ANTEBEQTEC TR RWY-INT OF
LAKE SEDEMEN'E'S '5'0. DETERMENE THE
EFFE T 0F FURG! 0N DECOMPOSITEON

Thesis for the Degree of M. S.
MECl-HGAN STATE UNWERSUY
STANLEY LEWES FLEGLER
1972

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ABSTRACT

ANTIBIOTIC TREATMENT OF LAKE SEDIMENTS
TO DETERMINE THE EFFECT OF
FUNGI ON DECOMPOSITION

BY

Stanley Lewis Flegler

To determine the role of fungi in decomposition I treated
sediment from a eutrOphic lake and a bog with antibacterial anti-
biotics or with antifungal antibiotics.

Sediment treated with antibacterial antibiotics showed a de-
crease in biochemical oxygen demand and a decrease in direct and
pour-plate bacterial counts when compared with controls.

Sediment treated with antifungal antibiotics showed an in-
crease in biochemical oxygen demand and an increase in pour-plate
and direct bacterial counts when compared with controls.

Several species of endemic lake fungi inhibited the growth
of a mixed culture of lake bacteria on agar plates.

The results infer that fungi inhibit the growth of bacteria

in aerobic lake sediment.

ANTIBIOTIC TREATMENT OF LAKE SEDIMENTS
TO DETERMINE THE EFFECT OF

FUNGI ON DECOMPOSITION

BY

Stanley Lewis Flegler

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1972

ACKNOWLEDGEMENTS

I wish to thank Dr. Clarence McNabb for serving as my major
professor, for giving guidance and direction to my research, and for
helping in the preparation of the final manuscript.

I wish to thank Dr. Niles Kevern for first mentioning fungal
decomposition as a research area and for helping to set up my graduate
program within the Fisheries and Wildlife Department.

I would especially like to thank Dr. William Fields for his
excellent instruction which first interested me in the fungi, for
providing laboratory and office facilities, and especially for the
advice and help with the multitude of small problems encountered in

this research project.

ii

Introduction .

Methods and materials

Results . . .

Discussion .

Literature cited

TABLE

OF

iii

CONTENTS

Page

11

14

LIST OF TABLES

Table

1. Mean BOD of dilution water plus antibiotics and
of controls with only dilution water . . .

2. Mean BOD of lake water treated with antibiotics
and of controls with no antibiotics . . .

3. Mean BOD and mean bacterial counts of Rose Lake

Bog water with antibiotics added and of
controls with no antibiotics added . . . .

iv

Page

LIST OF FIGURES

Figure Page

1. Inhibition of a mixed Species culture of Lake
Lansing bacteria by a fungus, A5pergillus s23,
recovered from Lake Lansing . . . . . . . . . 10

 

INTRODUCTION

In 1941 the renowned aquatic mycologist William H. Weston
wrote an excellent monograph (Weston, 1941) summarizing the knowledge
of the role of fungi in the aquatic ecosystem. Professor Weston
stated that the fungi are ubiquitous in waters, capable of surVival
in almost all conditions, and are able to break down and metabolize
a wide variety of substrates. It would seem that the fungi should
possess an excellent capability as decomposers. Yet in 1941, due to
lack of data about fungi, their role in decomposition was usually
ignored. Professor Weston believed that with further study the im-
portance of fungi would become evident. In the thirty years since
Weston's paper, there have been only a few papers on the role of fungi
in decomposition in aquatic habitats and our lack of knowledge is
still considerable. This paper attempts to add to our knowledge in
this area.

A major block to our understanding of decomposition by fungi
has been the lack of technique. How do you determine the role of
fungi in decomposition? I used antibiotics to selectively inhibit
either bacteria or fungi. As a criterion of decomposition I measured
biochemical oxygen demand (BOD) on the assumption that BOD is directly

related to rate of decomposition.

METHODS AND MATERIALS

I collected water samples from Rose Lake Bog, located in
section 26 TSN R1W and Lake Lansing, a eutrOphic lake, located in
sections 2,3,10, and 11 TSN R1W. Locations are referenced to the
Michigan meridian and base line. In each sampling, I disturbed the
bottom sediment in two feet of water, placed a bottle near the bottom
and allowed it to fill. I used the water within one hour after col-
lecting it.

To inhibit the growth of fungi, I added the antibiotics
nystatin and cycloheximide to water samples in biochemical oxygen
demand (BOD) bottles at a concentration of 50 ug/ml of each anti-
biotic. Williams and Davies (1965), testing a wide variety of soil
fungi, found that nystatin and cycloheximide (50 ug/ml of each) gave
total inhibition of 84% and partial inhibition of 16% of the species
tested.

