.
Soluble protein estimation was made according to the
method of Lowry et ~d. (1951) using Folin' s reagent. Total
727
-27' C. VENKOBACHAR. LEELA [YENGAR a nd A. V. S. PRABHAKARA R.~,o
Table i. Effect of chlorine on survival of E. coil
Chlorine dose Survival
mg l - t t~g of CI mg- t N of cells (%1
0.0 0.0 100
0.5 32.5 84
1.0 65.0 66
1.5 97.5 50
2.0 130.0 20
2.5 162.5 15
protein content of E. coli was determined according to
the method of Herbert et al. (1971). Determinations of
ribonucleic acid (RNA) and deoxyribonucleic acid were
made according to Hummer (197l).
Oxidative phosphorylating activity of crude cell free
extracts was measured by the method of Kashket and Bro-
die (t963). Oxygen uptake was measured using Warburg
respirometer at 30:C. The reaction mixture consisted of
lS#moles MgCI.,, 151~moles inorganic phosphate and
l mg yeast hexokinase in the main compartment of the
Warburg flask. The side arm contained 1001~moles KF.
25,tmoles mannose and 50 ltmoles of succinate. The cen-
tral well contained 0.2 ml of 10",; KOH solution absorbed
onto a filter paper. Final volume was adjusted to 1.5 ml.
Oxygen uptake was measured for 20 min. The reaction was
stopped by adding 1.0 ml of 5?0 TCA. After removing the
precipitate, phosphate was determined colorimetrically in
an aliquot of the supernatant (Wharton and McCarty,
I972).
RESULTS AND DI SCUSSI ON
E. coli cells were exposed to different chlorine doses
for 15 min and the results are shown in Tabl e l. This
table is mainly used to correlate the results of sub-
sequent experiments with bactericidal doses of chlor-
ine.
The effect of chlorine on zeta potential of E. coil
is shown in Fig. 1. A change in the potentials value
from - 4 0 to - 3 0 mV was observed corresponding
to the highest dose of chlorine ( 1000#g of Cl
mg- ' N). Bacterial cells can be visualised to be com-
prising of a number of concentric shells having an
ionic layer, cell wall and cell membrane. The outer-
most is ionic with side chains of ami no acids contri-
buting to the net negative charge on the surface of
E. coil (Salton, 1964).
4 0 Q
>
E
3c
o
N
\
F r [ I / I
150 4 5 0 7 5 0 1 0 5 0
Chlorine concentrotion, Mg Ct mg-'N
, 92
E
. 0
z
~ 3
12:.
75
z
Z5
0
8 C
~. ec
E
~ 4O
2C
f T t
- / . J
l I I I I ~ I
200 4.00 600 800 lOCX3 ~200 1400 ~600
Chlorine concentrotion, ~g CLrng"N
Fig. 2.
The bacterial cells after exposure to different chlor-
ine doses were spun down at 6000g. The collected
supernatant was subj ected to analysis of proteins,
ribonucleic acid ( RNA) and deoxyribonucleic acid
(DNA). Results on leakage of these macromolecules
are presented in Fig. 2. Proteins were detected in
supernatant when the cells were exposed to even low
doses of chlorine. The presence of RNA was detected
at 100,ug Cl mg - [ N. The presence of DNA was
observed only when high chlorine doses were used
against bacterial cells.
Studies on oxygen consumpt i on and phosphate
uptake of chlorinated cell-free extracts of E. coil were
conducted according to Kashket and Brodie (1963)
and the results are presented in Fig. 3. The uptake
of oxygen and that of phosphate decreased consider-
ably by exposure to low doses of chlorine. Interesting
feature is even in the presence of uptake of oxygen
there was a complete cessation of phosphate incor-
poration.
c
L
_oE I
O.E
Z E
Ro 0.20
o. , ,
~' ~ OK
o : o.o
r i O
I I ~ telincOrpOratiOn
20 40
Chlorine dose, ~,g Ct mg -~ N
Fig. I . Fig. 3.
Mechanism of disinfection 729
The decrease in oxygen uptake of chlorine treated
cell extract may be due to inhibition of enzyme(sl
which catalyse the oxi dat i on of substrate. In the
present experiments succinate was used as substrate
and hence the reduced oxygen uptake is partly due
to inhibition of succinic dehydrogenase ( Venkobachar
er al.. 1975). Further. chlorine may be exerting stress
on one or more component s of electron transporting
enzymes, thus affecting the oxygen uptake. Since
coupling of phosphorylation to electron transport is
obligatory, it is reasonable to assume any interruption
to the flow of electrons would affect phosphate uptake
also.
However, exami nat i on of Fig. 3 indicates phos-
phate incorporation is more sensitive to chlorine than
oxygen uptake. Compl et e cessation of phosphoryla-
tion occurred by a chlorine dose which produced only
0 j inactivation of bacterial cells. Chlorine seems to
"o
act as a decoupler and this may be due to inactivation
of trans-phosphorylases. The action of chlorine
appears to resemble the uncoupling action of carbon
tetrachloride on in uitro oxidative phosphoryl at i on by
rat liver mi t rochondri a (Artizu et aL, 1963).
Zeta potential, permeability and oxidative phos-
phorylation which are the i mport ant properties of cell
membrane are affected by exposure to chlorine. The
results presented show that oxidative phosphoryl at i on
is very sensitive to chlorine. Constant leakage of pro-
teins and RNA requires enhanced synthesis of these
components if the cell has to function normally. How-
ever, the energy needed for the synthesis of these
macromolecules is not available as energy rich ATP
molecules cannot be synthesised.
The first step in the interaction of chlorine with
E. coil is its reaction with cell membrane. Results
presented above indicate that physical, chemical and
biochemical changes occur to the cell membrane after
chlorine treatment affecting the vital functions of bac-
terium.
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w.p.. I | i 8- - 1

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