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Influence of temperature and u.v. light on disinfection with ozone
Water Research VO[. IL pp, 73 "r to 741 Pergamon Press 1977. Printed in Great Britain.
INFLUENCE OF TEMPERATURE AND U.V. LIGHT ON DISINFECTION WITH OZONE SHAUKAT FAROOQ,* RICHARD S. ENGELBRECHT and EDWARD S. K. CHIAN Department of Civil Engineering. University of Illinois at Urbana-Champaign. Urbana. Illinois 61801. U.S.A.
(Received 24 December 1976; in revised form 24 February 19771 Abstract--An inactivation study was performed with ozone in two laboratory-scale, continuous flow systems to determine the effects of temperature and u.v. light on the survival of Mvcobacteriumforruitum, a potential microbial indicator for disinfection efficiency. Four temperatures were investigated in the range of 9-37-'C. It was determined that a higher degree of inactivation occurred with ozone at elevated temperatures. The activation energy for M. fortuitttm was found to be 18.3 kcal. When u.v. light was employed as a catalyst with ozone disinfection, there was no apparent increase in the degree of inactivation of M. fortuitum in either the clean system (deionized water-phosphate buffer) or secondary wastewater effluent. However, u.v. light in itself exerted a strong disinfecting effect.
INTRODUCTION Few researchers, investigating ozone disinfection, have attempted to determine the effect of temperature on the inactivation of microorganisms. Leiguarda et al. (1949) reported that the bactericidal efficiency of ozone was not affected by temperature. K i n m a n (1972) studied ozone inactivation of bacteria at two temperatures, 25 and 39°C, and inferred that the rapid rate of destruction of bacteria was comparable at these two temperatures. A study of ozone inactivation was also performed by Zeff et al. (.1974) at 17, 25 and 40°C using Streptococcus faecalis, Klebsiella pneumoniae and Ancanthamoeba castellanii. It was concluded that the effect of t e m p e r a t u r e was insignificant. In the same study, ozone plus u.v. light was found to be more effective in destroying the test organisms than u.v. light alone in a stirred tank reactor. A review of the literature also reveals that no attempt has been made by previous researchers to correlate the degree of inactivation to the ozone residual, which in turn is affected by the temperature. Therefore, it was difficult in considering past studies to interpret the precise effect of temperature on disinfection. The purpose of this research was to investigate the effects of temperature and u.v. light on ozone residual and. consequently, the degree of inactivation of microorganisms, since ozone residual was found to be the most influential parameter in ozone disinfection at room temperature (Farooq et al., t976). Experiments also were performed to determine the effect of u.v. light o n disinfection with ozone.
as the primary organism in this study. A pure culture of M. fi,'tuitum was obtained from the study of Engelbrecht et ul. (1974), who proposed the acid-fast bacteria as a possible new microbial indicator of wastewater disinfection efficiency. In this study, M. fortuitum was used as the primary organism due to its greater resistance to ozone and ease of obtaining kinetic data (Engelbrecht et al., 1974: Farooq, 1976).
Medium and cultication method In preparation of inactivation experiments, M. Jbrtuitum was grown in a broth medium consisting of Middlebrook & Cohn 7H9 mineral base, 4.7 g; malachite green, 1.0 rag; and sodium propionate, 1.0 g l- t deionized water. The culture was incubated for 72 h at 37'~C in a water shaker bath before harvesting. Cells were harvested by means of a Sorval GLC-2 (DuPont Instruments, Newton, CT) general laboratory centrifuge at 1800 rev min - t for 15 min. They were then washed twice with a total of at least 150 ml of 0.0025 M phosphate buffer. Cells prepared in this manner were resuspended in phosphate buffer and were kept at 4°C until needed. M. fortuimm was enumerated using the 7H9 Middlebrook & Cohn medium indicated above but with 1.5~;; Bacto agar (Difco Lab, Detroit, MI) to solidify the medium. A spread plate technique was employed in this study to enumerate the surviving organisms. Samples were diluted with phosphate buffer solution such that a 0.l-ml aliquot of diluted sample would give at least 50 and no more than 200 colonies per culture dish. Incubation was accomplished at 37'C for 72-96 h. Detailed procedures for enumeration of M. fortuitum are given elsewhere (Engelbrecht et al., 1974; Farooq, 1976). Analytical technique The u.v. spectrophotomeiric method of Shechter (1973) was employed to monitor the aqueous concentrations of ozone, whereas the concentration of ozone in the gas phase was determined as described in Standard Methods (1971).
