Biotechnology Letters Vol 7 No 2 125-128 (1985)

PATHOGEN SURVIVAL DURING ANAEROBIC DIGESTION: FATTY ACIDS INHIBIT ANAEROBIC GROWTH OF ESCHERICHIA COLI

Pirshing Abdul, David Lloyd ~

Department of Microbiology, University College, Newport Road, Cardiff, CF2 1TA, Wales, U.K.

SUMMARY

The f a t e o f p a t h o g e n s i n a n a e r o b i c d i g e s t e r s has b e e n s t u d i e d i n a l a b o r a t o r y m o d e l s y s t e m i n w h i c h g l u c o s e - n u t r i e n t b r o t h c u l t u r e s o f genetically-defined strains of Escherichia coli received additions of fatty acids at concentrations similar to those attained during anaerobic treatment of farm wastes. Marked concentration-dependent inhibition of growth was observed for both antibiotic resistant and sensitive strains, and the effects increased with increasing chain lengths up to C 8. Survival of enteric organisms during anaerobic digestion may be limited by fatty acid toxicity.

INTRODUCTION

The reduction in viral, bacterial and protozoal pathogens present in

human and farm wastes that occurs during anaerobic digestion (Carrington,

et al., 1982) offers a method of pollution control as an added benefit

to biogas and fertilizer production. The mechanisms of pathogen

reduction during the process require investigation as it seems likely

that in many circumstances pathogen control may become the primary aim

in digester operation. Inhibitory effects of fatty acids on bacterial

growth have been reported (Eisler and Von Metz, 1968; Freese et al.~

1973; Salanitro and Wegener, 1971), but inconsistent results stem from

different strains and cultural conditions employed. It has been

suggested that Gram-negative bacteria are less sensitive to inhibition

by fatty acids than Gram-positives, being protected by their outer

lipopolysaccharide layer (Sheu and Freese, 1973). Mutants of

Escherichia coli lacking the polysaccharide portion of the coat are

much more sensitive to the effects of fatty acids than normal strains

(Elbein and Heath, 1965).

In the present paper we report concentration-dependent inhibition of

growth of three different defined strains of E. coli at fatty acid

concentrations often encountered in anaerobic digestion plant. This

suggests that fatty acid production is a major factor bringing about

pathogen reduction during digester treatment: similar results were

obtained for antibiotic resistant and sensitive strains.

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~ T E R I A L S AND }~THODS

O r g a n i s m s

S t r a i n s o f E. c o l i e m p l o y e d w e r e J 5 3 ( R 1 3 6 ) , t e t r a c y c l i n e r e s i s t a n t ; W3110T ( R 3 0 0 B ) , S t r e p t o m y c i n r e s i s t a n t ; MP1, a n t i b i o t i c s e n s i t i v e a n d c o n t a i n i n g n o R p l a s m i d .

Med ium a n d g r o w t h c o n d i t i o n s

A l l s t r a i n s w e r e g r o w n a n a e r o b i c a l l y i n 0 . 1 % g l u c o s e n u t r i e n t b r o t h (pH 8 . 0 ) u n d e r N 2 . G r o w t h c u r v e s w e r e p l o t t e d f r o m a b s o r b a n e e r e a d i n g s a t 6 0 0 nm t a k e n o n t h e 16 nun d i a m . s i d e a r m s o f 100 ml c o n i c a l f l a s k s . F a t t y a c i d s w e r e a d d e d a s p o t a s s i u m s a l t s w h e n a b s o r b a n c e a p p r o a c h e d 0 . 1 . V i a b l e c o u n t s w e r e d e t e r m i n e d b y s p i r a l p l a t i n g o n M a c C o n k e y a g a r .

RESULTS

T y p i c a l e f f e c t s o f f a t t y a c i d a d d i t i o n t o c u l t u r e s o f E. coli g r o w i n g

a n a e r o b i c a l l y a r e p r e s e n t i n F i g . 1 w h i c h s h o w s d e c r e a s e d g r o w t h r a t e s

a n d f i n a l p o p u l a t i o n s a t t a i n e d w i t h v a r i o u s c o n c e n t r a t i o n s o f i s o b u t y r a t e .

G r o w t h r a t e s o f b o t h a n t i b i o t i c r e s i s t a n t s t r a i n s , a s w e l l a s t h e a n t i -

b i o t i c s e n s i t i v e s t r a i n w e r e r e d u c e d b y 6 0 mM i s o b u t y r a t e ; s t r a i n s W3110T

( R 3 0 0 B ) a n d MP1 d i d n o t g r o w i n t h e p r e s e n c e o f 9 0 mM i s o b u t y r a t e a n d

s t r a i n J 5 3 ( R 1 3 6 ) s h o w e d n o g r o w t h w h e n t h i s f a t t y a c i d w a s p r e s e n t e d a t

180 mM. T a b l e 1 s u m m a r i z e s t h e e f f e c t s o f i n c r e a s i n g c h a i n l e n g t h f a t t y

a c i d s o n t h e t h r e e s t r a i n s o f E. c o l l . A l t h o u g h d i f f e r e n c e s a r e e v i d e n t

b e t w e e n s t r a i n s , i t i s c l e a r t h a t f a t t y a c i d t o x i c i t y i n c r e a s e s w i t h

i n c r e a s i n g c h a i n l e n g t h u p t o C8; c a p r a t e ( 2 . 5 mM) s h o w e d n o i n h i b i t o r y

p r o p e r t i e s . E s t i m a t i o n s o f v i a b l e c o u n t s b y s p i r a l p l a t i n g o f s a m p l e s

t a k e n f r o m f l a s k s i n w h i c h g r o w t h w a s c o m p l e t e l y i n h i b i t e d i n d i c a t e d

t h a t i n a l l c a s e s t h e r e w a s c o m p l e t e l o s s o f v i a b i l i t y .

