ABSTRACT

NITROGEN FIXATION AND PRODUCTIVITY IN A
EUTROPHIC HARD-WATER LAKE: IN_SITU
AND LABORATORY STUDIES

BY

Tran Phuoc Duong

Nitrogen fixing activity by planktonic organisms in
a shallow eutrophic hard-water lake in southwest Michigan
was measured at midday each week during the summer of
1970 and 1971 (July-September, 1970 and June-August, 1971)
using the acetylene reduction technique. Samples were
obtained from O, 1, 2, and 3 m and incubated with
acetylene at their original depth for 1 hr. Acetylene
reducing activity was confined to the aerobic 0-3 m zone

where heterocyst-bearing Aphanizomenon and Anabaena were

 

dominant in 1970 and 1971, respectively. Acetylene re-
duction was not detectable in the 4-5 m anaerobic zone

where Thiopedia and green sulfur bacteria dominated the

 

bacterial population. The population of Aphanizomenon

3

was more dense (3,197 x 10 filaments/l) than Anabaena

(355 x 103 filaments/l) but less efficient in reducing

9

acetylene (3 x 10- and 2 x 10-7 umole/filament/hr,

respectively.

Tran Phuoc Duong

Both algae reduced significant amounts of acetylene
at night with Aphanizomenon reducing more at night than
during the day. The peak activity during the day occurred
the third week of July both years.

From the weekly studies of acetylene reduction, it
was estimated that reduction in the water column to 3 m was
approximately 3,000 umole acetylene/mz/day in 1971 assuming
a fixation period of 12 hr. Using the conversion ratio 3
ethylene formed to 2 ammonia produced, this represents an
estimated quantity of 2 g/m2 ammonia produced during the
1971 bloom period.

An association between the blue-green algae
Anabaena, Calothrix, and Dichothrix and the duckweeds
Spirodela and 22mg; was shown to be active in reducing
acetylene. Dissection and microsc0py revealed that
microcolonies of the above algae were located in the
lower mesophyll and in the pockets of the duckweeds. The
acetylene reducing activity was associated with the fronds
(leaves, rather with the roots) of the plants and was light
dependent. The association reduced an average of 63 mumole

acetylene/g wet wt/hr when the assay was conducted in the

lake.

Weekly primary productivity and excretion of
organic carbon were also measured at 1 m intervals from

June to October, 1971 using 14

C methods. Net primary
productivity (light minus dark CO2 uptake) ranged from

300.0 to 2,752.5 mg C/mz/day, being mostly due to the

Tran Phuoc Duong

activity of blue-green algae which dominated the aerobic

zone (0-2 m). Dark CO2 uptake averaged 322.4 mg C/mZ/day
or 21.4% of net primary productivity. Total excretion
of newly fixed carbon was about 3.5% of the net primary
productivity. The excretion was highest in the aerobic
zone.

Strains of Rhodopseudomonas and Rhodomicrobium
isolated from this lake grew well on a variety of organic
carbon compounds such as organic acids, alcohols, carbo-
hydrates, and carbohydrate polymers in nitrogen-free

medium. Rhodomicrobium produced angular structures

 

similar to cysts in old cultures. The growth and

acetylene reduction by Rhodomicrobium were optimum at

 

30 C under 300 ft-c. Acetylene reduction by this
organism was suppressed by as low as 0.0005% combined
nitrogen and the induction of the acetylene reducing
activity was higher in argon or helium than in nitrogen
atmospheres.

in gig! acetate uptake was highest at 3 m where
Thiopedia and green sulfur bacteria represented 90% of
the bacterial population. In the laboratory, a 10 ml sample
of the natural pOpulation of these bacteria photodegraded to
C02 about 2.4% of 100 ug of uniformly labeled acetate/hr at

29 c under 600 ft-c.

Tran Phuoc Duong

in situ and laboratory data suggest that photo-
synthetic bacteria are capable of participating in all
steps of degradation of organic carbon but were not im-

portant nitrogen fixers in Wintergreen Lake.

NITROGEN FIXATION AND PRODUCTIVITY IN A
EUTROPHIC HARD-WATER LAKE: IN SITU

AND LABORATORY STUDIES

BY

Tran Phuoc Duong

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Crop and Soil Sciences

1972

"I \
\‘R
\

’ \

To the Vietnamese countryman to whom

I am in debt

ii

To my fathers and mothers
To my wife, Dai, Dan, Dong, and Dien
To professor Pham hoang Ho who inspires me

iii

ACKNOWLEDGMENTS

I am deeply thankful to Dr. J. M. Tiedje for his
interest and guidance during the course of this study.
The freedom he gave in the selection and execution of
research and his unlimited help in the research as well
as the preparation of this manuscript are appreciated.

Special thanks are due to Dr. A. R. Wolcott for
his guidance and hospitality particularly in the early
stage of the study.

I deeply appreciate Dr. G. H. Lauff, director of
the W. K. Kellogg Biological Station for permission to
use the research facilities of the station and for his
encouragement. I am also grateful to Dr. R. G. Wetzel
of the W. K. Kellogg Biological Station for allowing me
to use his research facilities, space and his cooperation
in the productivity measurements.

I am thankful to: Dr. M. J. Klug and Dr. J. L.
Lockwood for serving on my Guidance Committee, Dr. B. A.
Manny for providing his unpublished data of phosphorus,
ammonium, and nitrate for 1970-1971 from Wintergreen
Lake, and Mr. R. E. VanDeusen for permission to study

Wintergreen Lake.

iv

Special thanks are due to Miss Phung thi Nguyet
Hong and Mrs. Donna King for their patience and continuous
help throughout the study, Mrs. Mary Firestone for editing
and typing the manuscript, Mr. Douglas Caldwell and Miss
Sarah Chapman for helping with part of the photographic
and electron microsc0pe work, and my two cheerful friends:
Mr. Vu quoc Thuy and Miss Le thi Xuan.

Financial supports were provided by a scholarship
from the Agency for International Development No. 730-20121
to Tran Phuoc Duong, OWRR Grant No. 14-31-0001-3222
(A—046-Mich.) and USDA Regional Project NE-39 to Dr.
J. M. Tiedje. Use of the Michigan State University com-
puting facilities was made possible through support, in

part, from the National science Foundation.

TABLE OF CONTENTS

Chapter Page
I. INTRODUCTION . . . . . . . . . . . . 1
II. THE LAKE . . . . . . . . . . . . . 2

A. Location . . . . . . . . . . 2
B. Description of the Lake . . . . . . . 2

l. Topography . . . . . . . . . . 2
2. Morphometry. . . . . . . . . . 7

C. Climate . . . . . . . . . . . . 7
III. HYDROGRAPHY . . . . . . . . . . . . 10

A. Materials and Methods. . . . . . . . 10
B. Results and Discussion . . . . . . . ll

1. Temperature. . . . . . . . . . ll
2. Light and Water Transparency . . . . ll
3. Dissolved Oxygen . . . . . . . . l9
4. Total Alkalinity . . . . . . . . 25
5. pH. . . . . . . . . . . . . 25
6. Inorganic Elements . . . . . . 25

IV C THE BIOTA . C O O O O O O O O C O O 3 6

A. Introduction. . . . . . . . . . . 36
Materials and Methods. . . . . . . . 36

ED

1. Algal Enumerations . . . . . . 36

2. Photosynthetic Pigments. . . . . . 37

3. Multivariate Statistical Analysis . . 37

C. Results and Discussion . . . . . . . 39

V. PRIMARY PRODUCTIVITY . . . . . . . . . 50

A. Introduction . . . . . . . . . . 50
B. Definition 0 o o o o o o o O O O 50

vi

Chapter

VI.

VII;

VIII.

C. Materials and Methods . . . . . .
D. Results and Discussion. . . . . .

'EN SITU NITROGEN FIXATION . . . . . .

 

A. Introduction . . . . . .
B. Materials and Methods In Situ . . .
C. Results and Discussion. . . . . .

 

1. Combined Nitrogen Sources . . .
2. Fixation of Atmospheric Nitrogen .

(a) Heterocyst and Filament Den-
sities . . .
(b) Available Light .
(c) Phosphorous. . . .
(d) Oxygen . . .
(e) Combined Nitrogen.
(f) Other Factors . .
(g) Diurnal Acetylene Reduction

ACETYLENE REDUCTION BY NITROGEN-FIXING
ORGANISMS ASSOCIATED WITH DUCKWEEDS. . .

A. Introduction . . . . . . . .
B. Materials and Methods . . . . . .
C Q Results 0 O I O O O O O O O

. Composition of Duckweeds. .
. Distribution of Duckweeds .
. Nitrogen Fixation . . . .
. Anatomy Study . . . . .