MATERIALS A N D METHODS
Cell culture Mycohacterium fortuit,tm, an acid-fast bacteria, was used * Present address: Department of Civil Engineering, University of Miami, Coral Gables, Florida 33124, U.S.A. 737
EXPERIMENTAL TECHNIQUE The survival kinetics of M. fortuitum at various temperatures was determined in a continuous flow type reactor consisting of a 55 mm dia. and 270 mm long Pyrex glass column, having a volume of 50Oral (Fig. I). A gaseous
73S
SHAUKAT FAROOQ. RICHARD S. ENGELBRECHT and EDWARD S. K. CH1.-~X
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(6)
Fig. l. Flow diagram of the continuous flow reactor. mixture of ozone and air was supplied at the bottom of the reactor through a medium porosity, horizontal flitted glass diffuser. Feed water, consisting of deionized-buffered water (clean system) and inoculated with test organisms, was fed to the reactor at the top and was removed from the bottom under the force of gravity. A methylene blue tracer study showed that the reactor provided complete mixing (Farooq, 1976). Further, during all experiments, steady state was attained with respect to achieving a constant ozone residual at each detefition time prior to collecting samples for enumeration of organisms. An inactivation study was also performed in the reactor to determine the reproducibility of the experimental data in that the same experiment was repeated four times under identical conditions; good reproducibility was observed (Farooq. 1976). The ozone generator, Welsbach Model T-408 (Philadelphia, PA), was fed either oxygen or breathing air (dried to - 7 0 ° F dew point by passing through Drierite, i.e. CaCI2). The generator was operated at a pressure of 8 psig (55.2 kN m -2) and the feed pressure was maintained at 15 psig (103.5 kN m-'-). In performing the temperature studies, equal volumes of inoculated feed water (pH 7.0 and 24°C) and deionized water, buffered to pH 7.0 and maintained at the appropriate temperature, were pumped separately to the top of the reactor where mixing gave the desired final temperature within the reactor. The temperature was monitored intermittently during each experiment at both the inlet and outlet of the reactor. Because of the short detention time, i.e. 6-107 s. no provision for insulation of the reactor was made. The effect of u.v. light on disinfection with ozone was studied in a fermentor (New Brunswick Scientific Co., N J) consisting of a 15 cm dia. and 30 cm long Plexiglas reactor and having an effective volume of 41. This reactor was operated as a continuous flow system and was equipped with a 15 W low pressure mercury germicidal lamp (General Electric, Schenectady. NY), with the major u.v. light energy being emitted at a wavelength of 253.7 nm. Mixing was provided by three stainless steel impellers equally spaced on a shaft and operated at a speed of 380 rev min-t. The ozone-air gas mixture was introduced at the bottom of the reactor through a stainless steel sparger at a rate of I lmin-*. The gaseous ozone concentration was 16.8 mg 1- *. Inoculated feed water was pumped to the reac-
tor at the top and was removed from the bottom by another pump.
RESULTS AND DISCUSSION
Effect of temperature The effect of temperature was studied in order to determine whether the degree of microbial inactivation was affected by the rate of ozone decomposition at various temperatures under the same conditions of mass transfer. Two different series of experiments were designed to study the effects of temperature on the inactivation of M. fortuitum by ozone. In the first series of experiments, the partial pressure of gaseous ozone was kept constant at 8.26 x 10 -3 atm (i.e. 16.8mgl - t ) with an ozone-air gas flow rate of 0.5 1 m i n - t. The reactor temperatures studied were 9, 20. 30 and 40~C: the solution ozone residual was allowed to vary with temperature with different detention times. Figure 2 shows that the degree of inactivation of M. fortuitum is affected significantly by temperature for the same applied ozone dosage. The degree of inactivation of M. fortuitum, as a function of temperature, can be observed to be less dependent on the ozone residual, s h o w n in the parenthesis (Fig. 2). An increase in temperature resulted in a higher degree of inactivation of M. forttdt,m, even though the ozone residual was considerably less (Fig. 2). Using the same reactor, a control experiment was also performed at 30 and 37:C in the absence of ozone but at the same air flow rate of 0.51rain-1. Results in Fig. 2 indicate,that perhaps a small degree of inactivation occurred in these systems; this may have been due to the slight increase in temperature to which the cells were subjected, i.e. 30 and 37'C as c o m p a r e d to a prior cultivation and feed water temperature of 24:C.
Influence of temperature and u.v. light
739
tion for M.fortuitum in the reactor was made according to the following equation: input - output + generation = accumulation. (1) The accumulation of M. fortuitum inside the reactor will be zero at steady state. Therefore, equation (1) may be written as input - output + generation -- 0.
