Enrichment, isolation and characterization of anaerobic oxalate-degrading bacteria from the rumen

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Iowa State University Capstones, Theses and
Retrospective Theses and Dissertations
Dissertations
1979
Enrichment, isolation and characterization of
anaerobic oxalate-degrading bacteria from the
rumen
Karl A. Dawson
Iowa State University
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Recommended Citation
Dawson, Karl A., "Enrichment, isolation and characterization of anaerobic oxalate-degrading bacteria from the rumen" (1979).
Retrospective Theses and Dissertations. 6639.
https://lib.dr.iastate.edu/rtd/6639
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DAWSON# KARL A,
ENRICHMENT, ISOLATION AND CHARACTERIZATION OF
ANAEROBIC, OXALATE-OEGRAOIN6 BACTERIA FROM
THE RUMEN,
IOWA STATE UNIVERSITY, PH.D, 1979
Universi^
A/licrpfilms
intenTctfKXVll 300N. ZEES ROAD, ANN ARBOR, MI 48IO6
Enrichment, isolation and characterization of anaerobic,
oxalate-degrading bacteria from the rumen
by
Karl A. Dawson
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements of the Degree of
DOCTOR OF PHILOSOPHY
Major: Bacteriology
Approved :
Signature was redacted for privacy.
In Charge of Major Work
Signature was redacted for privacy.
For the Major Department
Signature was redacted for privacy.
For the Graduate College
Iowa State University
Ames, Iowa
1979
ii
TABLE OF CONTENTS
Page
INTRODUCTION 1
MATERIALS AND METHODS 13
Inocula 13
Organisms 13
Media 13
Serum bottle cultures 18
Qualitative test for oxalate 19
Organic acid concentrations 19
Gas analysis 20
Enrichment from the fermenter 20
Growth conditions and maintenance of cultures 20
Isolations 21
Growth experiments 21
Oxalate degradation rates 22
Chemostat cultures 23
Dry weight determinations 25
1 A
Distribution of C label in end products 26
RESULTS 28
Primary enrichment cultures 28
Methanogenic oxalate-degrading populations 32
Enrichment in continuous culture 42
Uncoupled population 47
Effects of the oxalate concentration 48
iii
Page
Enrichment in the coliform-depleted medium 51
Isolation of an oxalate degrader 54
Colony morphology of the isolates 54
Morphology of the isolates 55
Storage of the oxalate-degrading isolates 55
Characteristics of strain OxB 55
Minimal medium 56
Dry weight yields of OxB 61
End products from strain OxB 61
Distribution of label in end products 65
Substrate range of OxB 67
Antibiotic susceptibility 69
DISCUSSION 71
LITERATURE CITED 85
ACKNOWLEDGMENTS 92
1
INTRODUCTION
Plants from many different parts of the world are known to contain
exceptionally high concentrations of oxalic acid. In most instances,
these plants are of little consequence to man. However, livestock
grazing in areas where these plants are the predominant flora can in­
gest large amounts of oxalic acid and are subject to oxalate intoxication.
Many plants which grow on the cold desert ranges of the western
United States contain potentially toxic levels of oxalic acid (James and
Cronin, 1974; James, Williams and Bleak, 1976). One of these plants,
halogeton (Halogeton glometratus), is probably the most frequent cause
of oxalate intoxication in range animals (James and Cronin, 1974).
Halogeton is not part of the natural vegetation of the west, but is
well-adapted to the arid desert environment and quickly establishes it­
self in areas where the natural vegetation has been disturbed. As a
result, grazing animals may be exposed to areas containing dense popu­
lations of this weed. Acute oxalate intoxication and death can occur
after animals have ingested exceptionally large amounts of halogeton.
Chronic oxalate poisoning does not appear to be a problem in areas where
this plant grows (James and Butcher, 1972). Losses due to deaths from
oxalate intoxication are usually small, but occasional large scale in­
toxications have been reported (James, 1972; Wadsworth, 1971).
Another plant, known as soursob (of the Oxalis genus), dominates
certain grazing areas of South Australia (Watts, 1957; Dodson, 1959).
These plants are quite palatable and also contain exceptionally high
levels of oxalate. Ingestion of these plants by sheep results in many
2
acute poisonings and deaths in sheep each year (Dodson, 1959; Smith,
1951). In contrast to halogeton, investigators have reported problems
with chronic poisoning on soursob pastures (Mcintosh, 1972; Smith, 1951).