TABLE 1 R e d u c t i o n i n g r o w t h r a t e o f E. c o l i s t r a i n s ( e x p r e s s e d a s % o f c o n t r o l s ) b y a d d e d f a t t y a c i d s .

F a t t y a c i d C o n c . (mM) J 5 3 ( R 1 3 6 ) E. c o l i s t r a i n MP1 W3110T ( R 3 0 0 B )

A c e t a t e 60 71 * 7 0 . 5 1 2 0 0 0 30

P r o p i o n a t e 52 4 9 100 91 104 0 0 0

I s o b u t y r a t e 60 32 86 53 90 21 0 0

180 0

Caprylate 2.5 0 0 61

H e p t a n o a t e 2 . 5 0 0 N .D.

C a p r a t e 2 . 5 1 0 0 N.D. 10O

ina1~its growl, h s177 Z . o n only. N.D, n o t d~Lermined.

126

E r -

0 0 r v

.'M_

m

I I I

0

[ 0.4 ~- J53(R 1 3 6 ~ l ~ l l e 4 i l i ~ Control

0.2 - ~ _ _ _ _ , , , , M ~ o . ~

~., I PO '1 O0 200 ........

0"4/l | ~ ~ 1 ~ Cl~ontr,ol

t , - / - o., ~ ~ ~ ~ . ~ ~ u ~ , , o ~ , .L. I I 0 .......... I 0 0 _ ~

. ~ Control 0 . 4 - MP I . = ~ _ ~ o o -0 56mM

/~ ~.cr~O ~U- ~-

0 . 2 -

O. I 1 8 0 m M I_L_ 1 t 0 tOO 200

Time (rain)

Fig, 1. Effects of isobutyrate on growth of E.coli strains

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DISCUSSION

This paper demonstrates that fatty acids inhibit the growth of E. coli

in glucose nutrient broth. \ Toxicity shows strain dependence, thus

strains J53 (R136) and W3110T (R300B) were both highly sensitive to

2.5 mM caprylate, whereas ~1 was relatively insensitive to the same

concentration. Sensitivity tO fatty acid inhibition increased with

chain length up to C 8. In all cases the growth rates decreased with

increasing concentrations e.g. for E. coli J53 (R136) the normal rate

of 0.69 h -I was decreased to 0.34 h -I and to 0.I h -I with 56 and 90 mM

isobutyrate respectively. All these data were obtained in cultures at pH 8.0;

this value was chosen as it is typical of conditions which prevail in

pig waste digesters. The persistence of pathogens in anaerobic treatment

plant is of great importance because of potential transmission to man and

farm animals. Although enteric bacteria have special protective mechanisms

that enable them to survive fatty acids produced by digestion of fats in the

intestinal tract (Sheu and Freese, 1973), much higher concentrations of

fatty acids are often encountered in anaerobic digester systems. For

instance, Van Velsen (1981) has reported 180 mM acetate and 113 mM propionate

in working processes, and locally operated pilot plant often contained i0,000

ppm total volatile fatty acids (i.e. equivalent to 167 mM acetate or 114 mM

isobutyrate). Tappouni (1984) has shown that addition of fatty acids to

digesters markedly decreases survival rate of Salmonella sp. Clearly the

survival of enteric organisms during anaerobic treatment may be limited by

fatty acid toxicity, although in such a complex ecosystem multifactorial

dependence on many environmental influences is inevitable.

REFERENCES

CARRINGTON, E.G., HAR~L4N, S.A. AND PIKE, B. (1982). J. App, Bacteriol. 95, 1767-1773.

EISLER, D.M. AND VON METZ, E.K. (1968). J. Bacteriol. 95, 1767-1773. ELBEIN, AoD- AND HEATH, E.C. (1965) o J. Bacteriol. 210, 1919-1925. FREESE, E., SHEU, C.W. AND CALLIERS, E. (1973). Nature 241, 321-325. SALANITRO, J.P. AND WEGENER, W.S. (1971). J. Bacteriol. 108, 885-892. SHEU, C.W. AND FREESE, E. (1973). J. Bacteriol. 115, 869-875. TAPPOUNI, Y. (1984). "The Fate of Salmonella in Anaerobic Digestion".

Thesis, University of Wales. VAN VELSEN, A.F.M. (1981). "Anaerobic digestion of piggery waste".

Thesis, University of Wageningen, The Netherlands.

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