To inhibit the growth of bacteria, I added penicillin and
streptomycin to water samples in BOD bottles at a concentration of
1.5 ug/ml of each antibiotic.

All four of the above antibiotics will inhibit growth in a
few species of algae and protozoans, however their main action is

against the fungi in the case of nystatin and cycloheximide or against

the bacteria in the case of penicillin and streptomycin (Gottlieb
and Shaw, 1967; Lampen g£_§l,, 1959).

I measured the BOD of the water samples with a five-day incu-
bation in standard BOD bottles at 20 C using procedures outlined in
Standard Methods for the Examination of Water and Wastewater (Anon.,
1971). I used the Alsterberg (Azide) modification of the Winkler
method for measuring dissolved oxygen (92: ElEs) with 0.0250 normal
phenylarsene oxide substituted for sodium thiosulfate.

I made pour-plate bacterial counts using plate count agar
(containing glucose, yeast—extract, and peptone) and a 24-hour incu-
bation at 35 C as described in Standard Methods for the Examination
of Water and Wastewater (Anon., 1971).

I made direct microsc0pic bacterial counts on 0.22 u pore-size
filters (Millipore Corp.) using procedures described by Jannasch (19581
For each filtered sample I counted the number of bacteria visible in
ten randomly-chosen fields of view and averaged this value to give the
final count.

To test the effectiveness of the antifungal antibiotics, I
diluted Lake Lansing water to a 60% concentration, poured it into
six BOD bottles, and incubated the bottles using the same procedures
as in a BOD analysis. I added antifungal antibiotics to three of the
bottles. I determined the fungi present initially and after incu-
bation by plating the water on half-strength corn meal agar (Difco
Laboratories) plates and by baiting the water with Split sterile

hemp seeds (Alex0poulos and Beneke, 1962).

To test for fungal inhibition of bacteria, I grew the fungal

species Saprolegnia ferax (Gruith) Thuret, Penicillium s23, Candida

 

 

g2}, ASpergillus sp,, and Trichoderma ER: on half-strength corn meal

 

 

agar (Difco Laboratories) plates. I obtained all these fungi from
Lake Lansing. For a mixed species bacterial suspension, I used the
filtrate from the filtration of Lake Lansing water through a 5 u pore-
size filter (Millipore Corp.). When the fungi showed good growth on
the agar plates, I streaked each plate with the bacterial suspension

and looked for a zone of bacterial inhibition around the fungi.

RESULTS

To determine if the antibiotics I was using could contribute
to the BOD of water samples, I added antibiotics to dilution water
and measured BOD. My results (Table 1) show that only nystatin con-
tributes to BOD. To compensate for BOD by nystatin, I added 0.6 ug/l
to the final dissolved oxygen reading in all experiments testing the
effect of nystatin on BOD of lake water.

I collected water from Lake Lansing and Rose Lake Bog and
made dilutions of 60% and 15% reSpectively. Measurements of oxygen
following incubation (Table 2) show that the BOD increased with both
antifungal antibiotics (nystatin and cycloheximide) and decreased
with antibacterial antibiotics (penicillin and streptomycin).

Next I took Rose Lake Bog water and diluted it to a 10% con-
centration. I used ten bottles for each set, five for BOD analysis
and five for bacterial counts. I treated the bottles for bacterial
counts the same as a BOD analysis. The results (Table 3) show that
BOD, direct bacterial counts, and pour-plate bacterial counts all
increased when antifungal antibiotics (nystatin and cycloheximide)
were added. The BOD, direct bacterial counts, and pour-plate
bacterial counts all decreased when antibacterial antibiotics (peni-
cillin and streptomycin) were added. The direct bacterial counts

were 20 to 30 times greater than pour-plate bacterial counts.

TABLE 1. Mean BOD of dilution water plus antibiotics and
of controls with only dilution water

 

 

Set BOD, mg/l
Control 0.1 (4)
Penicillin and streptomycin 0.1 (4)
Nystatin and cycloheximide 0.7 (4)
Nystatin 0.7 (2)
Cycloheximide 0.1 (2)

 

*The number of samples for each mean is shown in
parentheses; the standard deviation for each mean is zero.