DWNH
00.0

D. Discussion . . . . . . . . .
PHOTOSYNTHETIC BACTERIA. . . . . . .

A. Introduction . . . . . . . .
B. Materials and Methods . . . . . .

. van Niel's Modified Medium . . .
. Medium "98" . . . . . . . .
. Isolation . . . . . . .
. Preparations for Electron Micro-
scope Studies . . . . . . .
Anaerobic Gas Flushing System . .
Photometabolism of Rhodopseudomonas

1
2
3
4

mm

 

and Rhodomicrobium. . . . . .
7. Acetylene RedUctIOn Assay . . .

 

vii

Page

51
53

80

80
81
87

87
89

98
100
105
108
108
112
112

133

133
134
136

136
136
137
139

142
149

149
151

151
151
152

154
155

158
159

Chapter

C.

Results and Discussion . . . . . .

1.
2.

3
4
5

Enrichment and Isolation . . .
Some Characteristics of the Isolated
Rhodomicrobium . . . . . . .
Photometabolism of Organic Compounds
Acetylene Reduction. . . . . .
Conclusion. . . . . . . . .

IX. PHOTOMETABOLISM BY NATURAL POPULATIONS OF
PHOTOSYNTHETIC BACTERIA . . . . . . .

A.
B.
C.

REFERENCES.

Introduction . . . . . . . .
Materials and Methods . . . . . .
Results and Discussion . . . . . .

viii

Page

166
166
168
173
175
203
206
206
206
210

221

10b.

11a.

11b.
12.

13.

LIST OF TABLES

Morphometric Parameters of Wintergreen Lake .

Diurnal Distribution of Temperature and pH
on 4-5/8 1970 o o o o o o o o o o

Conductivity (u ohms) of Wintergreen Lake
Water During the Summer of 1971 . . . .

Annual Range in Concentrations of Various
Chemical Components in Wintergreen Lake
water from 1 m in 1970-71 . . . . . .

Total Algae Population (Filaments + Colonies/
l x 10 ) During the Summer of 1971 . . .

Multivariate Statistical Analysis . . . .
Multivariate Statistical Analysis . . . .
Multivariate Statistical Analysis . . . .
Multivariate Statistical Analysis . . . .
Distribution of Net Primary Productivity in
Wintergreen Water Column During the Summer
of 1971 (mg C/m3/day) . . . . . . .
Chemosynthesis (Dark Bottle, mg C/m3/day). .
Distribution of Rate of Organic Excretions
in Wintergreen Lake During the Summer of
1971 (Transparent Bottle, mg C/m3/day) . .
Dark Excretion (Dark Bottle, mg C/m3/day). .

Multivariate Statistical Analysis . . . .

Multivariate Statistical Analysis . . . .

ix

Page

14

26

35

41
46
47
6O

64

66

66

69
69
77

97

Table Page

14. Diurnal Variation of Rate of Ethylene Produced
per Heterocyst . . . . . . . . . . . 99

15a. Effect of Light, Temperature on Rate of
Acetylene Reduction by a Natural Population
of Blue-Green Algae Obtained at Midnight . . 104

15b. Effect of Added Phosphorous on Rate of
Acetylene Reduction . . . . . . . . . 104

16a. Ig_Situ Rate of Acetylene Reduction by Whole
PIants of Lemna and Spirodela Collected from
Wintergreen LaEe in 1971’ . . . . . . . 138

 

16b. Effect of Plant Part and Light on In Situ
Acetylene Reduction by Plants of_Lemna and
Spirodela (Samples taken from Wintergreen
Lake on51/9/7l). . . . . . . . . . . 138

17. Growth of Rhodomicrobium and Rhodopseudomonas
on a Variety Of Organic Compounds. . . . . 174

 

18. Effect of Light and Temperature on Growth and
Rate of Acetylene Reduction by Rhodomicro-
bium O O O O I O O O O O O C O O 187

 

 

19. Effect of Light Quality on Acetylene
Reduction by Rhodomicrobium. . . . . . . 202

 

20. Effect of Wavelength on Growth and Acetylene
Reduction by Rhodomicrobium. . . . . . . 204

 

LIST OF FIGURES

Figure Page

1. Bathymetric Map of Wintergreen Lake Showing
Location of Sampling Stations A and B
Used in this study (Figure courtesy of
Dr. B. A. Manny) . . . . . . . . . . 4

2. Soil Map of the Wintergreen Lake Watershed
Area 0 O O O O O O O I O O O O 0 6

3. Distribution of Temperature in Wintergreen
Lake in the Summer of 1970 and 1971 (A) and
(B): Air Temperature 0.5 m Above Water . . l3

4. Percentage of Total Surface Light at Various
Depths in the Water Column During the Summer
Of 1971 O O O O O O O I O O O O I 16

5. Light Transmittance in the Water Column of
Wintergreen Lake in July, 1971 . . . . . 18

6. Dissolved Oxygen Concentration (mg/1) Iso-
pleths During the Summer of 1970 and 1971. . 21

7. Oxygen Concentration (mg/l) a; Rate of Change
in Oxygen Concentration (mg Oz/l/hr) b; and
Temperature (C) c during the Diurnal Samp-
ling Period of 27-28/7/71 . . . . . . . 24

8. Water Transparency, Water Level, and Alka-
linity O O O O O I O O O O I O O 28

9. pH of Wintergreen Lake Water at Various Depths
During the Summer of 1970 and 1971 . . . . 30

10. The Concentration (mg/l) of Dissolved Phos-
phorous (A), Nitrate (B), and Ammonium (C) in
Wintergreen Lake Water from March, 1970, to
October, 1971 . . . . . . . . . . . 33

xi

Figure Page

11. Photographs of Representative Algae in
Wintergreen Lake . . . . . . . . . . 43

12. The Concentration ( g/l) of Chlorophyll a
(O ) and Carotenoids (O ) at 0 m (A), l m
(B), 2 m (C), and 3 m (D) in Wintergreen Lake
During the Summer of 1971 . . . . . . . 45

13. Net Primary Productivity at 0 m, 0.5 m (A);
lm,2m(B);3m,4m,and5m(C) in
Wintergreen Lake During the Summer of 1971 . 57

14. The Distribution with Depth of Chlorophyll a
(Hg/l), (A); Net Primary Productivity —
(mg C/l/day), (B); and Net Primary Pro-
ductivity Per Unit Chlorophyll a (mg C/mg
chl a/day), (C) During the SummEr of 1971 . 62

15. The Density of Aphanizomenon plus Anabaena
Populations (FiIaments/l x 105, Q ) and
Total Heterocysts (numbers/l x 105, O )
at 0m (A), 1 m (B), 2m (C), and 3m
(D) During the Summer of 1971. . . . . . 72

 

16. Production of 1971 . . . . . . . . . . 74

17. Procedure for In Situ Acetylene Reduction
Assay in the Lake. . . . . . . . . . 84

18. Rates of Acetylene Reduction at 0, l, 2, and
3 m in Wintergreen Lake in 1970 When
Aphanizomenon was Dominant. . . . . . . 91

 

19. Rates of Acetylene Reduction at 0, 1, 2, and
3 m in Wintergreen Lake in 1971 When
Anabaena was Dominant . . . . . . . . 93

20. Density (Numbers/1 x 105) of the Aphanizomenon
Filaments (O ) and Heterocysts ( QTTn
1970 at 0 m (A), l m (B), 2 m (C), and 3 m
(D) . . . . . . . . . . . . . . 96

 

21. Distribution with Depth and Date in 1971 of
the Following: (A) Heterocystous Algae
(Filaments/l x 105); (B) Heterocysts (Number/
1 x 105); (C) Net Primary Productivity (mg
C/l/day); (D) Rate of Acetylene Reduction
(mu mole/l/hr x 102). . . . . . . . . 102

xii

Figure Page

22. Diurnal Rates of Acetylene Reduction (Left)
and A hanizomenon Filament Densities
(Right) During the Diurnal Sampling
Period of 4-5/8/70 . . . . . . . . . 114

 

23. Densities of Aphanizomenon Heterocysts During
the Diurnal SampIing Period of 4-5/8/70 . . 116

 

24. Diurnal Solar Radiation Received (Relative
Units, Top), Rates of Acetylene Reduction,
Aphanizomenon Plus Anabaena Population
Densities, and Total Heterocyst Densities
During the Diurnal Sampling Period of
5-6/7/71 . . . . . . . . . . . . 119

 

 

25. Diurnal Rates of Acetylene Reduction at l m
During the Diurnal Sampling Period of
27-28/7/71 When Anabaena was Dominant. . . 122

 

26. Densities of Anabaena Filaments (l),
Anabaena Heterocysts (2), and Microcystis
Colonies (3) at 1 m During the Diurnal
Sampling Period of 27-28/7/71 . . . . . 124

 

 

27. Recovery of Acetylene Reduction Ability in a
Light-Starved Culture of Aphanizomenon . . 128

 

28. Recovery of Acetylene Reduction Ability in a
Light-Starved Anabaena Taken from Winter-
green Lake at 1:00 AM and Kept in the Dark
for Approximately 17 hr . . . . . . . 130

29. Effect of Light and Temperature on the Rate
of Acetylene Reduction by Natural Samples
of Lemna and Spirodela Incubated in the
Laboratory. . . . . . . . . . . . 141

 

30. Algae Associated with Lemna and Spirodela . . 144

 

31. Continuous Culture of Rhodomicrobium. . . . 157

 

32. Standard Curve Relating Optical Density at
680 mu of Culture to Dry Weight of
RhOdomicrObium C O O O O O O C O O 165

 

33. Isolated Photosynthetic Bacteria . . . . . 170

xiii

Figure

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

Acetylene Reduction by Rhodomicrobium Versus

 

Acetylene Concentration (left), Time
Course of Ethylene Production (right),
Both in Assay Bottles with Argon as the

Dominant Head-Space Gas . . .

