CURRENT MICROBIOLOGY VO1. 19 (1989), pp. 277-281 Current Microbiology �9 Springer-Verlag New York Inc. 1989

Toxicity of Volatile Fatty Acids at Rumen pH Prevents Enrichment of Escherichia coli by Sorbitol in Rumen Contents

R. John Wallace, Margaret L. Falconer, and Padma K. Bhargava

Rowett Research Institute, Bucksburn, Aberdeen, UK

Abstract. Attempts were made to establish Escherichia coli ML308 in the sheep rumen by inoculating it in combination with the slowly metabolized sugar alcohol, sorbitol. Numbers were determined by plating dilutions on nutrient agar containing the chromogenic fl-galactoside, 5- bromo-4-chloro-3-indolyl-fl-D-galactoside. This strain, a lac-constitutive mutant, produced dis- tinctive blue colonies. E. coli ML308 failed to grow in rumen fluid, despite being able to grow rapidly on sorbitol and in rumen fluid at pH 7.0. Its growth rate was depressed by relatively small drops in pH in the presence of volatile fatty acids (VFA), such that normal pH's of 6.2-6.6 in rumen contents were inhibitory. The indigenous remen bacterium, Streptococcus boris, was much more resistant to the combination of high VFA concentrations and low pH. The success of this and similar strategies for the introduction of new organisms with beneficial new proper- ties will, therefore, depend on the organism's having a tolerance to VFA over a range of rumen pH that enables them to survive in the same way as native species.

It has been proposed that the selective disadvantage that many new and/or genetically modified organ- isms would have in establishing in the rumen [9] might be overcome by adding an otherwise slowly metabolized compound that the organism could fer- ment, thus creating an ecological niche for its sur- vival and growth [11]. Among several compounds identified to be suitable for this purpose was sorbi- tol.

Escherichia coli grows rapidly on sorbitol under anaerobic conditions [11] and has other advantages that might be considered useful for the proposed manipulation strategy. It is a facultative organism and would, therefore, present fewer technical prob- lems than a strict anaerobe if continued reinocula- tion became necessary. Furthermore, the vast knowledge of its molecular biology and genetics that already exists would accelerate the progress of any genetic manipulation work for strain improve- ment.

The present paper describes attempts to estab- lish E. coli in the sheep rumen with sorbitol as an enrichment factor. Although the experiments did not result in successful colonization, they offer use- ful pointers for future work.

Materials and Methods

Animals . Four mature sheep were fitted with permanent rumen cannulae. Sheep A received a ration containing 67% grass hay + 33% barley-based concentrate. Sheep B received 67% grass hay + 33% grass cubes. The others were fed 70% grass hay (contain- ing 1% urea) + 30% grass cubes. The sheep were fed at 0800 and 1600 h.

Introduction and detection of E. coli in rumen fluid in vitro. Escherichia coli ML308 (ATCC 15224), constitutive for the lac operon, was grown aerobically overnight in nutrient broth (Ox- oid) or in a simple salts medium [13] containing 16 mM sorbitol. Rumen fluid was removed from sheep A and B via the rumen cannulae at 1100 h. Culture (1 ml) was added to 9 ml of strained rumen fluid containing no addition or 20 mg of sorbitol, and the mixture was incubated under CO2 at 39~ Samples were re- moved, and a portion was acidified with trichloroacetic acid (5%, wt/vol), centrifuged at 11,600 g for 10 min, and analyzed for sorbitol [15]. One milliliter of sample was also serially diluted in sterile 50 mM potassium phosphate buffer, pH 7.5. Samples (10 /xl) of each tenfold dilution were spread on nutrient agar (Oxoid)

containing 5-bromo-4-chloro-3-indolyl-fl-D-galactoside (BCIG; 50/xg/ml) and incubated at 37~ for 18 h. This mutant produced blue colonies in BCIG-containing medium.

Introduction and detection of E. coli in rumen fluid in vivo. Sheep C and D were inoculated at 0900 h with 250 ml of nutrient broth culture via their rumen cannulae. Samples were removed periodically, and the survival of lac-constitutive organisms was

Address reprint requests to: Dr. R.J. Wallace, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK.

278 C U R R E N T M I C R O B I O L O G Y V o [ . 19 (1989)

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Fig. 1. Survival ofEscherichia coli ML308 in rumen contents in vitro. E. coli was grown in nutrient broth, and 1 ml of culture was incubated with 9 ml of stained rumen fluid taken from a sheep receiving a mixed hay-concentrate diet (�9 or another sheep fed all roughage (0). Viable counts of E. coli were done as described in Materials and Methods.

determined on BCIG plates as before. The influence of sorbitol on the survival of E. coli ML308 was determined in repeat exper- iments in which 10 g of sorbitol in 50 ml aqueous solution was added 1 h after the inoculum and at the 1600 h feed. Remaining sorbitol was determined as before in samples of rumen fluid re- moved hourly thereafter.