TABLE 2. Mean BOD of lake water treated with antibiotics and of
controls with no antibioticsa (standard deviation shown)

 

 

 

BOD, mg/l
Set
Rose Lake Bog Lake Lansing
Control 7.9 2 0.5 3.6 i 0.1
Penicillin and streptomycin 6.5 i 0.6 2.7 i 0.0
Nystatin and cycloheximide 10 i 1.0 4.2 i 0.3

 

aEach mean is significantly different from other means in
the trial at the 0.005 level of confidence using the Students' t
test; each mean had five samples.

TABLE 3. Mean BOD and mean bacterial counts of Rose Lake Bog water
with antibiotics added and of controls with no antibiotics
addeda (standard deviation shown)

 

Bacterial counts

 

 

Set
BOD, mg/l Direct? x lOG/ml Pour-plate? x 105/ml
Control 29* i 1 3.9** i 0.4 0.97* i 0.14
Penicillin and
streptomycin 27* i l 2.2** i 0.4 0.73* i 0.09
Nystatin and
cycloheximide 34** i 1 10.4** i 0.3 5.5** i 1.0

 

aEach mean had five samples except the pour-plate bacteria
count with nystatin and cycloheximide which had four samples and both
initial bacterial counts which had three samples.

bThe initial count was 3.5 i 0.0 x 106/ml.

CThe initial count was 0.68 i 0.02 x 105/ml.

*
This mean is significantly different from other means in the

trial at the 0.05 level of confidence using the Students' t test.

**
This mean is significantly different from other means in the

trial at the 0.005 level of confidence using the Students' t test.

In the test of antifungal antibiotic effectiveness, I ob-

tained Candida s23, Penicillium EEx' Pythium EB)! and Saprolegnia

 

 

ferax (Gruith) Thuret initially in Lake Lansing water. After the
water was incubated without antibiotics, I obtained Candida s23,

Pythium s23, Cladosporium sp,, and Saprolegnia ferax (Gruith) Thuret.

 

I obtained no fungi from the water after incubation with the anti-
fungal antibiotics nystatin and cycloheximide.

In the test for fungal inhibition of endemic Lake Lansing
bacteria on agar plates, two endemic Lake Lansing fungal species
produced a zone of bacterial inhibition on the agar plate. Pepi:

cillium g2, and Aspergillus s2, (Figure 1) both produced well-defined

 

zones of inhibition.

10

 

FIGURE 1.

Inhibition of a mixed Species culture of Lake
Lansing bacteria by a fungus, Aspergillus s23,
recovered from Lake Lansing. The zone of
bacterial inhibition around the fungus is
indicated by an arrow.

 

 

DISCUSSION

The results of the antibiotic BOD experiments show that
nystatin contributes to dissolved oxygen depletion and thus has a
BOD of its own, possibly in the form of a chemical oxygen demand
(COD). Nystatin is a conjugated polyene molecule and is subject to
oxidation (Kinsky, 1967). I observed that the initial dissolved
oxygen concentration of a mixture containing nystatin was always
slightly lower than mixtures of other antibiotics or of the control.
Nystatin apparently exerts a COD immediately upon addition to the
sample. In regard to the use of this molecule as an organic sub-
strate for metabolism, Lampen g£_31, (1959), Lampen and Arnow (1959),
Kinsky (1962), and Shockman and Lampen (1962) showed that bacterial
growth is neither stimulated nor suppressed by nystatin. The bac-
terial cells will not significantly alter the nystatin concentrations
in culture media. On these grounds, a correction factor of 0.6 mg/l
is justified for the COD of nystatin added in these experiments.

Each of the direct bacterial counts I made were at least
an order of magnitude higher than the counts obtained by the pour-
plate method. This is due to several factors. The agar used can be
very selective and not all bacteria grow on any one type of agar

(McCoy and Sarles, 1969). There is often a clumping effect,

11

12

especially prevalent in waters with much particulate organic matter,
whereby many bacteria may be clumped together and show up as one
colony on a pour-plate count (Jannasch, 1958). Both of these factors
cause the direct method to give higher counts than the pour-plate
method. This difference is often in the order of ten times greater
(Jannasch, 1958).

The bacterial counts showed a decrease (approximately 40%)
when I added antibacterial antibiotics to the lake water. This degree
of suppression of bacteria is consistent with the observations of
Ivarson and Sowden (1959) who found that large concentrations (0.5
to 1.0%) of antibacterial antibiotics gave only partial inhibition of
bacteria in decomposing forest litter. Pennak (1968) found a decrease
in dissolved oxygen depletion similar to mine over a 30-day period
when he added antibacterial antibiotics to lake water. The results
indicate that bacteria make a definite contribution to BOD, for when
bacteria are suppressed, the BOD decreases.

