Disinfection and Sterilization of Sewage by Ozone - Advances in

Disinfection and Sterilization of Sewage by Ozone SOL MILLER, BETTY BURKHARDT, and RICHARD EHRLICH Biological

Research Section, Armour Research Foundation,

ROBERT J O H N

Ill.

PETERSON

Chemical Corps, Fort Detrick, Frederick,

Downloaded by CORNELL UNIV on September 4, 2016 | http://pubs.acs.org Publication Date: January 1, 1959 | doi: 10.1021/ba-1959-0021.ch051

Chicago,

Md.

All microorganisms a n d toxin in raw sewage can be completely killed or inactivated in 30 minutes by treatment with ozone. The period of treatment is related to the efficiency of contact between ozone a n d the agent. Removal of suspended solids from sew a g e by filtration does not significantly decrease the time required to sterilize or the quantity of ozone used. Between 100 and 200 p.p.m. of ozone pro duced sterility under the experimental conditions used.

/ λ research p r o g r a m was conducted b y the A r m o u r Research F o u n d a t i o n for the C h e m i c a l C o r p s , F o r t D e t r i c k , Frederick, M d . , to study a n d evaluate methods for s t e r i l i z i n g sewage c o n t a i n i n g p a t h o g e n i c m i c r o o r g a n i s m s . I n a n y research i n s t i t u t i o n where infectious microorganisms are being studied a n d m a y be d i s c h a r g e d i n t o t h e sewage, i t is i m p o r t a n t , f r o m a p u b l i c h e a l t h v i e w , t o ensure t h e i n a c t i v a t i o n of these i n f e c t i o u s f o r m s b e f o r e sewage is d i s c h a r g e d t o n o r m a l d i s p o s a l c h a n n e l s . A t p r e s e n t h e a t is t h e m e t h o d of s t e r i l i z a t i o n m o s t c o m m o n l y used i n s u c h s i t u a t i o n s . O t h e r possible m e t h o d s of sewage s t e r i l i z a t i o n w e r e a n a l y z e d . O f t h e m e t h o d s a v a i l a b l e , t r e a t m e n t w i t h gaseous ozone w a s selected f o r f u r t h e r s t u d y . T h e t r e a t m e n t of sewage effluents w a s s t u d i e d b y b u b b l i n g ozone t h r o u g h u n t r e a t e d o r a u t o c l a v e d sewage s a m p l e s t o w h i c h h a d been a d d e d t r a c e r m i c r o o r g a n i s m s , i n f e c t i o u s b a c t e r i a l spores, i n f l u e n z a v i r u s , o r t h e t o x i n p r o d u c e d b y Clostridium botulinum. Review

of

Literature

T h e i n i t i a l l i t e r a t u r e s u r v e y d i s c l o s e d t h a t of t h e m e t h o d s of s t e r i l i z a t i o n , o t h e r t h a n h e a t , c h e m i c a l s t e r i l i z a t i o n w i t h ozone a p p e a r e d t o be m o r e p r o m i s i n g . S e v e r a l r e p o r t s i n d i c a t e t h a t ozone is b e i n g s u c c e s s f u l l y u s e d f o r w a t e r d i s i n f e c t i o n i n F r a n c e , w h e r e 136 m u n i c i p a l w a t e r p l a n t s s e r v i n g a p p r o x i m a t e l y 8,000,000 p e o p l e a r e i n use. O z o n i z a t i o n is a c c o m p l i s h e d b y i n j e c t i n g a n a i r s t r e a m c o n t a i n i n g 2.5 t o 5 g r a m s of ozone p e r c u b i c m e t e r of a i r i n t o w a t e r . T h e process of o z o n i z a t i o n o c c u r s i n selfc o n t a c t c o l u m n s a n d t h e r e t e n t i o n t i m e is 3 t o 5 m i n u t e s . P r i o r t o o z o n i z a t i o n , t h e w a t e r is u s u a l l y p r e t r e a t e d b y s e d i m e n t a t i o n , c o a g u l a t i o n , a n d f i l t r a t i o n t h r o u g h s a n d . A l l the pathogenic a n d saprophytic organisms usually found i n water are destroyed b y 381

OZONE CHEMISTRY AND TECHNOLOGY Advances in Chemistry; American Chemical Society: Washington, DC, 1959.

