The effect of oxalate on bacteria isolated from the rumen.

Abstract

. THE EFFECT OF OXALATE ON BACTERIA ISOLATED FROM THE RUMEN ByM.W. O'HALLORAN* Summary High oxalate levels showed no inhibitory effect on twenty-six apparently different strains of ruminal bacteria. One organism RO-16 decomposed oxalate. Examination of the filtrates and residues from the rumen content of sheep showed that the residues were more potent in oxalate decomposition than the filtrates. An organism resembling RO-16 was isolated from residues and filtrates which decomposed oxalate. I. INTRODUCTION It was suggested (Talapatra, Ray, and Sen 1948) that rumen microflora possess the ability to decompose oxalate. Evidence supporting this contention has since been obtained (Dodson 1959; Watts 1957, 1959). Recently a bacterium has been isolated (Michael 1959) which occurs in association with the leaves and bulbs of Ox&s pes-capyae and has the ability to metabolise oxalate. The object of the work presented here was as follows:(i) To determine by in vitro experiments the effect of high levels of oxalate on a number of bacteria isolated in pure culture from the rumen of sheep. (ii) To examine' the ability of rumen content to decompose oxalate with a view to isolating bacterial species capable of performing this function. II. EXPERIMENTAL Twenty-six apparently different strains of rumen bacteria were isolated from a total of 78 sheep slaughtered at Roseworthy throughout the whole of one year. These organisms have not been identified systematically. They have been catalogued on the basis of morphology, cultural characteristics, and biochemical activities. The effect of oxalate on the growth of these organisms was determined by adding 3 per cent. ammonium oxalate to the medium in which they were isolated (O' Halloran 1962). Growth of organisms was determined absorptiometrically at 650 rnp and comparisons were made with growth in the same medium without oxalate, but containing an identical inoculum of the organism under investigation. Rumen samples were collected aseptically at slaughter. Containers used to collect the rumen content were filled completely, then capped to maintain conditions of low oxygen tension. Samples were processed and inocula taken within one hour of slaughter. Samples were strained through sterile cheese-cloth which had previously been boiled three times in distilled water. For oxalate decomposition studies one ml of the filtrate and 1 g of the residue WeiX added to 9 ml of the medium described in Table 1. :> Agricultural College, Roseworthy, S.A. 18 TABLE 1 OXALATE ENRICHMENT MEDIUM Solution A Potassium oxalate (COOQ Ha0 5g Potassium di-hydrogen phosphate KHZPOJ, nz Sodium thio-glycollate (or Sodium Sulphide Na$ 9Hz0) 1 g Ammonium sulphate lg Magnesium sulphate MgSO* 7Hz0 0.2 g Ferrous sulphate FeSOI 7Hz0 0*05 g Calcium sulphate CaSOp 0.02 g Manganese sulphate MnS04, 4H20 0*002 g Sodium molybdate NaMoOa 2Hz0 O*OOl g Glass distilled water 1 litre pH 7 00 Stand overnight and filter Sterilize at 15 p.s.i. for 15 minutes Solution B Thiamine HC 1 Riboflavine Pyridoxine Calcium pantothenate Niacin p. Amino benzoic acid Folic acid Cyano cobalamin (Merk) Biotin Glass distilled water pH 6-O Sterilize at 10 p.s.i. for 15 minutes 10mg 10mg 10mg 10mg 10mg 10mg 10mg 200 E,Lgm lF 10ml Dispense 9 ml amounts of solution A prior to sterilization. Immediately before inoculation add O-05 ml of Solution B and place in boiling water bath for 15 minutes, allow to cool and inoculate. Decomposition of oxalate was determined by the method of Baker ( 1952). All were total oxalate determinations. Where oxalate decomposition was demonstrated the sample was plated out on tc solid media as described in Table 1, but containing 2 per cent. agar (BactoDifco Agar) . Plate cultures were incubated at 38�C for 3 days in both aerobic and micro-aerophilic environments (viz. 10 per cent. CO,, 89 per cent. H,, 1 per cent. 0,). Colonies developing were picked off and incubated at 38�C in the medium of Quayle and Keech ( 1959). The ability of the pure culture isolated to decompose oxalate was determined. 19 TABLE 2 THE PERCENTAGE DECOMPOSITION OF OXALATE BY RUMEN CONTENT --Sample No. RS-1 RS-2 RS-3 RS-4 RS-5 RS-6 RS-7 RS-8 RS-9 RS- 10 RS- 11 RS- 12 Residue * Filtrate 'r % 3.6 1.3 0 6.7 0 9.3 4.3 0 0 3.1 10-2 5.6 % 19=8 23.7 18.9 17-6 28.2 51.4 36.7 4.3 3: 279 73.0 46e7 --- * All determinations are for total oxalate. ? Values quoted are means of duplicate determinations. I Micro-organisms similar to RO-16 could not be isolated from this sample. III. RESULTS v&o studies of the effect of high oxalate levels on growth of 26 strains of rumen bacteria failed to show any significant effect. Of the 26 strains investigated only one effectively decomposed oxalate. On isolation in pure culture this organism was designated RO-16. Preliminary studies have shown RO-16 to be a facultatively aerobic, gram negative, motile rod growing well at temperatures between 38-40�C. When incubated aerobically at 38OC in the medium of Quayle and Keech ( 1959) oxalate is completely decomposed within 48 hours. In It is apparent from the results in Table 2 that the residues of the rumen contents tested when compared with the corresponding filtrates were much more potent in the destruction of oxalate. The differences between residues and filtrates were highly significant (P < 0 00 1) . Thus it appears that the enzymatic principle is associated with the residues rather than the filtrates. In all cases (except RS-9) where decomposition of oxalate has occurred an organism morphologically and culturally similar to RO-16 has been isolated. The following tentative conclusions have been drawn from these studies:(i) High levels of oxalate had no apparent effect on the growth of 2 6 strains of rumen bacteria studied. (ii) A facultatively aerobic, gram negative, rod decomposing oxalate has been isolated (RO-16). 20 (iii) Incubation of the solid and fluid portions of rumen content demonstrated that the solid components were significantly more active in the decomposition of oxalate than the fluid. (iv) Rumen samples decomposing oxalate have all yielded an organism similar to RO-16. IV. ACKNOWLEDGEMENTS The author is indebted to Mr. K. J. Hutchinson and Dr. B. J. Christie for helpful discussion and criticism during the preparation of this manuscript. V. REFERENCES BAKER, C. J. L. (1952).-halyst 77: 340-344. DODSON, M. E. (1959).-Aust. Vet. J. 35: 225-233. MICZIAEL, P. W. (1959).-Aust. Vet. J. 35: 431-432. O' HALLORAN, M. W. (1962) .-In preparation. QI.L+YLE. J. K., and KEECH, D. B. (1959).-Biochem. J. 72: 623-630 TALAPATKA, S. K., RAY, S. C., and SEN, K. C. (1948).--J. Agric. Sci. 38: 163-173. WATTS, P. S. (1957).-Aust. J. Agric. Res. 8: 266-270. WATTS, P. S. (1959) .-J. Agric. Sci. 52: 250-255. 21