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P e r g a m o n0043-1354 95 )00036-4

Wat. Res.Vol. 29, No. I 1, pp. 2598-2600 , 1995Elsevier Science Ltd . Printed in Great Britain

R E S E R C H N O T E

F T E O F F E C L C O L I F O R M B C T E R I I N W S T E

W T E R R E T E N T I O N R E S E R V O I R C O N T I N I N G

L E M N A G I B B A L

A H M E D D E W E D A R 1 a n d M A G D Y B A H G A T .2

~Suez Can a l Un ivers i ty, Facu l ty o f Sc ience , B otany D epar tm ent , I smai l i a , and 2Suez Can a l U nivers i ty,Facu l ty o f A gr icu l tu ra l and Env i ronm enta l Sc iences , A1 Ar i sh , Egypt

First received Mar ch 1994; accepted in revised for m January 1995)

A b s t r a c t - - T h e c o m p a r a t i v e s u r v i v a l o f t o t a l a n d f a e c a l c o l i f o r m b a c t e r i a w a s s t u d i e d i n a s t a b l e

was te -w ate r re ten t ion rese rvo i r. The sur face o f the wa te r rese rvo i r was covered wi th a macrop hyte ,Lemnagibba L. Se t s o f d ia lysi s sacs were suspen ded in an ex posed s i t e to the sun l igh t, whi le o ther se t s o f sacswere suspended benea th the th ick g reen f i lm ofL. gibba; where sun l igh t i s a lmos t absen t . To ta l co l i fo rmbac te r ia regrow in sun and in sacs unexposed to sun , whi le faeca l co l i fo rm ce l l s exposed to sun l igh td i sappe ared wi th a decay ra te 0 .1768 h -~ . In te res t ing ly, faeca l co l i fo rm in sacs suspended unde r the L .gibba green f i lm d id no t dec l ine dur ing the pe r iod o f the exper iment .

Key words--faecalco l i fo rm, to ta l co l i fo rm,Lemna gibbaL. , was te -wate r re ten t ion rese rvo i r s

INTRODUCTION

A n u n d e r s t a n d i n g o f t h e s u r v iv a l o f f ae c a l i n d i ca t o r s

i s b a s i c t o t h e m e a n i n g f u l i n t e r p r e t a t i o n o f s a n i t a r yw a t e r q u a l i t y d a t a . T h i s b e c a u s e t h e i s o l a t io n o fc o l i f o r m b a c t e r i a is c o m m o n l y u s e d t o in d i c a t e t h ep o t e n t i a l p r e se n c e o f i n t e s ti n a l p a t h o g e n s ( M c F e t e r set al . , 1 9 7 8 ) . B e c a u s e p a t h o g e n i c m i c r o o r g a n i s m su s u a l l y a p p e a r i n t e r m i t t e n t l y i n l o w c o n c e n t r a t i o n s ,a n d t h e n o n l y b r ie f l y t h e r o u t i n e e x a m i n a t i o n o fw a t e r n e e d s a m o r e d e p e n d a b l e a n d b r o a d e r b a s et h a n j u s t p a t h o g e n d e t e c t i o n . I d e a ll y , i t s h o u l d d e t e c to t h e r m i c r o - o r g a n i s m s w h i c h c o - ex i st w i t h t h e p a t h o -g e n s in th e f a e c a l e n v i r o n m e n t , a n d w h o s e p r e s e n c ei n a b o d y o f w a t e r w i ll s e rv e a s a n i n d i c a t o r o f f a e c a l

p o l l u t io n , p r i n c i p a ll y o f w a r m - b l o o d e d a n i m a l o r i g i n.F a e c a l c o li f o r m b a c t e r i a ( F C ) a r e k n o w n a s o n e o ft h e m o s t i m p o r t a n t i n d i c a t o rs o f p o t e n ti a l p u b l ich e a l t h h a z a r d d u e t o f a e c a l p o l l u t i o n .

C u l t i v a t i o n o f f l o a t in g a q u a t i c p l a n t s i n w a s t ew a t e r p o n d s i s in c r e a s i n g l y b e i n g p r a c t ic e d i n t h et r o p i c s a n d s u b t r o p i c s w i t h t h e d u a l o b j e c t i v e o fp u r i f i c a t i o n a n d r e s o u r c e r e c o v e r y.