A D V A N C E S IN CHEMISTRY SERIES

382

t h i s c o m b i n e d t r e a t m e n t . O z o n e i s also u s e d i n t h e U n i t e d S t a t e s i n w a t e r t r e a t m e n t p l a n t s a t P h i l a d e l p h i a , P a . , a n d W h i t i n g , I n d . P h i l a d e l p h i a uses a r e s i d u a l o f 0.13 t o 0.2 p . p . m . f o r d i s i n f e c t i o n ( 7 ) . D i c k e r m a n et al. (4) r e p o r t e d t h a t 1.5 p . p . m . o f ozone w i l l reduce a c o u n t of 70,000 s p o r e - f o r m i n g o r g a n i s m s p e r m l . o f w a t e r t o 0 w i t h i n 5 m i n u t e s . T h e y i n d i c a t e t h a t r a w s t r e a m w a t e r w i t h l o w o r g a n i c c o n t e n t w o u l d r e q u i r e a n ozone r e s i d u a l o f 2 p . p . m . a p p l i e d f o r a t least 5 m i n u t e s t o g i v e a c o m p l e t e k i l l . W h e n o r g a n i c c o n t e n t s are h i g h , l a r g e r c o n c e n t r a t i o n s a n d l o n g e r c o n t a c t t i m e are n e e d e d .


O z o n e h a s been suggested as a n agent f o r t r e a t i n g u s e d i n l i m i t e d e x p e r i m e n t a l r u n s as c o a g u l a t i n g agent o x y g e n d e m a n d ( B . O . D . ) i n sewage (5, 6). T h e r e i s also t h e effluent a f t e r c o a g u l a t i o n a n d filtration w i l l r e d u c e t h e h a v e been g r a n t e d f o r t r e a t m e n t a p p a r a t u s w h i c h uses flocculation a n d c o a g u l a t i o n of sewage {2,8).

sewage wastes, a n d h a s been a n d t o reduce t h e b i o l o g i c a l i n d i c a t i o n t h a t ozone a d d e d t o bacterial count. T w o patents ozone a n d f e r r o u s s u l f a t e f o r

A l t h o u g h ozone d e s t r o y s b a c t e r i a a n d h a s b e e n u s e d i n w a t e r d i s i n f e c t i o n , n o d a t a are a v a i l a b l e as t o i t s effectiveness as a s t e r i l i z i n g agent f o r r a w sewage c o n t a i n i n g pathogenic microorganisms. Therefore, studies were i n i t i a t e d t o o b t a i n experimental d a t a o n t h e b a c t e r i o l o g i c a l effectiveness of ozone s t e r i l i z a t i o n .

Experimental

Techniques

T h e first e x p e r i m e n t s w e r e p u r e l y f e a s i b i l i t y s t u d i e s , a n d , t h e r e f o r e , v a r i a t i o n s were u s e d i n t h e t e c h n i q u e s . F o r ozone p r o d u c t i o n , a W e l s b a c h l a b o r a t o r y o z o n a t o r , M o d e l T - 2 3 , w a s u s e d . T h e q u a n t i t y of ozone i n t h e g a s s t r e a m , w h i c h c o n t a i n e d f r o m 1.0 t o 5 % o z o n e , w a s r e g u l a t e d b y v a r y i n g t h e v o l t a g e , b y c h a n g i n g t h e i n p u t gas f r o m d r y a i r t o d r y o x y g e n , or b y v a r y i n g t h e flow r a t e o f t h e gas t h r o u g h t h e o z o n a t o r . T h e f o l l o w i n g o r g a n i s m s w e r e selected as r e p r e s e n t a t i v e o f g r o u p s f o r test p u r p o s e s : Bacillus anthracis T o x i n of Clostndium botulinum Influenza v i r u s Bacillus subtilis v a r . niger Bacillus subtilis v a r . niger w a s u s e d as t h e s i m u l a n t f o r p a t h o g e n i c m i c r o o r g a n i s m s . P r i o r t o i n o c u l a t i o n i n t o t h e sewage, t h e B. subtilis v a r . niger c u l t u r e s w e r e h e a t s h o c k e d a t 8 0 ° C . f o r 10 m i n u t e s t o e l i m i n a t e a l l v e g e t a t i v e f o r m s . K n o w n c o n c e n t r a t i o n s o f t h e c u l t u r e were a d d e d t o a u t o c l a v e d F o r t D e t r i c k sewage, o r d i s t i l l e d w a t e r , a n d t h e n t r e a t e d w i t h ozone. I t w a s possible t o u s e p a t h o g e n i c o r g a n i s m s a t F o r t D e t r i c k i n p l a c e o f s i m u l a n t s as i n o c u l a f o r t h e r a w sewage. V a r i o u s t y p e s o f a p p a r a t u s w e r e u s e d t o b r i n g ozone a n d l i q u i d i n c o n t a c t . I n t h e i n i t i a l e x p e r i m e n t s , t h e i n o c u l a t e d sewage w a s c i r c u l a t e d b y a s m a l l p u m p . Leaks i n t h e s y s t e m a n d o p e r a t i o n a l difficulties c a u s e d a change t o b a t c h t r e a t m e n t o f t h e sewage i n gas w a s h b o t t l e s . T h e sizes o f t h e g a s b u b b l e s w e r e r e d u c e d b y a t t a c h i n g f r i t t e d - g l a s s d i s k s , c y l i n d e r s , a n d t u b e s t o t h e d e l i v e r y t u b e i n t h e gas w a s h b o t t l e . I n t h e final e x p e r i m e n t s , t h e gas b o t t l e s w e r e r e p l a c e d b y glass c o l u m n s 2 i n c h e s i n d i a m e t e r a n d 2, 4, o r 8 feet h i g h . T h e d e l i v e r y t u b e a t t h e b o t t o m o f t h e c o l u m n w a s a f r i t t e d - g l a s s c y l i n d e r . T h e 4-foot c o n t a c t c o l u m n s were u t i l i z e d a t F o r t D e t r i c k a n d all pathogenic w o r k , i n c l u d i n g a l l equipment w h i c h could have been contaminated, was done i n biological safety cabinets. F i g u r e 1 illustrates the e q u i p m e n t necessary f o r o z o n a t i n g sewage i n t h e glass c o l u m n s . T h e q u a n t i t y o f ozone i n t h e gas s t r e a m , b e f o r e a n d a f t e r p a s s i n g t h r o u g h t h e sewage, w a s d e t e r m i n e d b y t h e i o d o m e t r i c m e t h o d . B a c t e r i a l c o u n t s o n t h e i n o c u l a t e d sewage a n d t h e t r e a t e d sewage w e r e d e t e r m i n e d b y a p p r o p r i a t e d i l u t i o n a n d p l a t i n g o n tryptose phosphate dextrose agar. T h e plates were c o u n t e d a f t e r 2 4 t o 4 8 h o u r s ' i n c u b a t i o n a t 37° C . I n a d d i t i o n , i n s e v e r a l e x p e r i -