When I added antifungal antibiotics to lake water, the fungi
decreased and the BOD and bacterial numbers increased. Neither
nystatin nor cycloheximide will stimulate bacterial growth (Lampen
and Arnow, 1959; Kinsky, 1962; Lampen et_al:, 1959; Shockman and
Lampen, 1962; Whiffen, 1948). Thus fungi apparently have an inhibi-
tory effect on bacteria in aerobic conditions for when fungi decrease
the bacteria increase and cause an increased BOD. Kaushik and Hynes
(1968) believed that fungi had an inhibitory effect on bacteria in de-
composing leaf litter in streams, although they had no direct evidence

to support this theory. Ivarson and Sowden (1959) reported an increase

13

in bacteria in decomposing forest litter treated with a 1% concen-
tration of the antifungal antibiotic cycloheximide. Saito (1958,
1960) has found inhibitory effects between various Species of bac-
teria and fungi, fungi and bacteria, and between fungi. The in-
hibition of a mixed species culture of bacteria from a lake by
non-selected Species of fungi from the same lake in my inhibition

experiment gives visual evidence that fungi can inhibit bacteria.

LITERATURE CITED

LITERATURE CITED

Alexopoulos, C. J., and E. S. Beneke. 1962. Laboratory manual for
introductory mycology. Burgess Publishing Co., Minneapolis,
Minn. 199p.

Anon. 1971. Standard methods for the examination of water and
wastewater. Thirteenth ed. American Public Health Assn.,
American Water Works Assn., Water Pollution Control
Federation. New York, N.Y. 874p.

Gottlieb, D., and P. D. Shaw. 1967. Antibiotics. Springer-Verlag.
New York, N.Y. 2v.

Ivarson, K. C., and F. J. Sowden. 1959. Decomposition of forest
litters. II. Production of ammonia and nitrate nitrogen,
changes in microbial population and rate of decomposition.
Pl. Soil. 11:237-248.

Jannasch, H. W. 1958. Studies of planktonic bacteria by means of a
direct membrane filter method. J. Gen. Microbiol. 18:609-
620.

Kaushik, N. K., and H. B. N. Hynes. 1968. Experimental study of the
role of autumn shed leaves in aquatic environments. J.
Ecol. 56:229-243.

Kinsky, S. C. 1962. Nystatin binding by protoplasts and a particulate
fraction of Neurospora crassa, and a basis for the selective
toxicity of polyene antifungal antibiotics. Proc. Natl. Acad.
Sci. U. S. 48:1049-1056.

 

Kinsky, S. C. 1967. Polyene antibiotics, p. 122-141. In D. Gottlieb
and P. D. Shaw, Antibiotics. Vol. 1. Mechanism of action.
Springer-Verlag. New York, N. Y.

Lampen, J. O., and P. Arnow. 1959. Significance of nystatin uptake
for its antifungal action. Proc. Soc. Exptl. Biol. Med.
101:792-797.

Lampen, J. 0., E. R. Morgan, A. Slocum, and P. Arnow. 1959. Absorp-

tion of nystatin by microorganisms. J. Bacteriol. 78:282-
289.

14

15

McCoy, E., and W. B. Sarles. 1969. Bacteria in lakes: populations
and functional relations, pp. 331-339. In_Eutrophication:
causes, consequences, correctives (proceedings of a symposium).
National Academy of Sciences. Washington, D. C.

Pennak, R. W. 1968. Field and experimental winter limnology of
three Colorado mountain lakes. Ecology 49:505-520.

Saito, T. 1958. The characteristic features of fungi taking part
in the decomposition of beech litter. Sci. Rep. Tokoku
Univ. Ser. IV (Biology) 24:73-79.

Saito, T. 1960. An approach to the mechanism of microbial decompo-
sition of beech litter. Sci. Rep. Tohoku Univ. Ser. IV
(Biology) 26:125-131.

Shockman, G., and J. O. Lampen. 1962. Inhibition by antibiotics of
the growth of bacterial and yeast protOplasts. J. Bacteriol.
84:508-512.

Weston, W. H. 1941. The role of the aquatic fungi in hydrobiology.
pp. 129-151. £2_A symposium on hydrobiology. Univ. of
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Whiffen, A. J. 1948. The production, assay, and antibiotic activity
of actidione, an antibiotic from Streptomycesjgriseus. J.
Bacteriol. 56:283-291.

Williams, S. T., and F. L. Davies. 1965. Use of antibiotics for
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