T h e p r e s e n c e o f a q u a t i c m a c r o p h y t e s i n a p o n da l t e rs t h e p h y s i o c h e m i c a l e n v i r o n m e n t o f th e w a t e r .D i f f e r e n t s t u d i e s h a v e s h o w n t h a t a q u a t i c p l a n t s ,s u c h a s w a t e r h y a c i n t h Echorn ia c rass ipes [ M a r t ]S o l m s ) , w a t e r l e t t u c e Pis t ia s t ra t io tes L . ) , c a t t a i lTy p h a l a t i f o l i a L . ) , a n d L e m n a g i bb aL . , a r e c a p a b l e

* A u t h o r t o w h o m a l l c o r r e s p o n d e n c e s h o u l d b e a d d r e ss e d .

o f t r e a t i n g w a s t e w a t e r ( R e d d y a n d D e B u s k , 1 9 85 ).A l s o , s t u d i e s h a v e d i s c u s s e d t h e p o t e n t i a l o f a q u a t i cp l a n t s , f o r r e d u c i n g N a n d P l e v e ls i n w a s t e w a t e r.M o s t o f s tu d i e s w e re l i m i t e d to t h e p h y s i o c h e m i c a lc h a r a c t e r i s t i c s o f t h e w a t e r, a t t e n t i o n h a s n o t b e e ng i v e n t o s t u d y o f t h e m i c r o b i o l o g i c a l c h a n g e s a c -c o m p a n i e d w i t h t h e in t r o d u c t i o n o f m a c r o p h y t e t ot h e w a t e r b o d y. P r e v i o u s s t u d i e s ( u n p u b l i s h e d d a t a )i n a w a s t e w a t e r r e t e n t i o n r e s e rv o i r , A b u - A t t w a ,E g y p t , s h o w e d t h a t t h e f a e ca l c o li f o r m n u m b e r sd e c l i n e d w i t h i n t h a t r e s e r v o i r .L e m n a g i b b a L . , af l o a t i n g p l a n t , h a s b e e n i n t r o d u c e d t o t h a t r e s e r v o i rc a u s i n g a c h a n g e i n t h e r a t e o f d ec l i n e w i t h e v i d e n c eo f r e g r o w t h .

T h e p u r p o s e o f t h i s s t u d y i s to d e t e r m i n e t h e e f fe c t

o f L e m n a g i b b a o n t h e r e m o v a l o f F C f r o m a w a s t ew a t e r r e t e n t i o n r e s e r v o i r .

MATERIALS AND METHODS

The inves t iga t ion was ca r r i ed ou t in a concre te was tewate r rese rvo i r (15 m3) . The wate r su r face w as covered w i tha dense thick f i lm ofLemna gibba L. and receiving sec-ond ary t rea ted was te w a te r d i scharged ou t o f a 200 mG r a v e l B e d H y d r o p o n i c ( G B H ) b e d f o r se w ag e t r e a t m e n t a tAbu-At twa , I smai l i a , Egypt . Descr ip t ion o f the sys tem wasexp la ined e l sewhere (But le r and Dewed ar, 1991) .

Pur i f i ed cu l tu res o f the faeca l co l i fo rm bac te r ia i so la tedf rom the s tud ied rese rvo i r were pur i f i ed by the methods

descr ibed in the Cowan and S tee l ' sMa nual for the Identifi-cation o f Medical B acteria(Co wa n an d Steel , 1 993 ). Allcu l tu res used in th i s s tudy were g row n for 24 h on n u t r i en tb ro th a t 37 C . Ce l l s were ha rves ted by cen t r i fuga t ion(6000 rpm ) for 10 min and was hed twice with s ter i le buffer

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Research Note

Table I. Some chemical characteristics of the reservoir water used in the study

2599

Determination Conductivity ~ Calcium Magnesium NH4-N b NO3b DO BOD5

Values of reservoir water 1000 44.9 26.2 11.05 1.4 7.5 27.4before applying L. gibba

Values of reservoir water 850 42.5 22.6 12.8 0.71 I.I 18.6after applying L. gibba

aMicron/cm at 25C.

bMilligram per liter.

solution. After the final wash, cells were suspended in thesterile buffer and diluted to the desired population densitywith the same diluent. Dialysis sacs were immersed in thereservoir which receives almost s teady flow o f water out ofa 200 m GBH system. Four sets of dialysis sacs were usedfor both total an d faecal coliform placed in exposed area tosunlight under Lemna gibbaL. forming a green film floatingon the water surface. One ho ur aft er the dialysis sacs weresuspended in the water one sac was removed from each setand coliform and faecal coliform counts performed. Thesecounts were used to de termine Co, the number of organismsat time O. Standard plate counts were used to enumeratethese bacteria on m-Endo Agar (APHA, 1985). The decayrate o f faecal coliform was determined from a plot of the logof the fractional decrease in bacteria concentration overtime, In Ct/Co) versus time. The slope of the line (fit by alinear regression) was equal to the decay rate, K. The largerthe number, the greater the decay rate. The decay rate ofsuch cells was determined using the model:

In Ct/Co) = - kt,

where K is the decay constant (h -j ) equal to the slope of theline (fit by a linear regression); C O is the initial faecalcoliform concentration; and C is the faecal coliform concen-tration at time t.