Effect of a Dark Period on the Rate of
Acetylene Reduction ( O ) and Growth ( O )

by Rhodomicrobium . . . . .

 

Rates of Acetylene Reduction (0 )

and Growth

( O ) Observed in a Rhodomicrobium Culture.

 

Effect of Light and Temperature on

the Rate

of Acetylene Reduction by Rhodomicrobium .

 

Effect of Different Concentrations
Extract on the Rate of Acetylene
by Rhodomicrobium . . . . .

 

Effect of Different Concentrations
Nitrate on the Rate of Acetylene
by Rhodomicrobium . . . . .

 

Effect of Different Concentrations
Ammonium Chloride on the Rate of

of Yeast
Reduction

of Sodium
Reduction

of

Acetylene Reduction by Rhodomicrobium . .

 

The Induction of Nitrogenase Synthesis by
Argon or Nitrogenase in Rhodomicrobium

 

(Ethylene produced by Rhodomicrobium cells

 

Following Transfer from a Combined-Nitrogen
Medium to a Nitrogen-Free Medium-Grown
Cells of Rhodomicrobium was Incubated

Under an Argon or Nitrogen Atmosphere . .

 

Suggested Life Cycle of Rhodomicrobium . .

 

Absorption Spectra of Photosynthetic Pig-
ments from Natural Populations Extracted
by a Mixture of Acetone and Methanol

(7:2, v/V) . . . . . . .

14C -Acetate Assimilated in 30 min by 10 m1
oi the Natural Microbial Population (105

Cpm = approx. 1.6 ug Acetate) .

xiv

Page

178

180

183

190

193

195

197

200

205

213

215

Figure Page

45. Effect of Light and Temperature on Minerali-
zation of Added Acetate by a Natural Popu-
lation of Photosynthetic Bacteria Taken
from 3.5 m in Wintergreen Lake on 10/8/71
(ug Acetate Degraded/ 10 m1 Sample/hr) . . . 218

XV

I . INTRODUCTION

Photosynthetic phytOplankton and photosynthetic
bacteria constitute a great part of the photosynthetic
producing capacity in lakes (Hutchinson, 1967; Kondrat'eva,
1963; Wetzel, 1964; Culver and Brunskill, 1969). These
plankton serve as food for zooplankton and bottom fauna
which in turn are devoured by fish and so on through the
food chain (Prowse, 1955; Kondrat'eva, 1963).

The objective of this study was to evaluate the
productive capacity of the planktonic organisms in a
eutrophic lake. The work focused mainly on the primary
productivity, atmospheric nitrogen fixation, and the major
limnological parameters which affect these activities.

The studies were done both in_§itu_and under laboratory

conditions.

II. THE LAKE

A. Location

Wintergreen Lake (Fig. 1) which was used in this
study is a part of the Kellogg Bird Sanctuary which was
established and donated to Michigan State University by
Mr. W. K. Kellogg (Manny, 1971). Wintergreen Lake, T.1a,
R 9 W, is located in Kalamazoo County, Michigan about
twelve miles northwest of Battle Creek and fifteen miles

northeast of Kalamazoo.

B. Description g£_the Lake

 

 

l. Topography. Wintergreen Lake is situated in a

 

low ridge lying between Cooper Ridge and Kalamazoo River.
'The ridge contains a chain of lakes which run from north-
east to southwest, the largest of which is Gull Lake.

The basin of Wintergreen Lake is of glacial origin,
probably formed during the last ice invasion (Taube and
Bacon, 1952; Chandler, 1963). The soils of the area are
of the Gray Brown Podzolic group which have a thin organic
covering and a illuvial horizon (Whiteside 22 31., 1968;
Millar 35 31., 1966). The lake is surrounded by loamy

soils (Fig. 2). These soils are characterized by a porous,

Figure l. Bathymetric map of Wintergreen Lake show-
ing location of sampling stations A and B
used in this study (figure courtesy of
Dr. B. A. Manny).

 

 

WINTERGREEN LAKE

KALAMAZOO COUNTY, MICHIGAN

 

 

0
L

ELEVATION 271m, AREA 15.3 ha 51° "3° '?°

 

METERS

CONTOUR INTERVALS IN METERS

 

 

Figure 2.

Soil map of the Wintergreen Lake water-

shed area. Fox loam or silt loam (1); Fox
sandy loam (2); Oshtemo sandy loam (3);
Washtenaw loam (4); and Brady loam (5).

The map also shows small road running around
the lake (this map was adapted from the Use
Capability Map prepared in 1963 by Soil
Conservation Service, U.S. Department of
Agriculture).

 

 

 

 

 

 

calcareous subsoil. The underdrainage is excellent,
except for the Washtenaw loam (4) which has poor drainage
(Perkin and Tyson, 1926; Whiteside, personal communi-
cation). The limey, porous soils probably contribute
greatly to the hardness of the water in Wintergreen Lake.
Nearly 80% of the soils of the watershed area is culti-
vated or used for grazing cattle. The remainder is
covered by oak, hickory, maple, and aspen trees (Manny,

1971).

2. Morphometry. The morphometry of Wintergreen

 

Lake as described by Manny (1971) is given in Table l.

The lake is more or less rectangular. The area covered

by shallow water is more than 50% of the basin and is
dotted with marl deposits (Schneibner, 1958). The south-
west submerged shore is covered by plant remains (20-40 cm
thick); the opposite end has a thinner organic deposit.
The rest of the submerged shore is solid and sandy. The
major water sources are surface runoff and pipe drainage
originating from a dairy feed-lot. In the spring, water

overflows a small dam in Midland Park into Gull Lake.

C. Climate
The climate of the Gull Lake area alternates
.between continental and semi-marine (Eichmeier). When

th£me is little or no wind, the climate becomes

Table 1. Morphometric parameters of Wintergreen Lake

*

 

 

Parameters

Basin type 35
Length (m) 544
Width (m) 375
Area (ha) 14.98
Volume (m3) 530,584
Maximum depth (m) 6.3
Mean depth (m) 3.54
Relative depth (%) 1.44
Volume development 1.69
Shore development 1.15

 

*
From Manny (1971)

continental. A strong wind from Lake Michigan transforms
the weather into the semi—marine type.

The annual mean air temperature (9.11 C) has not
changed in the last 46 years; the range is -20 C to 30 C.
Annual precipitation and snowfall in the Gull Lake area

averages 88.62 and 170.50 cm, respectively (USCOMM-NOAA,
1971).

III. HYDROGRAPHY

A. Materials and Methods

 

Sampling was conducted each Monday. Temperature,
dissolved oxygen, pH, and transparency of the entire water
column were measured at one meter intervals from July to
November, 1970, and from June through October, 1971. In
1971 alkalanity, conductivity, and light were also
measured. Temperature was measured in_§i£g using a
thermistor (Model 43 TB, Yellow Springs Instr. Co.,
Yellow Springs, Ohio). Incident and reflected light
were measured with a custom underwater photometer (Rich
and wetzel, 1969). Other physical and chemical analyses
of water obtained with a three-liter Van Dorn sampler
were performed within one hour of sampling. Oxygen was
estimated by a modified Winkler technique (Mackereth,
1963); pH was measured using a Beckman pH meter (Model
76-A). Total alkalinity was determined by titration
using H2804 and a mixture of bromcresol green and methyl
red as the indicators. Conductivity was measured with
a conductive bridge (Model 31, Yellow Springs Instr. Co.,
Yellow Springs, Ohio). The transparency of water was

followed with a white secchi disc.

10

11

B. Results and Discussion

 

1. Temperature. Wintergreen Lake is temperate,

 

dimictic, and second order (Hutchinson, 1957; Ruttner,
1960; Manny, 1971). Turnover is completed within 2-5 days
in October-November and April. Figure 3 shows the dis-
tribution of temperature of air and water during the
summer stratification; the thermocline stretches from

2.0 to 3.0 m (see also Fig. 45). In both years the mud
reached a maximum of 12.8 C in early October, then was
cooled during the fall mixing. The diurnal variation of
temperature and pH on 4-5 Aug., 1970, are shown in Table 2.
The nocturnal isotherm reported by Manny and Hall (1969)
in Lake Michigan, did not occur in Wintergreen; this was
probably due to the weak wind which failed to mix the
epilimnion. Temperature data for the diurnal measurement
on 27-28 July, 1971, is shown in Figure 7. Temperature
increased after sunrise, reached a maximum in the after-
noon, then decreased to a minimum in the morning of the

next day.