Growth of bacteria in pure culture. The influence of VFA on the growth rate of E. coli ML308 and Streptococcus boris C277, a proteolytic rumen isolate [12], was determined with the defined media and procedures described by Russell et al. [10]. The VFA content of the medium was varied by adding 0%, 0.17% or 0.34% of a mixture of 1.7 vol acetic acid, 0.6 vol propionic acid, and 0.4 vol n-butyric acid.

Results and Discussion

D e t e c t i o n o f E. coil M L 3 0 8 in r u m e n f luid. Escheri- chia coli ML308, a lac-const i tu t ive mutan t , hydro- lyzes the ch romogen ic f l -galactoside, BCIG, in the absence of inducer , p roduc ing blue colonies on solid med ium. The induc ib le wild type E. coli does no t hyd ro lyze BCIG. Thus , expe r imen t s were done to de t e rmine whe the r it was poss ib le to enume r a t e E. coli ML308 added to r u m e n fluid. The n u m b e r s of in tense ly b lue colonies s imilar to E. coli ML308

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Fig. 2. Survival of E. coti ML308 in rumen contents in the pres- ence of added sorbitol. E. coli was grown on sorbitol, and 1 ml of culture was incubated with 9 ml of strained rumen fluid from a roughage-fed sheep in the absence (O) or presence (O) of 20 mg of sorbitol. Viable counts of E. coli and estimation of sorbitol (2x) were carried out as described in Materials and Methods.

a l ready p re sen t in r u m e n fluid f rom the four sheep var ied f rom 0.1 x 105 to 1.8 x 105 per ml (average, 4.7 x 104). N u m b e r s of whi te colonies were an or- der of magn i tude grea ter (1 .1-4 .7 • 105 per ml; av- erage, 3.2 • 105). H e n c e the ind igenous facul ta t ive

popu la t ion did no t in terfere wi th the e n u m e r a t i o n of E. coli ML308 w h e n it was i n t roduced in subse- quen t expe r imen t s at a v iable cell dens i ty of abou t 108 ce l ls /ml .

Surv iva l o f E. coli M L 3 0 8 in r u m e n fluid in v i tro . W h e n E. coli ML308 was added to r u m e n fluid in vi tro, n u m b e r s were ma in t a ined at 2 x 108/ml for 4 h in r u m e n fluid f rom the roughage-fed sheep B (pH 6.8), bu t dec l ined rapidly in a sample f rom sheep A, which rece ived one- th i rd c o n c e n t r a t e in its diet (pH 6.2; Fig. 1). Thereaf te r , inves t iga t ions were con- fined to sheep rece iv ing roughage a lone. It should be no t e d here that the i ncuba t ions were done with- out shaking. Thus ciliate p ro tozoa set t led at the bot- tom of the tubes , p r e s u m a b l y min imiz ing the effects

of p r eda t ion on bacter ia l lysis [14]. S ince E. coli ML308 grows rapidly on sorbi tol

in a complex anae rob ic m e d i u m con ta in ing 20% clarified r u m e n fluid [11], it s eemed reasonab le that

R.J. Wallace et al.: Toxicity of VF A to E. coli in R u m e n Fluid 279

Table 1. Survival of Escherichia coliML308 in rumen fluid in vivo

E. coliML308 per ml"

No sorbitol Added sorbitol Time after inoculation (h) Sheep C Sheep D Sheep C Sheep D

1 4.4 • 107 4.6 x 107 2.0 • 107 4.7 • 107 6 1.1 )< 107 0.5 x 107 0.8 • 107 1.0 x 107

24 2.6 • 105 6.2 x 106 3.4 • 10 ~ 2.1 x 106

a A 250-ml nutr ient broth culture was poured into the rumen of two roughage-fed sheep via their rumen cannulae 1 h after feed- ing, and the survival o fE . coli ML308 was followed as described in Materials and Methods . The influence of introducing sorbitol was determined by adding 10 g of sorbitol 1 h after inoculation and with the feed 6 h later. Resul ts are the averages of two exper iments .

its numbers might be enhanced by the addition of sorbitol to rumen fluid. However, the sorbitol addi- tion failed to stimulate growth of E. coli ML308 despite the persistence of finite sorbitol concentra- tions for more than 6 h (Fig. 2).