(2)
In this case, input is the number of M. fortuitum cells added to the reactor, whereas output is the number of organisms in the effluent on a unit time basis. The generation term is the rate of inactivation of organisms in the reactor having a volume of V; thus, the term becomes negative. Hence, equation (2) can be written as follows: Noq - Nq - k N ° V = 0,
(3)
where
0
20
40
60
80
I00
120
Detention Time, sec
Fig. 2. Effect of temperature on the survival of M. fort,aturn at a constant rate of applied ozone. Another series of experiments were performed by maintaining a constant ozone residual at a given detention time for four different temperatures, i.e. 9, 20, 30 and 37~C by changing the partial pressure of applied ozone in the ozone--air gaseous mixture. However, the ozone-air flow rate was kept constant at 0 . 5 1 m i n - t . In contrast to the results of Kinman (1972) and Leiguarda (1948). the survival data for M. Jbrtuitum (Fig. 3) clearly shows the true effect of temperature on ozone disinfection in which the degree of inactivation increases significantly with an increase in temperature at a given level of ozone residual, i.e. approximately 0.6 mg I- 1. Results of this series of experiments indicate that the effect of temperature on disinfection with ozone is similar to that with chlorine in that, with both, there is a higher efficiency of disinfection at higher temperatures. Approximate values of the activation energy for ozone disinfection of M. fortuit=,m were calculated from the temperature data given above. The magnitude of the activation energy provides insight into whether the disinfection process is limited by mass transfer rate, e.g. diffusion, or the chemical reaction rate between ozone and cellular materials. In developing a kinetic expression for a completely mixed continuous flow system, it is assumed that the concentration of ozone residual is essentially constant at all detention times and temperatures, i.e. 9, 20, 30 and 37°C (Fig. 3). In actuality, this assumption may not be entirely valid as ozone residual actually varied between 0.49 and 0.61 mg I- t as shown in the parenthesis (Fig. 3). Regardless, a basic mass balance equa-
No = initial density of M. fortuitum entering the reactor (cells m l - ') N = density of M. forttdtum in the effluent (cells m l - t) V = volume of the reactor occupied by the liquid (ml) q = volumetric flow rate of fluid (ml m i n - t ) k = reaction rate constant of inactivation a= order of the reaction. Dividing both sides of equation (3) by q and rearranging, (4)
(No - N)/t --- k N =, (St)
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20
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40
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(s
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Detention Time,DT, sec
Fig. 3. Effect of temperature on the survival of M. fortuiturn for a constant ozone residual at a given detention time.
740
SHAUKAT F'AROOQ. RICHARD S. ENGELBRECHT and EDWARD S. K. CHIAN
in which t is the detention time and equals (V/q). By taking the logarithm of both sides of equation (4). log (No - N)/t = log k + a log N.
(5)
A log-log plot of (No - NVt for various detention times vs N for a given temperature will give a straight line. having a slope a and an intercept k (Fig. 4). Therefore, various values of a and k can be determined for different temperatures. The value of the natural log k, i.e. In k, for a number of temperatures can then be plotted vs the reciprocal of the absolute temperature, 1/T (Fig. 5). The slope of the straight line of best fit is eo..ual to - E / R according to the well-known Arrhenius equation; E is the activation energy and R is the gas constant. Using this method of analysis, the activation energy determined in this study was approximately 18.3 kcal. while the average value for a, i.e. order of the reaction with respect to the number of cells, was 0.54. The former value suggests that the rate-limiting step in ozone disinfection at a pH of 7.0 is the chemical reaction rate between ozone and bacterial cells, rather than the mass transfer rate of ozone across the cell wall materials. This agrees with the findings that inactivation of microorganisms improves with increasing ozone concentrations in the aqueous solution according to the reaction kinetics (Farooq et al., 1976). In a parallel study in this laboratory, Siiriicii (1975) determined the activation energies of a group of acidfast organisms (M. Jbrtuitum find four other acid-fast organisms, with M. fortuitum being the most resistant against chlorine) with free aqueous chlorine within a temperature range of 5 and 20~C at various pH values. The values obtained were 3.11, 12.8 and 15 kcal at pH values of 6.0, 7.0 and I0.0, respectively. Therefore, the mass transfer rate of chlorine to the cell appears to limit disinfection at low pH whereas the chemical reaction rate is limiting at high pH. Fair et al. (1968) also reported E values for E. coli to be 8.2, 6.4, 12 and 15 kcal at pH 7.0, 8.5, 9.5 and 10.7, respectively, with free aqueous chlorine. This broad
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l i t x 103 ( I / ~ ) Fig. 5. Plot for determination of activation energy (El.
comparison of E values provides some information about the response of acid-fast organisms and E. coil to chlorine and. further, of acid-fast organisms to chlorine and ozone. However, a note should be made that E values derived for the same organism but at various temperature ranges and concentrations of disinfectant are entirely different. In general terms, it may be noted that acid-fast organisms are more resistant than E. coil to chlorine, and that ozone is a comparable or a better disinfectant than chlorine with respect to acid-fast organisms (M. fi~rtuitum).
Effect of u.v. light A study was undertaken to investigate the effects of u.v. light alone, ozone plus u.v. light, and ozone alone on the disinfection of M. Jbrtuitum so as to determine whether the inactivation by ozone is augmented in the presence of u.v. light. The minimum detention time studied in these experiments was 72s. due to the limitations of the pump. as compared to 6s minimum used with the 500ml reactor in the other studies. The results of these experiments are shown in Fig. 6. It may be noted that comparable inactivation of M. fortuitum occurred with the three different conditions, i.e.u.v. light, ozone plus u.v. light, and ozone. When u.v. light was employed alone, the ozone-air mixture was replaced by an air flow rate of l l m i n - 1 in order to maintain identical mixing conditions. From Fig. 6, it would appear that u.v. light slightly improves the degree of inactivation when it is used with ozone, as compared to ozone alone. However, the importance of ozone residual, as demonstrated in previous experiments (Figs. 2 and 3), becomes less significant with respect to inactivation in the presence of u.v. light as it increases the degree of inactivation of M. fortuitum even though ozone residual is decreased. It can also be seen in Fig. 6 that u.v. light alone is a strong disinfecting agent; thus, the increased inactivation in the case of ozone plus u.v. light may be attributed to the application of u.v. light rather than to the catalytic effect of u.v. light with ozone. Experiments were also performed using an activated sludge effluent obtained from the local treatment plant. The effluent was brought to the laboratory. equilibrated at 24°C. inoculated with M. fortui-
Influence of temperature and u.v. light ,~U-.