2. Light and Water Transparency. Figure 4 shows

 

the per cent transmittance of total surface incident light
(A) and reflected light (B). More than 50% of total
surface incident light was absorbed by the first 1 m of
water. The quantity of light absorbed by the water

column varied with the wavelengths. Figure 5 shows that

12

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onma mo ADEEDm on“ Ca mxmq cmmnmumucflz cw musumumafimu mo :ofluonfluumflo

.m mudmflm

13

 

 

 

 

 

 

 

 

 

 

0&0—

 

 

 

 

 

 

M_/.\

39

 

(“‘)Hld3Cl

fl

 

 

 

 

 

 

 

l4

 

 

 

 

 

 

 

Table 2. Diurnal distribution of temperature and pH on
4-5/8 1970
Depth PM AM PM
(m) 8.15 11.30 5.15 8.02 10.53 2.05 5.0 8.0
Temperature

air 19.5 16.5 14.0 16.0 21.0 26.8 23.5 22.0
0 26.0 25.0 24.0 24.0 25.5 26.0 26.8 26.5
1 26.0 25.5 24.5 25.5 25.0 25.0 25.1 25.3
2 24.5 24.5 24.5 24.5 24.5 23.5 24.0 24.2
3 21.5 21.0 21.0 21.2 21.5 20.8 20.7 22.0
4 17.0 17.0 17.0 17.5 17.5 16.5 17.1 17.5
5 13.0 13.0 13.0 13.0 14.0 13.0 13.0 13.0
mud 11.0 11.0 11.0 10.5 11.0 10.5 10.5 11.0
(6)

pH

0 9.30 9.40 9.50 9.40
1 9.50 9.60 9.70 9.65
2 9.50 9.50 9.70 9.35
3 8.70 8.12 8.38 8.75
4 7.60 7.70 7.64 7.50
5 7.30 7.22 7.25 7.30

 

15

Dames mmuomammm Ame

Dames DammeocH Ame

.Hbma mo umfifidw Tau mcfiuoo GEDHOO

Hmumz on» cH msummc msownm> um unmfla mommusm Hmuou mo mmmucmoumm

.¢ musmflm

16

 

 

(w) Hidid

 

 

 

 

 

 

 

 

 

 

17

. 1 01 0:3
2! $05 1 19 68:9 MESH 0623 .23 .32, 5
mxmq cmmumumucwz mo GEDHOO Hmum3 on» a“ mocmuuHEmcmuu usofiq

.m musmflm

l8

 

bee7_e4_ a fiaeqqdflq T 4___14_ . _ #0

fi‘
I
In

 

 

 

& _ _P_L___ P . we..._._ _ 0

oo— o— _ _.o

 

19

more than 90% of the incident blue light was absorbed

by 1.0 m of surface water. This strong absorption was
probably due to the high organic matter content of the
water (Hutchinson, 1957; Ruttner, 1960). The absorption
of red and green light was of the same magnitude as that
of white light. Thus high absorption of incident light
by the water column appeared to be due to the high
plankton density, organic matter, and probably colloidal

suspension of CaCO (Wetzel, 1966).

3
The color of the water varied from a light blue in
winter and early spring to a yellow-green in summer. In
general the secchi disc transparency was inversely pro-
portional to primary productivity and acetylene reduction
(Fig. 8). However, the lowest values for transparency
occurred during die-offs of algal blooms (3/8/70, 6/8/71,
Fig. 8, 15, and 20) and did not coincide with maximum
acetylene reduction, primary productivity, or chlorOphyll
concentration. Thus, in Wintergreen Lake, secchi disc
transparency only served as an indicator of the concen-

tration of organic matter and not necessarily of the

activity of a biotic community.

3. Dissolved Oxygen. During the fall and spring

 

mixing periods, the oxygen distribution was uniform
throughout the lake profile (Fig. 6). The development

of a clinograde distribution of oxygen was rapid and

20

Figure 6. Dissolved oxygen concentration (mg/1)
isopleths during the summer of 1970 and
1971. The shaded area indicates the
anaerobic zone.

 

 

 

 

 

 

 

I970

 

 

 

I

/

 

 

U

 

 

I971

22

typical of a eutrophic lake. The depletion of oxygen in
the hypolimnion (within 3-5 weeks) following stratifi-
cation was probably due to loss of oxygen to lake sedi-
ment (Hutchinson, 1957) and bacterial respiration but
the later is believed to be the main cause of the oxygen
deficit in the hypolimnion of this lake. Figure 44
shows the clinograde distribution of oxygen during the
stagnant period. The high oxygen concentration at 2 m
was related to photosynthesis.

Figure 7 shows the concentrations of oxygen (a),
the rates of change of oxygen (b), and the diurnal vari-
ation of temperature (c) during the period 27-28 July,
1971. The concentration of oxygen decreased during the
night to reach a minimum value at 8 AM, then began
increasing to obtain a maximum at 5:30 PM. The curve
for the rate of change of oxygen was constructed from
the diurnal oxygen curve using Odum's method (Odum, 1956).
The curve shows a high rate of 02 production (photosynthe—
sis) in the morning as indicated by the positive slope,
passed an equivalence point (O2 produced was equal to O2
consumed) at 9 AM and reached a maximum at noon. In the
afternoon, the amount of O2 respired was greater than the
02 produced, causing a negative slope. This was probably
due to a stimulative effect of increasing temperature
(Fig. 7c) on the respiration of the entire community.

The stimulation of respiration by temperature was also

23

Figure 7. Oxygen concentration (mg/1) a; rate of
change in oxygen concentration (mg 0 /l/hr) b;
and temperature (C) c during the diurnal
sampling period of 27-28/7/71.

TEMP.

 

 

 

OO

 

 

: “0O

 

DAY

 

25

observed by Verduin (1956) and Manny and Hall (1969).
However, metabolite secretions resulting from a higher
rate of photosynthesis (F099, 1962; Lucas, 1947; Wetzel,
1969) may be the causal factor for the increased respir-

ation.

4. Total Alkalinity. Wintergreen is an alkaline

 

hard water lake, characterized by high concentrations of
HCOS, Ca, and Mg, as is commonly observed in basins of
glacial origin (Wetzel, 1966-1969; Rich gt 31., 1971;
Manny, 1971). The conductivity was high and proportional
to the total alkalinity (Table 3, Fig. 8C). Figure 8 shows
the water transparency (A, B1), water level (B2), and the

distribution of alkalinity (C) during the summer 1971.

5. EH. Wintergreen water was highly buffered by
the COz-HCOS-COS system. The pH was rarely below 7
during the period of study (Fig. 9). The alkaline
heterograde developed only during stagnant periods.
The low pH in the hypolimnion was probably due to
intense bacterial metabolic activity which produced CO2
and organic acids. Diurnal variation of pH at all depths

was insignificant (Table 2).

6. Inorganic Elements. Very limited information is

 

available on the inorganic nutrients in Wintergreen Lake

water except for the nitrogen and phosphorus data of

26

 

 

Table 3. Conductivity (u ohms) of Wintergreen Lake
water during the summer of 1971

Diޤh 22/6 6/7 20/7 3/8 24/8 7/9 21/9 5/10
0 270 268 270 258 270 281 305 291
l 280 273 271 261 273 280 310 299
2 298 280 274 264 271 283 308 303
3 330 310 319 298 308 313 310 321
4 370 355 365 360 418 358 312 350
5 372 430 430 422 483 460 505 437

 

Figure 8.

27

Water transparency, water level, and
alkalinity.

(A) and (B1): Transparency of the Secchi
disc «depth at which disc disappears from
view) during the summer of 1970 and 1971.
(Bz): Water level (m)

(C): Total alkalinity in the water column
during the summer of 1971 expressed as
mg/l of CaCO3. The shaded area indicates
the anaerobic zone.