MILLER, BURKHARDT, EHRLICH,

AND P E T E R S O N - S E W A G E DISINFECTION

383

Determination of Ozone in Influent Stream

Determination of Ozone in Effluent Stream

Figure 1.

Equipment for ozonating sewage in glass columns

m e n t s t h e m o s t p r o b a b l e n u m b e r ( M . P . N . ) p e r 100 m l . w a s d e t e r m i n e d (1). The s t e r i l i t y of r a w sewage i n o c u l a t e d w i t h B. anthracis spores w a s c h e c k e d b y i n o c u l a t i n g m i c e as w e l l as b y c u l t u r e . T h e i n a c t i v a t i o n of t h e b o t u l i n u m t o x i n w a s t e s t e d i n m i c e ; t h e i n a c t i v a t i o n of t h e i n f l u e n z a v i r u s w a s d e t e r m i n e d w i t h e m b r y o n a t e d eggs. Results T a b l e I shows s u r v i v a l of B. subtilis v a r . nicer spores i n sewage w i t h i n c r e a s i n g t i m e of t r e a t m e n t . R e s u l t s of e x p e r i m e n t s were i n c l u d e d i n t h e a v e r a g e , regardless Table I.

A v e r a g e Survival of B. Subtilis Var. Niger Spores in S e w a g e after O z o n e Treatment a

Period of Treatment, M i n . 1 2 5 15 30

No. of Samples 2 1 3 6 9

Survival, Log % -0.10 -0.66 -1.17 -1.57 -1.88

Approximately 250 ml. of sewage treated in 10-inch gas wash bottles. Initial count was 1 to 2 Χ 10 spores/ml. a

8

of v a r i a t i o n s i n f l o w r a t e o r c o n c e n t r a t i o n of ozone i n t h e s t r e a m . T h i s t a b l e i n d i c a t e s a m a r k e d r e d u c t i o n of o r g a n i s m s . T h i s w o u l d be c o n s i d e r e d a d e q u a t e f o r d i s i n f e c t i o n of sewage, b u t does n o t s a t i s f y t h e r i g i d 1 0 0 % s t e r i l i z a t i o n r e q u i r e m e n t f o r F o r t Detrick. T h e e x p e r i m e n t a l d a t a were e x a m i n e d t o d e t e r m i n e w h e t h e r a n y c o r r e l a t i o n exists b e t w e e n v a r i a t i o n s i n t h e e x p e r i m e n t a l p r o c e d u r e s a n d t h e efficiency of t h e ozone s t e r i l i z a t i o n s y s t e m . I t c a n be c o n c l u d e d t h a t t h e r e w a s n o definite c o r r e l a t i o n a m o n g t h e flow r a t e of t h e gas s t r e a m , p e r c e n t c o n c e n t r a t i o n of ozone i n t h e gas s t r e a m , a n d