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741
of M. jbrttdtum with ozone in both a clean system and secondary wastewater effluent.
( )=Ozone IResiduol,m(~/l CONCLUSIONS
Based on the findings of this research, the following conclusions may be drawn:
\\\
,
1, Temperature influences the rate of ozone disinfection; for a given dosage, an increase in temperature increases the rate of inactivation, even though the ozone residual is decreased. 2. Activation energy calculated for ozone disinfection of 3 f . J b r t , i t u m is approximately 18.3 kcal within a temperature range of 9-37~C at pH 7.0 and an ozone residual of 0.6 mg 1- t. 3. Ultra-violet light does not appear to catalyse or enhance disinfection with ozone; however, u.v. light exerts a strong disinfection effect when applied alone.
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Acknowled,qements--This investigation was funded jointly by the U.S. Army Medical Research and Development Command and the U.S. Environmental Protection Agency.
Fig. 6. Effect of u.v. light on the survival of M. fortuit,m at a constant rate of applied ozone.
REFERENCES
turn, and tested at a detention time of 72 s. Two different conditions were evaluated, i.e. ozone plus u.v. light, and ozone alone. The percentage survival of M. fortuittLm observed was 6.2 and 6.7 with ozone plus u.v. light and ozone alone, respectively. When u.v. light was employed in combination with ozone, the ozone residual decreased to 0.3 mg 1-z as compared to 0.78 mg 1-L in the case of ozone alone, when the partial pressure of ozone in the gas phase was kept constant in both cases. In these experiments, oxygen was used to generate the ozone as a higher concentration of ozone was required in order to meet the high ozone demand of the wastewater. However, although ozone was applied at a flow rate of 1 I rain- ~, the concentration of gaseous ozone was increased to 26 mg 1- t due to the use of oxygen instead of air. Hewes et al. (1974) combined ozone with u.v. radiation to determine the rates of oxidation of difficult to oxidize, "refractory', chemical species which were found ubiquitously in secondary effluent and were not removed by conventional secondary treatment plus carbon adsorption. Five "model compounds', i.e. glycine, ethanol, acetic acid, glycerol and palmitic acid, were chosen for their investigation, Their results indicated that the organic compounds were not oxidized by ozone alone, but were readily oxidized by ozone when the reaction was 'activated' by u.v. radiation. However, their data cannot be compared with the findings of this study, because chemical oxidation of these ozone refractory compounds generally takes hours as compared to seconds for the inactivation
Engelbrecht R, S., Foster D. H., Greening E. O. & Lee S. H. (1974) New microbial indicators of wastewater chlorination efficiency. EPA Rep. No. G70/2-73-082. Fair G. M., Geyer J. C. & Okun D. A. (1968) Water and Wastewater En,qqineerin,q, Vol. II. Water P~lrification and Wastewater Treatment and Disposal, pp. 31-11. John Wiley, New York. Farooq S. (1976) Kinetics of inactivation of yeasts and acid-fast organisms with ozone. Ph.D. thesis, Department of Civil Engineering, University of Illinois at Urbana-Champaign. Farooq S.. Engelbrecht R. S. & Chian E. S. K. (1976) The effects of ozone bubbles on disinfection. Paper presented at the 8th Int. Ass. Wat. Pollut. Res. Conf., Sydney, Australia. Hewes C. G., Prengle H. W., Mauk C. E. & Sparkman O. D. (1974) Oxidation of refractory organic materials by ozone and ultraviolet light. Final Report for U.S. Army Mobility Equipment R & D Center, Fort Belvoir, VA, Contract DAAK02-74-C-0239. Kinman R. N. (1972) Ozone in water disinfection. In O-one in Water and Wastewater Treatment, (Edited by Evans F. L.), pp. 123-143. Ann Arbor Science Publishers, Ann Arbor. Leiguarda R. H., Peso O. A. & Palazzola A. Z. R. (1949)' Bactericidal action of oxone. An. Asoc. quire, argent. 37, 165, Water Pollut. Abs. 22, 268. Shechter H. (1973) Spectrophotometric method for determination of ozone in aqueous solutions. Water Res. 7, 729-739. Standard MethodsJbr the Examination of Water and Wastewater (1971) 13th edn. American Public Health Association. Washington. Si.iriicli F. (1975) Kinetics of inactivation of proposed indicator organisms by free available chlorine species. M.S. special problem, Department of Civil Engineering, University of Illinois at Urbana-Cbampaign. Zeff D. J.. Baron R. & Reuter L. H. (1974) UV-ozone water oxidation-sterilization process. Westgate Res. Corp., Marina del Rey, CA (unpublished).
INFLUENCE OF TEMPERATURE AND U.V. LIGHT ON DISINFECTION WITH OZONE SHAUKAT FAROOQ,* RICHARD S. ENGELBRECHT and EDWARD S. K. CHIAN Department of Civil Engineering. University of Illinois at Urbana-Champaign. Urbana. Illinois 61801. U.S.A.