28

 

p -

IO

I970

 

.> MI. ”mm—{.3

 

 

 

 

0
I

I97I

 

— q

 

I30

 

 

 

 

 

 

 

 

 

1
[\I/ J
8 47
m 00H.
‘5'
I
p p
0 2

1...... been

 

I971

29

.Hhma cam onma mo Hmfifism
mnu mcflusp mzummp msowum> um Hmum3 Oxmq cwmumumucflz mo mm

.m musmflm

31

Manny (1971). The following discussion is based on his
data for the last three years. Ammonium and nitrate had
similar seasonal distribution patterns (Fig. 10). The
concentrations of ammonium and nitrate at 0.0 m and 0.5 m
increased following the fall turnover and reached their
maxima in winter. Apparently the low temperature did not
totally inhibit nitrification during the winter, as some
of the ammonium freed by the fall turnover was eventually
oxidized to nitrate. Romanova (1957) reported that the
distribution of nitrifying bacteria in Lake Baikal followed
a yearly cycle. Their numbers increased from spring to
summer, reaching a maximum in fall. The lag of the
nitrate maximum in winter (Fig. 10B) may be due to slow-
growing nitrite oxidizers (Peck, 1968; Alexander, 1967)
and are more sensitive to high concentrations of ammonium
in an alkaline environment (Alexander, 1967). Nakos

(1969) found the activity of Nitrobacter to be suppressed

 

by as low as 1.0 mg/l of ammonium; the concentrations of
ammonium in Wintergreen were greater than 1.0 mg/l from
November to February (Fig. 10C). The low levels of
nitrate and ammonium in the epilimnion in June-August
(0.0—19.0 ug/l and 0.0-36.0 ug/l respectively) coincided
with the highest nitrogen fixation (Fig. 18, 19) by the
blue-green algal bloom. The total Kjeldahl nitrogen

and total organic nitrogen per unit algal cell volume

32

A.>uwmum>flco mumum savanna: .cowumum

HmOflmoHOHm mmOHHOM mo accmz .d .m .Ho an omcfl>oum mum3 sumo Hadv
.AOV E o.m IIV E o.m 20V E m.o Ho o.o "mHoQEnm

.Hema .Amnouoo

Ou .onma .sonmz Eoum nmumz wxmq cmwumumucflz GH ADV EDHGOEEm can
.Amv mumuuflc .Amv moouonmmonm UO>HOmch mo AH\mEV cowumuucmocoo one

.oa mesmem

33

.50—

 

 

.okS

 

 

 

- 2!..th

r.

 

O

 

 

N. o o m o
. . . ..:\I..I:1§.u.m\ufiqd.lll
.u. \. ./
.. \. 1. End A
1 .A.\.w../\.\ -u N
1. \uo\\ L H
. .7
I. u .1 Q
.. .....IE m @ I
.. o
fiduonJll 31.1.0341)? 1 1 \uL-Idlvuisu. . .w . 5.4.4.»... O N
Av... Em. .o\.’.. . so

../\.-

 

clue-'19“ 010401515

/\ <

.6,

 

.\/_.VL./.\.I./ \.

_.J_

can"!

fil‘rooor cacao...—

\

/

.1.

Li):

 

34

(Manny, 1971, Fig. 17 and Fig. 19) were also low (about
300 ug/l) during this time.

Dissolved phosphorus (filtered water) and total
phosphorus (unfiltered water) (Fig. 10A and Manny, 1971,
Fig. 9) had similar annual distribution patterns to
ammonium and nitrate. Their concentrations increased
to reach a maximum in winter and decreased in summer.

As soon as the stratification began in early
summer, the odor of H28 was detected at 5 m and soon
moved up to 4-4.5 m where it remained for the duration
of the stagnant season.

Table 4 summarizes the concentrations of the
other elements in Wintergreen Lake as reported by
Manny (1971).

In summary, the physical and chemical properties
of Wintergreen Lake are those of a dimictic, alkaline,

hard water, eutrophic lake.

35

Table 4. Annual range in concentrations of various
chemical components in Wintergreen Lake water
from 1 m in 1970-71*

 

 

Parameter Concentration (mg/1)
UV-labile DON 0.50 -l.37
UV-refractory DON 0.22 -0.52
TDON 0.62 -l.60
NO3 0.005-1.34
NO2 0.012-0.040
NH4 0.005-2.32
Total dissolved P 0.02 -0.13
Total dissolved C 6.0 ~9.1
Ca 10.0 -48

 

*
From Manny (1971)

IV. THE BIOTA

A. Introduction

 

The producers which utilize light energy in
Wintergreen Lake are composed of three physiologically
distinct groups--macrophytes, algae, and photosynthetic
bacteria--which occupy different habitats within the
lake basin. In the following study, all effort was con-
centrated on these communities although others are equally

important in cycling matter in the lake.

B. Materials and Methods

No attempt was made to measure quantitatively the
macrophyte population and activity. Study of their dis-
tribution and identification of genera (sometimes species)
was achieved by sporadic field observations. Water
samples for algal enumeration and photosynthetic pigment
analysis were taken directly from the water used for

acetylene reduction assays or primary production measure-

ments.

1. Algal Enumerations. Algal samples were pre-
served immediately with Lugol's solution in brown poly-

ethylene bottles and stored at 10 C. Blue-green algal

36

37

filaments (trichomes) and heterocysts were counted micro-
scopically in a one-m1 Sedgewick-Rafter counting chamber
under a 10X objective; reported data are the average of

20 fields. Microcystis were counted as number of colonies.

 

2. Photosynthetic Pigments. (All laboratory work

 

in this section was conducted by Dr. Wetzel's laboratory.)
The samples for pigment analysis were prepared as follows:
40 to 200 m1 of water (depending on the photosynthetic
plankton density) were filtered onto Millipore HA mem-
brane filters under low vacuum (1/3 atm). The filters
were then homogenized in a Teflon glass homogenizer

(Model 4288-C, A. H. Thomas Co., Philadelphia, Pa.) in
90% basic acetone. The extracts were centrifuged and

the absorption of the supernatants were measured in a
spectrophotometer (Hitachi-Perkin Elmer, Model UV-VIS 139).
Concentrations of chlorophylls were calculated by using
the trichromatic equations of Parson and Strickland (1963)
as modified by Westlake (1969); chlorophyll 2 also was
corrected for pheopigments using the modified equation
(Wetzel and Westlake, 1969) of Parson and Strickland's

(1963).

3. Multivariate Statistical Analysis. The

 

relationship between living organisms and their environ-

ment is complex (Lindeman, 1942). Therefore it is

38

difficult to single out and weigh the effect of each
factor (Singh, 1955; Saunders, 1963; Wetzel, 1966; Brown,
1971) on others.

As an approach to this problem, multivariate sta-
tistical analysis was used (Green, 1971). A computer
program was used which permits stepwise deletion of
independent variables from a least squares solution until
some stopping criterion is fulfilled (Rafter and Ruble,
1966). The criterion used was a minimum significance
probability (Sig Fbi) of 10% for the regression coefficient
for the current candidate for deletion (MINSIG = .10).

By this sequential process, independent variables
were eliminated which probably (p > .10) did not account
for variation in the dependent variable (above that
accounted for by the remaining independent variables
and the overall mean of the dependent variable). The
final solution printed out, after this criterion was
met, included statistics whereby the relative importance
of independent variables retained can be estimated:
constant (b0), regression coefficients (bi), Fbi'

Sig Fbir partial correlation coefficients, and "R2
deletes." The last statistic is the coefficient of
multiple determination when the independent variable
under consideration is left out of the solution. The
"R2 delete" for each independent variable can be com-

2

pared to R for the solution which includes the variable.

39

The difference R2 - "R2 deletei" is an estimate of the

percentage of total variation in the dependent variable
which was associated with the independent variable under
consideration.

The facilities of the Michigan State University

Computer Laboratory were used.

C. Results and Discussion

 

Macrophytes were composed largely of free floating

duckweeds: (two species of Lemna, Spirodela, and Wolffia),

 

 

submerged CeratoPhyllum demersum L., two species of

 

Potamogeton, Myriophyllum, Najas, and emergent Nuphar
advena Aiton, two species of Nymphaea. The emergent
macrophytes occupied approximately 72% (Manny, 1971)

of the Wintergreen basin. Vigorous growth of macrophytes
occurred in the period from April to June when the temper-
ature increased. Submerged leaves became heavily coated
with organic matter, epiphytes, and white materials,
presumably marl deposits during the stagnant period.

The population of planktonic algae in Wintergreen Lake,
as in other aquatic habitats (Olivier, 1955; Singh, 1955;
Vertebnaya, 1957; Wetzel, 1964; Caspers, 1964; urbaéek,
1964; Billaud, 1968; Stewart 35 31., 1971) followed a
seasonal cycle. The development of these algae was
limited to 0-3 m water layer during stratified periods.

According to Manny (1971), the algal population during

40

the 1970-1971 period was dominated by Chlorophyta in
the cold months (November-April).

Data collected during the summers 1970-1971 indi-
cated that the algal population of Wintergreen Lake water
was also subjected to a shift of species (Table 5).

Aphanizomenon and Microcystis dominated the algal popu-

 

 

lation in the summer and early fall of 1970. However,

in summer, 1971, Aphanizomenon developed earlier (May-

 

June) then died off in late June. The Aphanizomenon-

 

Microcystis bloom was succeeded by an Anabaena-Microcystis

 

 

bloom which reached its maximum in late July. Photographs
of representative algae are shown in Figure 11. The

Anabaena-Microcystis bloom died off in late August, 1971,

 

to give way to a short-lived bloom of green filamentous
algae which developed at 3-4 m. At this time, the
thermocline was below 4 m.

Table 5 and Figure 12 illustrate total numbers of
algae, concentration of chlorophyll a, and carotenoids
per liter during summer of 1971.