Chemical characteristics

Conductivity, calcium, magnesium, ammoniacal-N(NHa-N) and, nitrate-N (NO3-N), dissolved oxygen (DO)and Biological oxygen demand (BODs) conc entratio ns weremeasured according to APHA (1985).

RESULTS

Water quality para meter s (Table 1) were stable andonly minor variati ons in temper atures (34-35C) werenoticed during the experiment in the studied wastewater retention reservoir.

S u r v i va l o f To t a l C o l i fo r m T C )

The survival of natural populations of total coli-form (TC) bacteria isolated from domestic sewagewas studied to obtain informati on on the populat iondynamics on water either exposed to sunlight orunder a thick film of L . g i b b a L. where sunlight isalmost nil. Figure 1 is showing survival of mixednatural populations of total coliform bacteria ex-posed and unexposed to sunlight.

No linear relation was found between the naturallogarith m of the number of the surviving bacteria andtime by the simple regression analysis using themodel:

l n y = l n a + b x

where y = estimated bacterial counts (cfu/ml):a = intercept; b = slope; x = time of the experiment

in days. No decay was recorded in case of the totalcoliform cells.

S u r v iv a l o f F a e c a l C o l i fo r m F C )

The survival of pure cultures of FC was studied inthe same meth od used for the total coliform (Fig. 2).

No decline in counts were recorded in case of FCpresent in the set of dialysis sacs suspended under theL . g i b b a , whereas, FC in dialysis sacs exposed to sunsho wed a n initial rise in gro wth in the first 24 hfollowed by a decline to the end of the experiment.The rate constant for decay of faecal coliform cellsexpo sed to the sun was 0.1768 h -~.

DISCUSSION

The result of the experiment describing the survivalof indicator bacteria in the waste water retentionreservoir demonstrated that the total and faecalcoliform bacteria remained viable except FC bacteriain dialysis sacs which were exposed to the sun. It iswell documented that total coliform bacteria can

regenerate in a natural environment, and manyworkers criticized the use of the total coliform coun tas an indi cator o f feces-oriented water poll ution(Dutka, 1973).

Previous studies of bacteria survival that were

.1

8.0

7.8

7.6

7.4

7.2

7.0

6.8

6 . 6 L

6.4 t6.2

6.00

/ \

TC coveredra TC sun

I I I I I

2 3 4 5

Time (days)Fig. I. Survival of indica tor Bacteria (TC) in reservoirwater. Total coliform cells in dialysis sacs suspended under

the L. gibba thick film exposed to sunlight.

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2600 R e s e a r c h N o t e

8 - -

/

7 -

61

_~. 5

4

FC o,,er~d \- .1 3 - [ ] F C sun q

1

0 I I I I ~h0 1 2 3 4 5

Ti m e d a y s )

F ig. 2 . Surv iva l o f ind ica to r bac te r ia FC) in rese rvo i r wa te r.Faeca l co l i fo rm ce l ls in d ia lys i s sacs suspended und er the L .gibba th ick f ilm, on the wa te r su r face , exposed to sun l igh t .

l a n d s 0 . 0 2 9 h ~) G e r s b e r get al., 1 9 8 7 ) . T h i s i sp r o b a b l y d u e t o s u n l i g h t e f fe c t w h i c h h a v e a l e t h a le f f e c t o n c o l i f o r m s .

CONCLUSIONS

T h e c o n c l u s i o n s d r a w n f r o m t h i s s t u d y o b t a i n e d

u n d e r c o n t r o l l e d e x p e r i m e n t a l c o n d i t i o n s , c o m p a r e dw i t h t h c a c t u a l c o n d i t i o n s i n t h e f ie ld s h o u l d b ea p p r o a c h e d w i t h g r e a t c a u t i o n . T h i s is b e c a u s e t h e r ea r e m a n y v a r i a b l e s i n t h e n a t u r a l a q u a t i c e n v i r o n -m e n t s u c h as p h a g e , a n d c o m p e t i n g o r p r e d a t o r yo r g a n i s m s , o n l y o n e o f w h i c h m a y i n f l u e n c e b a c t e r i a ls u r v i v a l . E v e n s o , s o m e g e n e r a l c o n c l u s i o n c a n b ee x t r a c t e d t h a t c o u l d b e o f v a l u e i n th e i n t e r p r e t a t i o no f d a t a i n t h e e n v i r o n m e n t . A m o n g t h e se is t h ef i n d i n g t h a t t h e a p p l i c a t i o n o fLemna gibba L . , w h i c hi s a f l o a t in g m i n u t e p l a n t , t o w a t e r r e s e r v o i r s c o u l db e a s o u r c e o f n u t r i e n t s a s w e ll a s s h e l t e r w h e r eb a c t e r i a c a n p r o l i f e r a t e f o r e x t e n d e d p e r i o d s . A l s o ,t h e e x p e r im e n t d e m o n s t r a t e s t h c ne e d t o u s e m o r et h a n o n e i n d i c a t o r f o r p o l l u t i o n b y w a s t e w a t e r i n t h ee n v i r o n m e n t s tu d ie s .