(Received 24 December 1976; in revised form 24 February 19771 Abstract--An inactivation study was performed with ozone in two laboratory-scale, continuous flow systems to determine the effects of temperature and u.v. light on the survival of Mvcobacteriumforruitum, a potential microbial indicator for disinfection efficiency. Four temperatures were investigated in the range of 9-37-'C. It was determined that a higher degree of inactivation occurred with ozone at elevated temperatures. The activation energy for M. fortuitttm was found to be 18.3 kcal. When u.v. light was employed as a catalyst with ozone disinfection, there was no apparent increase in the degree of inactivation of M. fortuitum in either the clean system (deionized water-phosphate buffer) or secondary wastewater effluent. However, u.v. light in itself exerted a strong disinfecting effect.
INTRODUCTION Few researchers, investigating ozone disinfection, have attempted to determine the effect of temperature on the inactivation of microorganisms. Leiguarda et al. (1949) reported that the bactericidal efficiency of ozone was not affected by temperature. K i n m a n (1972) studied ozone inactivation of bacteria at two temperatures, 25 and 39°C, and inferred that the rapid rate of destruction of bacteria was comparable at these two temperatures. A study of ozone inactivation was also performed by Zeff et al. (.1974) at 17, 25 and 40°C using Streptococcus faecalis, Klebsiella pneumoniae and Ancanthamoeba castellanii. It was concluded that the effect of t e m p e r a t u r e was insignificant. In the same study, ozone plus u.v. light was found to be more effective in destroying the test organisms than u.v. light alone in a stirred tank reactor. A review of the literature also reveals that no attempt has been made by previous researchers to correlate the degree of inactivation to the ozone residual, which in turn is affected by the temperature. Therefore, it was difficult in considering past studies to interpret the precise effect of temperature on disinfection. The purpose of this research was to investigate the effects of temperature and u.v. light on ozone residual and. consequently, the degree of inactivation of microorganisms, since ozone residual was found to be the most influential parameter in ozone disinfection at room temperature (Farooq et al., t976). Experiments also were performed to determine the effect of u.v. light o n disinfection with ozone.
as the primary organism in this study. A pure culture of M. fi,'tuitum was obtained from the study of Engelbrecht et ul. (1974), who proposed the acid-fast bacteria as a possible new microbial indicator of wastewater disinfection efficiency. In this study, M. fortuitum was used as the primary organism due to its greater resistance to ozone and ease of obtaining kinetic data (Engelbrecht et al., 1974: Farooq, 1976).
Medium and cultication method In preparation of inactivation experiments, M. Jbrtuitum was grown in a broth medium consisting of Middlebrook & Cohn 7H9 mineral base, 4.7 g; malachite green, 1.0 rag; and sodium propionate, 1.0 g l- t deionized water. The culture was incubated for 72 h at 37'~C in a water shaker bath before harvesting. Cells were harvested by means of a Sorval GLC-2 (DuPont Instruments, Newton, CT) general laboratory centrifuge at 1800 rev min - t for 15 min. They were then washed twice with a total of at least 150 ml of 0.0025 M phosphate buffer. Cells prepared in this manner were resuspended in phosphate buffer and were kept at 4°C until needed. M. fortuimm was enumerated using the 7H9 Middlebrook & Cohn medium indicated above but with 1.5~;; Bacto agar (Difco Lab, Detroit, MI) to solidify the medium. A spread plate technique was employed in this study to enumerate the surviving organisms. Samples were diluted with phosphate buffer solution such that a 0.l-ml aliquot of diluted sample would give at least 50 and no more than 200 colonies per culture dish. Incubation was accomplished at 37'C for 72-96 h. Detailed procedures for enumeration of M. fortuitum are given elsewhere (Engelbrecht et al., 1974; Farooq, 1976). Analytical technique The u.v. spectrophotomeiric method of Shechter (1973) was employed to monitor the aqueous concentrations of ozone, whereas the concentration of ozone in the gas phase was determined as described in Standard Methods (1971).