The results of the statistical analysis are shown
in Table 6 and 7. In general, the chlorophyll a concen-
tration (algal mass) in Wintergreen during the period
June-September, 1971, was correlated to the combination
of total alkalinity, temperature, pH, and dissolved
phosphorus. The combination of these parameters

explained about 81% of the variation of chlorophyll a

41

mmmHm msoucmamHHm cmoum UO>HHIuHonm
k.

 

.1

ucmcHEmm mfiummoouoaz + mammnmcd »m\th\m

 

 

ucmcHEOU mwumNOOHOAE + mammnmc4 + cocmSoNHcmsmd u>\m
k.

 

 

om om «tmma on ma ma hm I o MMH em m
we om e.Ceomm «Ha I ma mm mmm 0 ma mmv v
mm mm mm 0mm hvm mom mmw mm mm mmm mmmea m
oma mm mm oma 0mm vmv mom 0mm vmv HNM.H mom N
om mm om new mmm ham who mow mmm nvm amata H
mm om mm «Ha 0mm mmm mam va Ohm NNB mmv.H o
oa\m m\HN m\h m\oa m\m h\hm h\om h\ma b\m m\mN m\mm SMMWQ

 

.Hema mo

umEEdm map mcfluso Amoa x H\mwflcoHoo + mucmfimaflmv cowumaomom mmmHm Hmuoe .m manna

42

1 OH ucmmmummu mumm "mamom

.Ocsb CH comon>m©
AEdHcomoomov mamas cmwum oaumnmammIaEmm "Rev
mammnmcm "amv
unwooumumn
cam A3ouumv gunman mcfl3onm mammamcd "Amy
A3OHMMV
unmooumuwn mcfi3onm cocmfioNHcmnm< "AHV

 

 

 

"Oxmq cmmumumucfiz
CH mmmam m>flumucmmmumwn mo magnumouosm .HH whomwm

43

 

 

 

 

 

44

Figure 12. The concentration (Hg/1) of chlorophyll a
(O ) and carotenoids ( Q ) at 0 m (A), l m
(B), 2 m (C), and 3 m (D) in Wintergreen
Lake during the summer of 1971.

 

4O-

 

 

 

 

 

 

 

 

 

 

 

20 \\ e
\o,” \
\\ /.
,9
07‘8”
O— 1 L ' I
_ 226.17 1
120- @ /'\ ~
- . 1
60? ’6‘ //’°\\\ 7
" o—-—-' I ‘\o\ / \ -
0;.0/1 \- I \\\‘O/I \\.‘
3O 3O 3O

46

*
Table 6. Multivariate statistical analysis

 

Dependent variable Y: chl a (0-2m)

 

 

 

 

 

 

 

_ 2
Y = -8008.084 - 1.680X - 7.090X + 1937.899X - 112.388x
l 2 3 3
+ 2.37lx4
(a) Overall Regression
Sum of df Mean of F Sig.
Squares Squares
Regression
(about mean) 2741.457 5 548.291 12.585 <0.0005
Error 609.938 14 43.567
Total
(about mean) 3351.396 19
(b) Multiple Correlation Coefficients
Mean = 23.461 ug/l R2 = 0.812 R = 0.904
Sig. Partial R2
Reg. Coef. Level Corr. Coef. Deletes
Constant -8008.084 0.011
Alkalinity (X1) -l.680 0.023 -0.562 0.734
Temperature (X2) -7.090 0.001 -0.761 0.567
pH (x3) 1937.899 0.008 0.636 0.694
(x3) -112.388 0.008 -0.668 0.693
Diss. P (x4) 2.371 <0.0005 0.823 0.437

 

*
Factors tested: Net primary productivity, available

light, total alkalinity, temperature, pH, nitrate,
ammonium, phosphate, carotenoids, and oxygen.

 

47

*
Table 7. Multivariate statistical analysis

 

Dependent variable Y: Carotenoids (0-2m) 2
Y = 338.323 - 0.657 X1 - 3.308 X2 - 1.063 X3 + 1.529 X4
- 0.013 X5

 

(a) Overall Regression

 

 

Sum of Mean of .
Squares df Squares F 819.
Regression
(about mean) 837.359 5 167.472 12.135 <0.0005
Error 193.206 14 13.800
Total

(about mean) 1030.566 19

 

(b) Multiple Correlation Coefficients

 

Mean = 15.474 ug/l

 

 

Sig. Partial R2

Reg. Coef. Level Corr. Coef. Deletes
Constant 338.323 0.003
Alkalinity (X1) -0.657 0.001 -0.732 0.596
TemperaSure (X2) -3.308 0.001 -0.726 0.604
(pH)2(x3) -l.063 0.083 -0.445 0.766
Diss. P (X4) 1.529 <0.0005 0.838 0.371
Light (X5) -0.013 0.037 -0.525 0.741

 

*

Factors tested: Net primary productivity, available
light, total alkalinity, temperature, pH, nitrate,
ammonium, phosphate, chlorophll g, and oxygen.

 

48

concentration. Table 6, part (b), column 4 also indicates
that the concentration of chlorophyll §_was negatively
related to total alkalinity and temperature. It also
shows that at certain values of pH, the chlorophyll a
concentration was inversely proportional to pH. The most
significant factor in this group was the dissolved phos-
phorus which when depleted from the group, caused the R2
value to drop in half (R2 = 0.437).

The concentration of carotenoids was also correlated
to light, total alkalinity, temperature, pH, and dissolved
phosphorus (Table 7). Again carotenoid concentration
was inversely related to total alkalinity, temperature,
pH, and available light. The relationship between caro—
tenoid and pH was nonlinear. Dissolved phosphorus was
highly correlated to carotenoid concentration, thus it
seems that dissolved phosphorus was the most decisive
factor which limited the development of blue-green algae.

Photosynthetic bacteria in the hypolimnion (3memud)

were composed mainly of Thiopedia (purple sulfur bacteria),

 

Chromatium, Chlathrochloris sp., and Prosthecochloris sp.

 

 

These organisms constituted 90% of the bacterial popu-
lation above the mud (Caldwell, unpublished data, MSU).
These blooms also exhibited an annual cycle. The blooms
disappeared each year during fall turnover probably due
to the inhibitive effect of oxygen on these obligate

anaerobes. Some of these bacteria probably survived the

49

oxygenated period buried in the sediment. As discussed

in Chapter III, after the spring overturn, the hypolimnion
rapidly became anaerobic which favored the development

of various anaerobic bacteria. Small but significant

numbers of Thiopedia can be seen microscopically as

 

early as late May. The newly established anaerobic con-
ditions also favored the development of heterotrOphic
anaerobic bacteria Which fermented organic matter leading
to the formation of fatty acids, alcohols, etc., which
could serve as substrates for the photosynthetic and
methane bacteria.

As the stratification of the water column intensi-
fied, the metalimnion was permanently established at
2-3 m which limited oxygen movement from the surface
water into the hypolimnion during the entire stagnant
period (June-August). During this time, the hypolimnion
was continuously supplied with organic compounds syn-
thesized in the metalimnion and epilimnion. In the
hypolimnion some of those organic compounds were degraded
by a bacterial population presumably consisting of photo-
synthetic, acid-forming (Toerien 33 31., 1967), methano-
genic (Barker, 1956), nitrate-reducing and sulfate-

reducing bacteria.

V. PRIMARY PRODUCTIVITY

A. Introduction
The problems of relative primary productivity of
terrestrial and marine ecosystems have been of continuing
interest to limnologists and oceanographers. Odum (1963,
1971) believed that terrestrial and marine habitats
might be equally productive. According to Odum (1963):
. . . basic primary productivity is not necessarily
a function of the kind of producer organism or the
kind of medium (whether, air, fresh water, or salt
water), but is controlled by local supply of raw
materials, sun energy, and the ability of local
communities as a whole (and including man) to utilize
and regenerate materials for continuous reuse.
In this part of the study, the primary productivity of
phytoplankton in Wintergreen Lake was measured. To avoid

confusion, the terminology of productivity used in this

chapter will be briefly discussed and defined.

B. Definition

The definitions of productivity adopted here are
based on the definitions of Odum (1971) but have been
adopted to conform with this study. The primary pro-
ductivity of phytoplankton is defined as the rate at

which light energy is stored by photosynthetic activity

50

51

in the form of organic substances. It is important to
distinguish the terms in the photosynthetic production

process:

a. Gross productivity is the total rate of photo—
synthesis, including materials used in respiration and

excreted by phytoplankton during the measurement period.

b. Net primary productivity of phytoplankton or
net light CO2 fixation is the rate of storage of organic
cellular materials in excess of the respiratory utili-
zation and excretion by phytOplankton during the measure—

ment period.

C. Materials and Methods (All laboratory work was done by

 

Dr. R. G. Wetzel's laboratory.)