c a r r ie d o u t u s i n g e i t h er m e m b r a n e c h a m b e r sM c F e t e r s et al., 1 9 7 4 ) o r d i a l y s i s s a c s H e n d r i c k s

a n d M o r r i s o n , 1 9 67 ) f il le d w i t h s u s p e n s i o n s o f t h ei n d i c a t o r b a c t e r i a a n d i m m e r s e d i n s t r e a m s r e v e a l e dt h a t f l u c t u a t i o n s i n t h e w a t e r c o m p o s i t i o n i n f l u e n c e dt h e p o p u l a t i o n d y n a m i c s o b s e r v e d . M i n o r c h a n g e sw e r e r e c o r d e d i n t h e w a t e r q u a l i t y o f t h e s t u d i e dr e s e r v o i r s .

A n i n i ti a l r i se i n c o l if o r m b a c t e r i a w a s n o t i c e d i nm a n y s t u d i e s b o t h i n s o il Va n D o n s e let al., 1 9 6 7 )a n d i n w a t e r D u t k a , 1 9 73 ). F a e c a l c o l i f o r m b a c t e r i aa r e a t h e r m o t o l e r a n t g r o u p , t h a t d e c l i n es in n a t u r a le n v i r o n m e n t s M c F e t e r s et al., 1 9 7 4 ) . H o w e v e r ,C o o k a n d R u p l e , 1 9 8 9) , r e p o r t e d i n c r e a s e i n t h el e ve l s o f f ae c a l c o l i f o r m b a c t e r i a a c c o m p a n i e d b yi n c r e a s e s i n Vi b r i o n a c e a e i n p o s t - h a r v e s t s h e l l s t o c ko y s te r s d u r i n g c o m m e r c i a l t r a n s p o r t a n d d u r i n g s t o r-a g e a t t e m p e r a t u r e o f 1 0 , 2 2 a n d 3 0 C . A l s o , H e n -d r i c k s a n d M o r r i s o n , 1 9 6 7) , s h o w e d t h a t t h e a q u a t i ce n v i r o n m e n t a s s o c ia t e d w i t h a c l ea r m o u n t a i n s t r e a mc a n n o t o n l y m a i n t a i n p o p u l a t i o n s o f e nt e r ic b a c t e r iab u t a l s o c a n s u p p l y s u f f i c i e n t n u t r i e n t s t o i n i t i a lm u l t i p l i c a t i o n a n dde novo p r o t e i n s y n t h e s i s .

T h e Lemna gibba L . is e x p e c t e d t o c r e a t e a n u t r i -e n t - r ic h e n v i r o n m e n t t h r o u g h p o p u l a t i o n i n c re a s ea n d e x c r e t i o n o f p h o t o a s s i m i l a t e d o r g a n i c c o m -p o u n d s . T h e a b i l i ty t o r e m a i n w i t h i n t h i s e n v i r o n -m e n t e n a b l e s la r ge n u m b e r s o f h e t e r o t r o p h i cb a c t e r i a , i n c l u d i n g c o l i f o r m s , t o p r o l i f e r a t e e v e n i ne n v i r o n m e n t s u n s u i t a b l e f o r s u r v iv a l H a a c k a n dM c F e t e r s , 1 9 8 2 ) .

F a e c a l c o l i f o r m c e l l s i n d i a l y s i s s a c s e x p o s e d t o

s u n l i g h t s h o w e d t y p i c a l d ie - o f f p a t t e r n e x p e r i e n c e d i nd i f f e r e n t e n v i r o n m e n t s . T h e d e c a y r a t e c a l c u l a t e d i nt h e p r e s e n t s t u d y 0 . 17 6 8 h 1) w a s h i g h c o m p a r e dw i t h d e c a y r a t e c a l c u l a t e d i n a n a t u r a l c y p r u s w e t -

Acknowledgement--The a u t h o r s w i s h t o t h a n k D r A .EI-Sayed, Suez Canal Universi ty for helpful discussions.

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