Survival of E. coil ML308 in vivo. The survival of E. coli ML308 in vivo was investigated in sheep C and D. When E. coli ML308 was inoculated in the ab- sence of added sorbitol, only an average of 18% of the number present at 1 h survived at 6 h (Table I). This decline is equivalent to a death + washout rate of 0.34 h -], which is much greater than the rumen outflow rate of 0.03-0.15 h 1 in sheep [3]. Predation by protozoa was almost certainly responsible, the rate of loss correponding to the highest rates of breakdown seen with a range of different bacterial species in faunated rumen fluid [14]. After 24 h, only 0.6% of the original BCIG--positive colonies remained in sheep C. A higher proportion (13%) survived in sheep D. No net growth occurred when sorbitol was added (Table 1). Indeed, not only did not growth occur, but survival was hardly affected by sorbitol addition, yet sorbitol was detected in rumen contents for at least 2 h after its administra- tion.

Influence of V F A on growth of E. coli. In order to identify the reasons for the failure to establish E. coli ML308 in the rumen, growth on a defined me- dium containing different concentrations of VFA at different pH was investigated. Rumen fluid has pre- viously been shown to be toxic to E. coli under some circumstances in vitro, particularly at pH 6.0 rather than pH 7.0 [4, 16].

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Decreasing the pH of the medium to 5.8 had no effect on the specific growth rate (/x) of E. coli ML308 when no VFA were added, but E. coli ML308 proved sensitive to high concentrations of VFA, similar to those which are found in rumen contents, at lower pH values (Fig. 3). The specific growth rate was halved at pH 6.6 by the addition of VFA, and growth virtually ceased at pH 6.2. Inter- mediate VFA concentrations had correspondingly less effect, but growth was still abolished at pH 5.8. These results are consistent with those of Wolin [16] and add more detail about the sensitivity of E. coli to VFA. In particular, it becomes clearer from the effects on/x why E. coli ML308 should fail to survive in the rumen at intermediate pH's.

The poorer survival ofE. coli ML308 in vitro in rumen fluid from the sheep on the concentrate-con- taining diet (Fig. 1) can, therefore, be explained by its almost complete inability to grow at pH 6.2, the pH of the rumen fluid, in the presence of high VFA concentrations. The same phenomenon was proba- bly responsible for the poor survival and absence of growth on sorbitol in the roughage-fed animals in vivo, despite the higher pH. Growth rate was still depressed at pH 6.6 in vitro and, in any case, the

280 CURRENT MICROBIOLOGY Vol. 19 (1989)

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r u m e n p H of r o u g h a g e - f e d sheep was va r i ab le , such tha t a r u m e n p H o f 6.2 was s o m e t i m e s o b s e r v e d .

The r u m e n b a c t e r i u m , Streptococcus boris, was a f f ec t ed v e r y m u c h less b y high V F A conc e n - t r a t ions (Fig. 4). I t s g r o w t h ra te dec l ined as the p H fell , bu t on ly to 70% o f its va lue in the a b s e n c e o f V F A at p H 5.8. S e v e r a l o t h e r spec i e s o f t u r een bac- t e r ia we re s imi la r ly l i t t le a f f ec t ed by V F A at l o w e r p H (not shown) .

R u m e n b a c t e r i a w o u l d be e x p e c t e d to have e v o l v e d to g r o w in the cond i t i ons p reva i l ing in the rumen , inc lud ing the p r e s e n c e o f high c o n c e n t r a - t ions o f V F A . V F A ac t as u n c o u p l e r s o f the p r o t o n e l e c t r o c h e m i c a l g r a d i e n t a c r o s s the bac t e r i a l cell m e m b r a n e , e s p e c i a l l y at high c o n c e n t r a t i o n s and as the p H falls [5]. R u m e n bac t e r i a , p r e s u m a b l y in re- s p o n s e to the cha l l enge o f l iv ing in an e n v i r o n m e n t con t a in ing high c o n c e n t r a t i o n s o f V F A , regu la te in- t r ace l lu l a r p H on ly p o o r l y and main ta in low A p H ' s o f 30 mV or less at an e x t e r n a l p H o f a r o u n d 6.5 [6 - 8]. In con t r a s t , i n t r a ce l l u l a r p H in E. coli is s t r ic t ly r egu la t ed , g iv ing r i se to va lue s o f ApH of a b o u t 70 and 105 m V dur ing g r o w t h at p H 6.5 and 6.0 r e s p e c - t ive ly [17]. Thus , E. coli will be m u c h m o r e sensi - t ive than S. boris to the u n c o u p l i n g effects o f V F A , resu l t ing in i ts fa i lu re to c o l o n i z e the sheep r u m e n in

the p r e s e n t e x p e r i m e n t s and p r o b a b l y a lso the low n u m b e r s (10Uml o r less) o f E. coli found in r u m e n c o n t e n t s [1, 2].