Primary productivity of photosynthetic plankton was
measured by a modification (cf. Wetzel, 1964; Goldman,
1960; Goldman, 1963) of the 14C technique of Steemann
Nielsen (1951). All calibrations were done in terms of
absolute activity as determined by gas-phase analysis,
to eliminated errors associated with counting of radio-
activity (Goldman, 1968). The concentrations of absolute
activity were determined by Dr. R. G. Wetzel (3.24 or
3.665 uCi/ml). The sampling schedule coincided with that
described in Chapter III for 1971. The primary produc-

tivity was measured at 1 m intervals throughout the

entire water column. Water of each depth was taken

52

using an opaque 3—liter Van Dorn sampler and transferred
to two clear and one opaque (with black polyethylene tape)
ground-glass-stoppered Pyrex bottles (125 ml). A cor-
rection was made for the variation in bottle volumes.
Remaining water was collected into brown polyethylene
bottles for pigment analysis. All of the sample bottles
were kept in a black box to prevent physiological changes

caused by direct sun light. A l-ml solution of 14CO

2:
predominantly NaH14CO3, was injected into each bottle.
The dark bottles were secured then with an extra layer
of aluminum foil. The samples were then resuspended at
their original depths. Time of sampling was carefully
planned so that the incubation could begin at around 10 AM
to ensure maximum photosynthesis (Vollenweider and Nau-
werck, 1961) and uniform timing. After a 3-hour incubation
period, samples were placed in a black box and taken
immediately to the laboratory. Usually about 10-15 min
elapsed between sample removal and laboratory analysis.
A 25 m1 sample from each bottle was filtered through a
Millipore filter (HA, 0.45 p) on a multiple millipore
filter unit (a vacuum of 1/3 atm was used). A 10 ml
sample from each bottle was filtered through the same
type of Millipore filter (HA, 0.45 u) attached to a
syringe and the filtrate was collected and acidified to
3 4 and flushed

14

with nitrogen for 2 min to drive off dissolved C02.

approximately pH 2 with 0.4 ml of 3% H PO

53

Photosynthetic Millipore filters and 14C02-free filtrates

(duplicated) were dried and exposed to fumes of HC1 for

10 min to remove any 14

CO2 contaminant. Radioactivity
was determined (with a minimum of 1,000 counts) by a
gas-flow Geiger-Mueller counter (Nuclear Chicago,
Model 6919, with micromil window D-47). The net primary
production was calculated following the standardized
procedures of Goldman 33 $1., (1969). A correction of
6% for isotopic discrimination was made in the net
primary productivity data. A diurnal factor was calcu-
lated from the total solar energy and the portion
received during the in situ incubation period (Belfort
pyrheliometer, Belfort Instr. Co., Baltimore, md.).
Total available inorganic carbon was calculated from
total carbonate alkalinity and pH measured at the same
time (see Chapter III).

The radioactivity of the filter and filtrate were
plotted versus depth. The daily net primary productivity

and net excretion were calculated using a planimeter to

estimate plot area and the diurnal factor.

D. Results and Discussion

 

The 14C method is considered one of the best
available techniques to measure photosynthesis, although

there are uncertainties inherent to it, such as:

(a) There is no way to determine whether this

technique is measuring gross production, net production,

54

or some value between these two (Steemann Nielsen, 1963;
Vollenweider and Nauwerck, 1961). The radioactivity
measured may not represent the true photosynthesis due
to assimilation of dissolved organic materials by algae

(Wright, 1964; Pearce and Carr, 1967, 1968).

(b) Interferences result in the loss of 14C, due
to lowering or inhibition of photoactivity by nutrient
exhaustion (Talling, 1957); alteration of the chemical
composition of the enclosed water (Gessner and Pannier,
1958), etc., could be great during prolonged incubation

periods ( > 8 hr, Vollenweider and Nauwerck, 1961).

(c) Enclosure of water sample eliminates turbu-
lence (Verduin g£_31., 1959) causing sedimentation of

algae at the bottom of the bottles.

There is sufficient available information which
indicates that photoassimilation of organic matter by
algae is negligible because of an exceedingly small
amount of available organic carbon in lake water (Wright
and Hobbie, 1965; Wetzel, 1967, 1968). Other possible
errors were minimized by incubating for a short period
(3 hr, Vollenweider and Nauwerck, 1961) at a fixed daily
time.

The net primary production rate reported here was

the radioactivity computed from C0 fixation by light

2
minus dark samples with the assumption that the above

errors were negligible.

55

Net primary productivity is shown in Figure 13.
Net photofixation rates of CO2 in the anaerobic zone ,
3-5 m, were negligible, as compared to those in the
epilimnion. This low primary productivity was observed
elsewhere in marl lakes, but where the mineralization
of organic matter is almost completed in the aerobic
zones (Kuznetsov, 1956), the productivity may be higher
(Culver and Brunskill, 1969). Culver and Brunskill
reported that primary production of green sulfur bacteria
in deep water of Green Lake, New York, accounted for
almost 83% of the annual productivity (290 gC/mZ/year)
of the lake. Total annual primary productivity including
planktonic organisms and macrophytes in Wintergreen Lake
would rank far above its neighboring Lawrence Lake (Rich
gt 21., 1971) and most of the lakes studied so far (see
wetzel, 1964, Table 10).

The weekly fluctuation of net CO fixation (Fig. 13)

2
was probably due to (1) vertical movement of algae and
(2) daily fluctuation of solar radiation received. The
first hypothesis is justified by the diurnal vertical

density variations of Aphanizomenon (1970), or Anabaena

 

and Microcystis (1971) shown in Figures 22 and 26.

 

Similar observations were reported by Talling (1957),

who observed that Anabaena flos-aguae Born & Flahvar.

 

intermedia f. spiroides Woron accumulated in the surface

 

water during the day but was evenly distributed in the

56

Figure 13. Net primary productivity at 0 m, 0.5 m
(A); 1m, 2m (B); 3m, 4m, andSm (C)
in Wintergreen Lake during the summer of
1971.

57

“('5
o
o

 

m3/d cy)
1'6
CI
9 .
(9
\O
U
3

 

600 .—-. . .\\\ ’O‘\ :
0'“ °%0/°\ .
1 l I l I l M
0 3O 3O 30 3O
1

O
\
,/
\

14co2 FIXED (mgC/
8 8

. //~\ "wm1~§“////"\::\

 

 

C l 1 \OTo/OT—h‘O‘ F~g/ l /.\ _|—:-‘
3001 7._\ 3
m
G) I Y
200 , \
i \.
100 .\\ .\ i] \ ’.’.,.-O\\
/' ' h‘f4NI 5q1\b’

JUNE3 JULY AUG.
1971

30

,0

30

SEPT

 

 

q

 

58

water column during the nocturnal mixing. Both Anabaena
and Microcystis seemed to display diurnal distributions
(Fig. 24, 26) in Wintergreen Lake. It should be noted
that heterocyst density was consistently higher during
the day than the night. The dynamics and mechanism of
the movement of algae were not studied. There is evidence
which indicates that gas vacuoles found in procaryotic
organisms (bacteria and blue-green algae, Cohen-Bazire
gg‘al., 1969) probably play a major role in the daily
ascent of algae (Talling, 1957) against turbulence (Gess-
ner, 1948) and passive sinking which tend to drive the
algae to lower zones. Oxygen bubbles formed by intense
photosynthesis also play an important role in keeping
algae in the trophogenic zone. Visible bubbles of oxygen
can be seen attached to filamentous algae on a quiet day.
Increasing body density of algae resulting from accumu-
lation of photosynthetic products may play an important
role in the nocturnal downward movement of algae.

The sudden high net primary productivity at 3 m on
August 10 (Fig. 13, C) coincided with the loss of the
previous floating capability of the algae which now
accumulated at 3 m. At this time the majority of algae
was probably dying; filaments began to break into shorter
segments.

Figure 13 supported the arguments that favorable

temperature and high available solar radiation in the

Go

Sci

th.

in!

59

summer are not necessary for the highest biological
activities. But, the combination of a whole set of
chemical, physical, and biological factors determine the
activity and composition of the algal population. The
multivariate statistical analysis shown in Table 8 indi-
cates a significant correlation between the net primary
productivity in the epilimnion (0-2 m) and the combination
of available light, ammonium, and chlorophyll a, The
net photosynthetic rate was positively correlated to an
increasing amount of available light up to a certain
level, however, strong light inhibited photometabolism
of C02. Available light during the study period (1971)
varied from 277.94 to 648.53 g cal per cm2 per day (at
the Kellogg Gull Lake Laboratories). No attempt, how-
ever, was made to determine the saturation value of
light. The general effect of light on CO2 uptake can

be visualized from the graphs of net CO2 uptake versus
depth (Fig. 14). The CO2 uptake was seemingly inhibited
by the strong light at the surface. This partial inhi-
bition of photosynthesis at the surface by strong solar
radiation has been commonly observed during the growing
season (Edmondson, 1956; Verdium, 1956; Talling, 1957;
Goldman, 1960; Goldman and Wetzel, 1963; Wetzel, 1964;
Schindler and Holmgren, 1971). The photoinhibition of
the photosynthetic apparatus by strong light probably
involves photoinactivation of their enzymes (Steemann

Nielsen, 1962; Steeman Nielson and Jorgensen, 1962).

