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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.
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.
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.
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. REFERENCES Artizzu M.. Haccbino P. M. & Dianzani M. V. (1963) The action of carbon tetrachloride on mitrocbondria in L'itro Biochem. biophys. Acta 78, 1-11. Bringmann G. {19531 Electron microscopic findings of the action of chlorine, bromine iodine, copper, silver and hydrogen peroxide on E. coli. Z. Hy 9. l nj ektiKrankh. 130, 155-166. Chem. Abs. 48, 34401. Fair G. M., Morris J. C., Chang S. L. & Burden R. B. (1948) The behaviour of chlorine as a water disinfectant, J. Am. Wat. Wks Ass. 40, 1051-1061. Frigberg L. (1957i Further quantitative studies on the reac- tion of chlorine with bacteria in waste disinfection. Acta path. microbiol, stand. 40, 67-80. Green D. E. & Stumpf P. K. {1946) The mode of action of chlorine. J. Am. War. Wk s Ass. 38, 1301-1305. Greening E. O. (1971~ Microbial indicators for the biologi- cal quality of treated waste water effluents. Dissertation submitted for M.S. University of Illinois, Urbana. Herbert D., Phippe P. J. & Strange J. E. (1971) Chemical analysis of microbiol cells. In: Methods Microbiology 5B, (Edited by Morris & Ribbons), Academic Press, New York. Kashket E. R. & Brodie A. F. [1963) Oxidative phosphory- lation of fractionated bacterial system. Biochim. biophys. Acta 78, 55-65. Knox W. E.. Stumpf P. K., Green D. E. & Auerbach V. H. (1948) The inhibition of sulfhydryl enzymes as a basis of the bactericidal action of chlorine. J. Bact. 55, 451M.58. Lowry O. H., Rosebrough N. J., Farr A. L. & Randall R. J. (1951) Protein measurement with folin phenol re- agent. J. biol. Chem. 193, 265-275. Plummer D. T. (1971) An lntrod,~ction to Practical Bio- chemistry. McGraw-Hill, London. Salton M. R. J. (19641 The Bacterial Cell Wall. Elsevier, Amsterdam. Standard methods for examination of water and waste- water (1971) American Public Health Association, Wash- ington, D.C. 13th edn. Venkobacher C, lyengar L. & Prabhakara Rap A. V. S. (1975) Mechanism of disinfection. Water Res. 9, 119- 124. Wharton D. C. & McCarty E. (1972) Experiments and Methods in Biochemistry. Macmillam, New York. White G. C. (1972) Handbook o f Chlorination. Van Nos- trand Reinheld, New York. w.p.. I | i 8- - 1