Concluding observations. D e s p i t e the fac t tha t w e did not s u c c e e d in e s t ab l i sh ing E. coli ML308 in the r u m e n b y the c o - a d d i t i o n o f sorb i to l , we be l i eve tha t the bas i c r e a s o n i n g b e h i n d these e x p e r i m e n t s is sound . The c h o i c e of su i t ab le c o m p o u n d s is fa i r ly wide [1 l ] , and it shou ld b e p o s s i b l e to iden t i fy ap- p r o p r i a t e o r g a n i s m s tha t f e r m e n t some o f the c o m - p o u n d s . W h a t is r e q u i r e d for the s t r a t egy to suc- c eed is a m o r e r o b u s t o r g a n i s m , p e r h a p s S. boris or a n o t h e r gut a n a e r o b e , tha t f e r m e n t s any of the s lowly m e t a b o l i z e d c o m p o u n d s r e a s o n a b l y r ap id ly u n d e r r u m e n c o n d i t i o n s . W e a re n o w a t t emp t ing to ident i fy su i tab le o rga n i sms .

ACKNOWLEDGMENTS

We thank Drs H.J. Flint and C.S. Stewart for their helpful criti- cism.

Literature Cited

1. Brownlie LE, Grau FH (1967) Effect of food intake on growth and survival of Salmonellas and Escherichia coli in the bovine rumen. J Gen Microbiol 46:125-134

2. Flint HJ, Duncan SH, Stewart CS (1987) Antibiotic resis- tance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides rnultiacidus. Appl Microbiol Biotechnol 26:450-455

3. Harrison DG, McAllan AB (1980) Factors affecting micro- bial growth yields in the reticulo-rumen. In: Ruckebusch Y, Thivend P (eds) Digestive physiology and metabolism in ru- minants. Lancaster: MTP Press, pp 205-226

4. Hollowell CA, Wolin MJ (1965) Basis of the exclusion of Escherichia coli from the rumen ecosystem. Appl Microbiol 13:918-924

5. KeU DB, Peck MW, Rodger G, Morris JG (1981) On the permeability to weak acids and bases of the cytoplasmic membrane of Clostridium pasteurianum. Biochem Biophys Res Commun 99:81-88

6. Robertson JD (1986) The energetics of end product excretion from a rumen bacterium, Selenomonas ruminantium. Ph.D. thesis, University of Aberdeen

7. Russell JB (1987) Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulo- lytic rumen bacterium. Appl Environ Microbiol 53:2379- 2383

8. Russell JB, Hino T (1985) Regulation of lactate production in Streptococcus boris: a spiraling effect that contributes to rumen acidosis. J Dairy Sci 68:1712-1721

9. Russell JB, Wilson DB (1988) Potential opportunities and problems for genetically altered rumen microorganisms. J Nutr 118:271-279

10. Russell JB, Sharp WM, Baldwin RL (1979) The effect of pH on maximum bacterial growth rate and its possible role as a determinant of bacterial competition in the rumen. J Anita Sci 48:251-255

R.J. Wallace et al.: Toxicity of VFA to E. coli in Rumen Fluid 281

11. Wallace RJ (1989) Identification of slowly metabolized sug- ars and sugar derivatives that could be used to establish new or modified microbial species in the rumen. Curr Microbiol 19:271-274

12. Wallace RJ, Brammall ML (1985) The role of different spe- cies of bacteria in the hydrolysis of protein in the rumen. J Gen Microbiol 131:821-832

13. Wallace RJ, Holms WH (1986) Maintenance coefficients and rates of turnover of cell material in Escherichia coli at differ- ent growth temperatures. FEMS Microbiol Lett 37:317-320

14. Wallace RJ, McPherson CA (1987) Factors affecting the rate

of breakdown of bacterial protein in rumen fluid. Br J Nutr 58:313-323

15. West CD, Rapoport S (1949) Modification of colorimetric method for the determination of mannitol and sorbitol in plasma and urine. Proc Soc Exp Biol Med 70:141-142

16. Wolin MJ (1969) Volatile fatty acids and the inhibition of Escherichia coli growth by rumen fluid. Appl Microbiol 17:83-87

17. Zilberstein D, Schuldiner S, Padan E (1979) Proton electro- chemical gradient in Escherichia coli cells and its relation to active transport of lactose. Biochemistry 18:669-673