MATERIALS A N D METHODS
Cell culture Mycohacterium fortuit,tm, an acid-fast bacteria, was used * Present address: Department of Civil Engineering, University of Miami, Coral Gables, Florida 33124, U.S.A. 737
EXPERIMENTAL TECHNIQUE The survival kinetics of M. fortuitum at various temperatures was determined in a continuous flow type reactor consisting of a 55 mm dia. and 270 mm long Pyrex glass column, having a volume of 50Oral (Fig. I). A gaseous
73S
SHAUKAT FAROOQ. RICHARD S. ENGELBRECHT and EDWARD S. K. CH1.-~X
l
__J
(3) *
"78") t R2
I
RI
To drain
~
(21
I If lf I J
(11 (2) (3) (4) (5) (6) (7) (8) (9) R
Pressure guoge
Drierite Ozonotor Reoctor Pump Magnetic stirrer Feed reservoir Cotfen plug Diffuser Rotometer
(6)
Fig. l. Flow diagram of the continuous flow reactor. mixture of ozone and air was supplied at the bottom of the reactor through a medium porosity, horizontal flitted glass diffuser. Feed water, consisting of deionized-buffered water (clean system) and inoculated with test organisms, was fed to the reactor at the top and was removed from the bottom under the force of gravity. A methylene blue tracer study showed that the reactor provided complete mixing (Farooq, 1976). Further, during all experiments, steady state was attained with respect to achieving a constant ozone residual at each detefition time prior to collecting samples for enumeration of organisms. An inactivation study was also performed in the reactor to determine the reproducibility of the experimental data in that the same experiment was repeated four times under identical conditions; good reproducibility was observed (Farooq. 1976). The ozone generator, Welsbach Model T-408 (Philadelphia, PA), was fed either oxygen or breathing air (dried to - 7 0 ° F dew point by passing through Drierite, i.e. CaCI2). The generator was operated at a pressure of 8 psig (55.2 kN m -2) and the feed pressure was maintained at 15 psig (103.5 kN m-'-). In performing the temperature studies, equal volumes of inoculated feed water (pH 7.0 and 24°C) and deionized water, buffered to pH 7.0 and maintained at the appropriate temperature, were pumped separately to the top of the reactor where mixing gave the desired final temperature within the reactor. The temperature was monitored intermittently during each experiment at both the inlet and outlet of the reactor. Because of the short detention time, i.e. 6-107 s. no provision for insulation of the reactor was made. The effect of u.v. light on disinfection with ozone was studied in a fermentor (New Brunswick Scientific Co., N J) consisting of a 15 cm dia. and 30 cm long Plexiglas reactor and having an effective volume of 41. This reactor was operated as a continuous flow system and was equipped with a 15 W low pressure mercury germicidal lamp (General Electric, Schenectady. NY), with the major u.v. light energy being emitted at a wavelength of 253.7 nm. Mixing was provided by three stainless steel impellers equally spaced on a shaft and operated at a speed of 380 rev min-t. The ozone-air gas mixture was introduced at the bottom of the reactor through a stainless steel sparger at a rate of I lmin-*. The gaseous ozone concentration was 16.8 mg 1- *. Inoculated feed water was pumped to the reac-
tor at the top and was removed from the bottom by another pump.
RESULTS AND DISCUSSION
Effect of temperature The effect of temperature was studied in order to determine whether the degree of microbial inactivation was affected by the rate of ozone decomposition at various temperatures under the same conditions of mass transfer. Two different series of experiments were designed to study the effects of temperature on the inactivation of M. fortuitum by ozone. In the first series of experiments, the partial pressure of gaseous ozone was kept constant at 8.26 x 10 -3 atm (i.e. 16.8mgl - t ) with an ozone-air gas flow rate of 0.5 1 m i n - t. The reactor temperatures studied were 9, 20. 30 and 40~C: the solution ozone residual was allowed to vary with temperature with different detention times. Figure 2 shows that the degree of inactivation of M. fortuitum is affected significantly by temperature for the same applied ozone dosage. The degree of inactivation of M. fortuitum, as a function of temperature, can be observed to be less dependent on the ozone residual, s h o w n in the parenthesis (Fig. 2). An increase in temperature resulted in a higher degree of inactivation of M. forttdt,m, even though the ozone residual was considerably less (Fig. 2). Using the same reactor, a control experiment was also performed at 30 and 37:C in the absence of ozone but at the same air flow rate of 0.51rain-1. Results in Fig. 2 indicate,that perhaps a small degree of inactivation occurred in these systems; this may have been due to the slight increase in temperature to which the cells were subjected, i.e. 30 and 37'C as c o m p a r e d to a prior cultivation and feed water temperature of 24:C.
Influence of temperature and u.v. light
739
tion for M.fortuitum in the reactor was made according to the following equation: input - output + generation = accumulation. (1) The accumulation of M. fortuitum inside the reactor will be zero at steady state. Therefore, equation (1) may be written as input - output + generation -- 0.