In these cases, the animals appear normal, but may collapse and die when
placed under stress.
Many of the toxic effects of oxalate can be explained by the forma­
tion of calcium oxalate crystals. These crystals can interfere with
calcium absorption, deplete blood calcium levels and damage the kidney,
rumen wall and other organs (James, 1972). After ingesting large amounts
of oxalate, animals rapidly go into shock, followed closely by a coma
and death. In chronic intoxication, there is a gradual deterioration of
the body, accompanied by stiff legs and a nasal discharge (Smith, 1951;
James, 1972). However, the problem of oxalate poisoning is far from
being totally understood because oxalates also have been shown to influ­
ence cellulose degradation in the rumen, rumen pH, andb lood urea nitrogen
levels (James, Street and Butcher, 1967; James and Butcher, 1972;
Talapatra, Ray and Sen, 1948; Singh et al., 1975). This has led some in­
vestigators (James et al., 1967) to believe that rumen dysfunction may be
as important as altered calcium metabolism and kidney damage in contributing
to the death of the animal. The severity of the poisoning thus reflects
such factors as water status, nutritionals tatus, and the amount of oxalate
ingested. The fact that oxalate intoxication is not completely understood
makes treatment difficult. Although some investigators have suggested
that calcium supplementation may reduce acute oxalate intoxications
(James et al., 1967), the most successful approach to decreasing losses
3
has been to control oxalate intake through management (James and Cronin,
1974).
It has long been known that after extended periods of oxalate in­
gestion, animals become more resistant to the toxic effects of this acid.
Although some attempts have been made to attribute this adaptation to
physiological changes in the animal (Nicolaysen, Eeg-Larsen and Malm,
1953), most of the current evidence suggests that resistance is caused
by increased rates of oxalate degradation by bacteria in the gastro­
intestinal tract of these animals.
The first qualitative evidence of such oxalate degradation in the
rumen was presented by Talapatra and his associates (1948) as a result
of calcium assimilation studies in cattle. These studies showed that
ingested soluble oxalates were not recovered in the blood, urine or
feces, but did alter the calcium balance and urinary carbonate levels.
These authors suggested that oxalate salts are broken down by the ruminai
flora before they enter the stomach. Morris and Garcia-Rivera (1955)
provided the first direct quantitative evidence for ruminai oxalate deg­
radation. In these in vitro experiments, oxalate concentrations decreased
when fresh rumen ingesta was incubated with sodium oxalate.
No attempts were made to correlate oxalate degradation in the rumen
with diet until Watts (1957) demonstrated that the ability of rumen con­
tents to degrade this acid was greater in sheep which had been adapted by
regularly adding small quantities of oxalic acid to the rumen. In a more
comprehensive study, Dodson (1959) also showed that the rumen is re­
sponsible for detoxifying oxalate. This study correlated the ability to
degrade oxalate with the seasonal changes in the sheep pastures, since
4
oxalate degradation was most complete during periods when the oxalate-
containing plant, soursob, was predominant in the grazing area. Dodson
suggested that a microorganism associated with oxalate-containing plants
was responsible for ruminai oxalate degradation, because degradation was
greatest when these plants were ingested. James and his associates (1967)
demonstrated similar ruminai changes in sheep fed halogeton. The in
vitro degradation of oxalate by rumen ingesta was significantly increased
after sheep were fed halogeton.
More recent studies by Allison and his associates (1977) have used
the production of carbon dioxide from ^^C-labeled oxalate to measure the
in vitro rates of oxalate degradation in rumen contents. Measurable
amounts of oxalate were degraded by rumen contents from sheep and cattle
maintained on an alfalfa diet. The addition of halogeton to these diets
resulted in a 12-fold increase in the ruminai oxalate degradation rate.
In examining the rumen contents of these adapted animals, these investi­
gators found that oxalate degradation was associated with theb acterial
fraction obtained during centrifugation and that oxalate degradation was
greatest under oxygen-free conditions. These studies were consistent with
the hypothesis that adaptation to oxalate was due to the selection of an
anaerobic, oxalate-degrading population from microbes present in the
normal rumen population and not to the introduction of new oxalate-
degrading organisms, as was suggested by Dodson (1959).
The proposal that changes in the bacterial populations in ther umen
are responsible for the increased rates of oxalate degradation in adapted
animals is supported by studies with vitro cultures of rumen bacteria
(unpublished work by M. J. Allison and H. M. Cook). These investigators