384

A D V A N C E S IN

CHEMISTRY SERIES

p e r cent s u r v i v a l of spores. T h e flow r a t e v a r i a t i o n s r a n g e d b e t w e e n 0.005 a n d 0.16 c u . f o o t p e r m i n u t e , a n d t h e c o n c e n t r a t i o n of ozone i n t h e gas s t r e a m w a s i n t h e range of 1.5 t o 5 % . O n t h e o t h e r h a n d , t h e h e i g h t of t h e c o l u m n of l i q u i d b e i n g t r e a t e d h a d a m a r k e d effect o n t h e s u r v i v a l of B. subtilis v a r . niger spores. N o definite s t a t e m e n t o n t h i s difference c a n be m a d e . T h e d a t a o b t a i n e d w i t h 2-, 4-, a n d 8-foot c o l u m n s are s u m m a r i z e d i n T a b l e I I . Table II.

Effect of Column Height on Survival of B. Subtilis Var. Niger Spores after O z o n e Treatment


a

Height of Column, F t . 2 2 4 4 8 8

Treatment, Min. 15 30 15 30 20 30

No. of Samples, Sterile/Total 0/2 4/10 0/4 14/15 5/5 5/5

Log Av. % Survival -2.00 -3.63 -3.13 -5.00 Sterile Sterile

Experiments conducted at A R F with autoclaved Fort Detrick sewage inoculated with 1 to 2 Χ 10 B. subtilis var. niger spores/ml. 8

8

I n c r e a s i n g h e i g h t gives d e c r e a s i n g l o g a v e r a g e p e r cent s u r v i v a l . A l t h o u g h t h e t o t a l n u m b e r of s a m p l e s o z o n i z e d i n t h e 8-foot c o l u m n is s m a l l e r t h a n t h e n u m b e r i n t h e 2 - or 4-foot c o l u m n , t h e f a c t t h a t a l l s a m p l e s i n t h e 8-foot c o l u m n a r e s t e r i l e a f t e r 20 as w e l l as a f t e r 30 m i n u t e s ' t r e a t m e n t i n d i c a t e s t h a t t h e 8-foot c o l u m n is s u p e r i o r to t h e 2 - or 4-foot c o l u m n . T o d e t e r m i n e t h e effect of s u s p e n d e d solids, s a m p l e s of sewage were t r e a t e d w i t h ozone u n d e r i d e n t i c a l c o n d i t i o n s , e x c e p t t h a t a p o r t i o n of e a c h s a m p l e w a s f i l t e r e d prior to inoculation a n d ozonization (Table I I I ) . T a b l e III.

Bacteriological Results of O z o n e Treatment on Filtered and Unfiltered S e w a g e * Log % Survival of B. subtilis var. niger Spores

O3 Treatment, Min. 20 30 45

Filtered -1.15 -1.69 -1.85

b

Unfiltered -0.16 -1.00 -1.80

Average of 3 lots of sewage in 3 separate experiments. In each experi ment the same lot of sewage was used to obtain filtered and unfiltered values. Initial count was 10 B. subtilis var. niger spores/ml. a

b

8

T h e l o g of a v e r a g e p e r cent s u r v i v a l i n d i c a t e s fewer s u r v i v o r s w i t h filtered t h a n w i t h u n f i l t e r e d sewage w i t h i n t h e first 30 m i n u t e s of t r e a t m e n t , b u t a f t e r 45 m i n u t e s the difference is i n s i g n i f i c a n t . T a b l e s I , I I , a n d I I I s h o w t h e t r e n d or p a t t e r n of t h e v a r i o u s p a r a m e t e r s r a t h e r t h a n p o r t r a y a n y definite r e p r e s e n t a t i v e e x p e r i m e n t . T a b l e s I V t h r o u g h V I I s h o w t h e results of b i o l o g i c a l w o r k p e r f o r m e d a t F o r t Detrick. Table IV.

. Type of Liquid m

Buffered distilled water

Results of Experiments with B. Subtilis Var. Niger Spores Inoculum B. subtilis var. niger Spores Surviving/100-M1. Exposure to O3, M i n .

Initial Count, Org./Ml.

30

22.1 X 10 6.6 X 10

7 7

Raw sewage count. 7.3 X 10 7

a b

8 X 10

7

>240 0 >240

60 7.6 10* >240

120

90

—0

0 240*

Disinfection and Sterilization of Sewage by Ozone - Advances in

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