(2)
In this case, input is the number of M. fortuitum cells added to the reactor, whereas output is the number of organisms in the effluent on a unit time basis. The generation term is the rate of inactivation of organisms in the reactor having a volume of V; thus, the term becomes negative. Hence, equation (2) can be written as follows: Noq - Nq - k N ° V = 0,
(3)
where
0
20
40
60
80
I00
120
Detention Time, sec
Fig. 2. Effect of temperature on the survival of M. fort,aturn at a constant rate of applied ozone. Another series of experiments were performed by maintaining a constant ozone residual at a given detention time for four different temperatures, i.e. 9, 20, 30 and 37~C by changing the partial pressure of applied ozone in the ozone--air gaseous mixture. However, the ozone-air flow rate was kept constant at 0 . 5 1 m i n - t . In contrast to the results of Kinman (1972) and Leiguarda (1948). the survival data for M. Jbrtuitum (Fig. 3) clearly shows the true effect of temperature on ozone disinfection in which the degree of inactivation increases significantly with an increase in temperature at a given level of ozone residual, i.e. approximately 0.6 mg I- 1. Results of this series of experiments indicate that the effect of temperature on disinfection with ozone is similar to that with chlorine in that, with both, there is a higher efficiency of disinfection at higher temperatures. Approximate values of the activation energy for ozone disinfection of M. fortuit=,m were calculated from the temperature data given above. The magnitude of the activation energy provides insight into whether the disinfection process is limited by mass transfer rate, e.g. diffusion, or the chemical reaction rate between ozone and cellular materials. In developing a kinetic expression for a completely mixed continuous flow system, it is assumed that the concentration of ozone residual is essentially constant at all detention times and temperatures, i.e. 9, 20, 30 and 37°C (Fig. 3). In actuality, this assumption may not be entirely valid as ozone residual actually varied between 0.49 and 0.61 mg I- t as shown in the parenthesis (Fig. 3). Regardless, a basic mass balance equa-
No = initial density of M. fortuitum entering the reactor (cells m l - ') N = density of M. forttdtum in the effluent (cells m l - t) V = volume of the reactor occupied by the liquid (ml) q = volumetric flow rate of fluid (ml m i n - t ) k = reaction rate constant of inactivation a= order of the reaction. Dividing both sides of equation (3) by q and rearranging, (4)
(No - N)/t --- k N =, (St)
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20
i
40
i
60
i
60
(s
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120
Detention Time,DT, sec
Fig. 3. Effect of temperature on the survival of M. fortuiturn for a constant ozone residual at a given detention time.
740
SHAUKAT F'AROOQ. RICHARD S. ENGELBRECHT and EDWARD S. K. CHIAN
in which t is the detention time and equals (V/q). By taking the logarithm of both sides of equation (4). log (No - N)/t = log k + a log N.
(5)
A log-log plot of (No - NVt for various detention times vs N for a given temperature will give a straight line. having a slope a and an intercept k (Fig. 4). Therefore, various values of a and k can be determined for different temperatures. The value of the natural log k, i.e. In k, for a number of temperatures can then be plotted vs the reciprocal of the absolute temperature, 1/T (Fig. 5). The slope of the straight line of best fit is eo..ual to - E / R according to the well-known Arrhenius equation; E is the activation energy and R is the gas constant. Using this method of analysis, the activation energy determined in this study was approximately 18.3 kcal. while the average value for a, i.e. order of the reaction with respect to the number of cells, was 0.54. The former value suggests that the rate-limiting step in ozone disinfection at a pH of 7.0 is the chemical reaction rate between ozone and bacterial cells, rather than the mass transfer rate of ozone across the cell wall materials. This agrees with the findings that inactivation of microorganisms improves with increasing ozone concentrations in the aqueous solution according to the reaction kinetics (Farooq et al., 1976). In a parallel study in this laboratory, Siiriicii (1975) determined the activation energies of a group of acidfast organisms (M. Jbrtuitum find four other acid-fast organisms, with M. fortuitum being the most resistant against chlorine) with free aqueous chlorine within a temperature range of 5 and 20~C at various pH values. The values obtained were 3.11, 12.8 and 15 kcal at pH values of 6.0, 7.0 and I0.0, respectively. Therefore, the mass transfer rate of chlorine to the cell appears to limit disinfection at low pH whereas the chemical reaction rate is limiting at high pH. Fair et al. (1968) also reported E values for E. coli to be 8.2, 6.4, 12 and 15 kcal at pH 7.0, 8.5, 9.5 and 10.7, respectively, with free aqueous chlorine. This broad
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~
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3.4
I 5.5
3.6
3.7
l i t x 103 ( I / ~ ) Fig. 5. Plot for determination of activation energy (El.
comparison of E values provides some information about the response of acid-fast organisms and E. coil to chlorine and. further, of acid-fast organisms to chlorine and ozone. However, a note should be made that E values derived for the same organism but at various temperature ranges and concentrations of disinfectant are entirely different. In general terms, it may be noted that acid-fast organisms are more resistant than E. coil to chlorine, and that ozone is a comparable or a better disinfectant than chlorine with respect to acid-fast organisms (M. fi~rtuitum).
Effect of u.v. light A study was undertaken to investigate the effects of u.v. light alone, ozone plus u.v. light, and ozone alone on the disinfection of M. Jbrtuitum so as to determine whether the inactivation by ozone is augmented in the presence of u.v. light. The minimum detention time studied in these experiments was 72s. due to the limitations of the pump. as compared to 6s minimum used with the 500ml reactor in the other studies. The results of these experiments are shown in Fig. 6. It may be noted that comparable inactivation of M. fortuitum occurred with the three different conditions, i.e.u.v. light, ozone plus u.v. light, and ozone. When u.v. light was employed alone, the ozone-air mixture was replaced by an air flow rate of l l m i n - 1 in order to maintain identical mixing conditions. From Fig. 6, it would appear that u.v. light slightly improves the degree of inactivation when it is used with ozone, as compared to ozone alone. However, the importance of ozone residual, as demonstrated in previous experiments (Figs. 2 and 3), becomes less significant with respect to inactivation in the presence of u.v. light as it increases the degree of inactivation of M. fortuitum even though ozone residual is decreased. It can also be seen in Fig. 6 that u.v. light alone is a strong disinfecting agent; thus, the increased inactivation in the case of ozone plus u.v. light may be attributed to the application of u.v. light rather than to the catalytic effect of u.v. light with ozone. Experiments were also performed using an activated sludge effluent obtained from the local treatment plant. The effluent was brought to the laboratory. equilibrated at 24°C. inoculated with M. fortui-
Influence of temperature and u.v. light ,~U-.
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of M. jbrttdtum with ozone in both a clean system and secondary wastewater effluent.
( )=Ozone IResiduol,m(~/l CONCLUSIONS
Based on the findings of this research, the following conclusions may be drawn:
\\\
,
1, Temperature influences the rate of ozone disinfection; for a given dosage, an increase in temperature increases the rate of inactivation, even though the ozone residual is decreased. 2. Activation energy calculated for ozone disinfection of 3 f . J b r t , i t u m is approximately 18.3 kcal within a temperature range of 9-37~C at pH 7.0 and an ozone residual of 0.6 mg 1- t. 3. Ultra-violet light does not appear to catalyse or enhance disinfection with ozone; however, u.v. light exerts a strong disinfection effect when applied alone.
(3Or}
(O~)
o
"~L_ Ozone* UlJ'roviolet Liqht
ca I ~ _
5-Ultraviolet
i~
0
I
40
I
80
LiQhl
I
120
I
160
I
200
DT, sec
Acknowled,qements--This investigation was funded jointly by the U.S. Army Medical Research and Development Command and the U.S. Environmental Protection Agency.
Fig. 6. Effect of u.v. light on the survival of M. fortuit,m at a constant rate of applied ozone.
REFERENCES
turn, and tested at a detention time of 72 s. Two different conditions were evaluated, i.e. ozone plus u.v. light, and ozone alone. The percentage survival of M. fortuittLm observed was 6.2 and 6.7 with ozone plus u.v. light and ozone alone, respectively. When u.v. light was employed in combination with ozone, the ozone residual decreased to 0.3 mg 1-z as compared to 0.78 mg 1-L in the case of ozone alone, when the partial pressure of ozone in the gas phase was kept constant in both cases. In these experiments, oxygen was used to generate the ozone as a higher concentration of ozone was required in order to meet the high ozone demand of the wastewater. However, although ozone was applied at a flow rate of 1 I rain- ~, the concentration of gaseous ozone was increased to 26 mg 1- t due to the use of oxygen instead of air. Hewes et al. (1974) combined ozone with u.v. radiation to determine the rates of oxidation of difficult to oxidize, "refractory', chemical species which were found ubiquitously in secondary effluent and were not removed by conventional secondary treatment plus carbon adsorption. Five "model compounds', i.e. glycine, ethanol, acetic acid, glycerol and palmitic acid, were chosen for their investigation, Their results indicated that the organic compounds were not oxidized by ozone alone, but were readily oxidized by ozone when the reaction was 'activated' by u.v. radiation. However, their data cannot be compared with the findings of this study, because chemical oxidation of these ozone refractory compounds generally takes hours as compared to seconds for the inactivation
Engelbrecht R, S., Foster D. H., Greening E. O. & Lee S. H. (1974) New microbial indicators of wastewater chlorination efficiency. EPA Rep. No. G70/2-73-082. Fair G. M., Geyer J. C. & Okun D. A. (1968) Water and Wastewater En,qqineerin,q, Vol. II. Water P~lrification and Wastewater Treatment and Disposal, pp. 31-11. John Wiley, New York. Farooq S. (1976) Kinetics of inactivation of yeasts and acid-fast organisms with ozone. Ph.D. thesis, Department of Civil Engineering, University of Illinois at Urbana-Champaign. Farooq S.. Engelbrecht R. S. & Chian E. S. K. (1976) The effects of ozone bubbles on disinfection. Paper presented at the 8th Int. Ass. Wat. Pollut. Res. Conf., Sydney, Australia. Hewes C. G., Prengle H. W., Mauk C. E. & Sparkman O. D. (1974) Oxidation of refractory organic materials by ozone and ultraviolet light. Final Report for U.S. Army Mobility Equipment R & D Center, Fort Belvoir, VA, Contract DAAK02-74-C-0239. Kinman R. N. (1972) Ozone in water disinfection. In O-one in Water and Wastewater Treatment, (Edited by Evans F. L.), pp. 123-143. Ann Arbor Science Publishers, Ann Arbor. Leiguarda R. H., Peso O. A. & Palazzola A. Z. R. (1949)' Bactericidal action of oxone. An. Asoc. quire, argent. 37, 165, Water Pollut. Abs. 22, 268. Shechter H. (1973) Spectrophotometric method for determination of ozone in aqueous solutions. Water Res. 7, 729-739. Standard MethodsJbr the Examination of Water and Wastewater (1971) 13th edn. American Public Health Association. Washington. Si.iriicli F. (1975) Kinetics of inactivation of proposed indicator organisms by free available chlorine species. M.S. special problem, Department of Civil Engineering, University of Illinois at Urbana-Cbampaign. Zeff D. J.. Baron R. & Reuter L. H. (1974) UV-ozone water oxidation-sterilization process. Westgate Res. Corp., Marina del Rey, CA (unpublished).