6O

*
Table 8. Multivariate statistical analysis

 

Dependent variable Y: Net primary productivity (0-2 m)
Y = -179.402 + 2.219x1 - 0.002xf - 1.646x2 + 14.559x3

 

(a) Overall Analysis

 

Sum of df Mean of

 

Squares Squares F 819'
Regression
(about mean) 1073933.145 4 268483.286 9.26 <0.001
Error 435007.176 15 29000.478
Total

(about mean) 1508940.321 19

 

(b) Multiple Correlation Coefficients

 

 

 

Mean = 448.067 mg C/m3/day R2 = 0.712 R = 0.844
Sig. Partial R2

Reg. Coef. Level Corr. Coef. Deletes

Constant -179.402 0.239

Light (x1) 2.219 0.018 0.564 0.577

xl -0.002 0.070 -0.499 0.639

Ammonium (X2) -l.646 0.554 -0.154 0.705

Chl 2 (x3) 14.559 <0.0005 0.773 0.282

 

*

Factors tested: Available light, alkalinity,
temperature, pH, nitrate, ammonium, total dissolved P,
chl a, carotenoids, oxygen, and depth.

 

61

Figure 14. The distribution with depth of chlorophyll a
(ug/l), (A); net primary productivity —
(mg C/l/day), (B); and net primary pro-
ductivity per unit chlorophyll 2 (mg C/mg
chl a/day), (C) during the summer of 1971.

DEPTH (m)

I“cog /chlc1

O

UN‘O

62

14C

600040

18> 8

 

E/

 

 

 

 

P I P
I I 1 l

 

l I
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2.
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I I 1
I '1 l
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2
3
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2
3

 

 

o.chlc1
50 IO

 

 

 

HA6"

 

 

 

V

7894/7

 

 

U

 

 

I

2 4l/8

 

 

 

63

The effect of the selective absorption of blue
light by the water column (Fig. 5) on photometabolism of
CO2 by in situ organisms was not studied. However,
Findernegg (1964) reported that in a eutrophic lake,
by the time algal blooms occurred, the decline of CO2
uptake did not conform to the decreased transmittance
of some wavelengths. It was, however, correlated with
the total energy of the whole photosynthetic active
spectrum. But recently, Kaushik and Kumar (1970) found
that green and blue light showed very little promotion

of growth of Anabaena doliolum and Fischerella mucicola

 

 

as compared to yellow, red, or white light. Wallen and
Geen (1971a, 1971b) also found that light of different
spectral quality, but the same intensity, produced dif-
ferences in cell growth rates, CO2 assimilation rates,
photosynthetic products, concentrations of various photo-
synthetic pigments, proteins, DNA, and RNA in two species
of marine plankton algae. The same authors (1971c) sub-
sequently found qualitative and quantitative differences
in photosynthetic carbon products between surface algal
samples and deeper algal samples in a lake even though
these algae were subjected to considerable vertical
transport. They suggested that the observed changes
were in reSponse to light quality rather than intensity.
Table 9 shows that net primary productivity at

3-5 m was highly correlated with available light, total

64

*
Table 9. Multivariate statistical analysis

 

Dependent variable: Net primary productivity (3-5 m)
Y = 190.513 + 1.623X1 - 0.171x2 - 24.500x3 + 0.981X§
- 70.895x4 + 18.928xi

 

(a) Overall Analysis

 

 

Sum of Mean of Sig.
df F
Squares Squares
Regression
(about mean) 28217.716 6 4702.953 41.37 <0.0005
Error 2273.770 20 113.688
Total

(about mean) 30491.486 26

 

(b) Multiple Correlation Coefficients

 

 

 

Mean = 26.91 mg C/m3/day R2 = 0.925 R = 0.9620
- - 2
Sig. Partial R
Reg. Coef. Level Corr. Coef. Deletes
Constant 190.513 0.003
Light (X1) 1.623 <0.0005 0.755 0.826
Alkalinity (X2) -0.l7l 0.062 —0.483 0.910
Temperature (X3) -24.500 0.003 -0.599 0.883
x3 0.981 0.001 0.658 0.868
Oxygen (X4) -70.895 <0.0005 -0.710 0.849
(X4) 18.928 0.003 0.597 0.884

 

*

Factors tested: Net primary productivity,
available light, alkalinity, temperature, pH, oxygen,
and depth.

 

65

alkalinity, temperature, and oxygen. Carbon dioxide
uptake was positively correlated with available light
and inversely correlated with oxygen concentration and
total alkalinity. Temperature became inversely related
to light CO2 fixation at certain high values of temper-
ature.

It is surprising that there was no significant
correlation between concentration of dissolved phosphorus
and net primary productivity (Table 8). This lack of
correlation seems contrary to the general belief that
phosphorus is probably important biogenic element
limiting primary productivity (Brock, 1966; Rich 35 31.,
1971; Kalff, 1971).

Table 10b shows dark CO2 fixation which could be due
to algal dark metabolism (Holm-Hansen, 1962), hetero-
trophic bacteria (WOod and Stjernholm, 1962), chemolith-
otrophic bacteria (Elsden, 1962) and animals.

During the period 22/6 to 5/10, 1971, dark CO2
fixation averaged 322.40 mg C per m2 per day or 21.40%
of total CO2 photofixation. Similarly significant rates
of dark CO2 fixation were reported by Kuznetsov (1956)
and Sorokin (1961) in stratified reservoirs.

Total Chemosynthesis (dark fixation) rates were
fairly constant during the beginning of summer until
the sudden die-off of the blue-green algal bloom in

late August. Intense degradation of organic debris

665

 

 

 

 

 

00.000 00.000 00.000 00.000 00.000 00.000 00.000 00.000 00.000 00.000 00.000 00.000 00060
00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.000 00.00 0
00.00 00.00 00.000 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.000 0
00.00 00.0 00.00 00.00 00.00 00.00 00.00 00.00 00.000 00.000 00.00 00.00 0
00.000 00.00 00.0 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.000 00.00 0
00.00 00.00 00.0 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 0
00.0 00.00 00.0 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 0.0
00.0 00.0 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 0

00\0 0x00 0x0 0x00 0x00 0x0 0x00 0x00 0\00 0x0 0\00 0x00 shuwo

0000\0exu 05 .600060 00000 00000000065600 .000 00000

00.000 00.000 00.0000 00.000 00.0000 00.0000 00.0000 00.0000 00.0000 00.0000 00.0000 00.0000 00060
00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.00 00.00 0
00.0 00.00 00.0 00.00 00.00 00.00 00.0 00.00 00.0 00.00 00.00 00.0 0
00.00 00.0 00.000 00.00 00.000 00.00 00.00 00.00 00.00 00.00 00.00 00.000 0
00.000 00.000 00.0 00.000 00.000 00.00 00.000 00.000 00.000 00.000 00.000 00.000 0
00.00 00.000 00.000 00.000 00.000 00.000 00.000 00.000 00.0000 00.000 00.000 00.000 0
00.0 00.0 00.000 00.000 00.000 00.000 00.0000 00.000 00.0000 00.000 00.000 00.000 0.0
00.00 00.0 00.000 00.000 00.000 00.000 00.000 00.000 00.0000 00.000 00.000 00.000 0
a

00\0 0x00 0\0 0x00 0\00 0x0 0x00 0\00 0x00 0x0 0x00 0\00 awnwo

 

30690.30 0.15 2.00 no .0983.— 003 900.006 55.000 noun: soouuuoufii 5.. 00000000005300“ gum van «0. 0003050000006 .060 0.33.

67

following the algal die—off seemingly stimulated dark
CO2 fixation, which exceeded photosynthesis in early
October. A similar observation following algal die-off
was reported by Kuznetsov (1958).

Kuznetsov (1956) rejected the Chemosynthesis idea
attributed to photosynthetic bacteria. He found no
connection between natural populations of photosynthetic
bacteria and anaerobic Chemosynthesis in reservoirs.

He also found that the productions of sulfate-reducing
and iron bacteria were negligible. On the contrary, he
found high Chemosynthesis in the anaerobic zone due to
the activity of hydrogen sulfide-oxidizing bacteria
(Thionic bacteria) which were found mostly at the inter-
face between the hydrogen sulfide and oxygenated layers
(Kuznetsov, 1968).

The metabolism of single carbon compounds has been
recently reviewed by Ribbons 32 31. (1970). Methane-
oxidizing bacteria are known to oxidize methane to CO2
and then assimilate it gig the carboxydismutase
reaction. Thus the presence of methane-oxidizing
bacteria could contribute to the chemosynthetic activity
in oxygenated zones of natural waters. Sorokin (1961)
observed the coincidence between the highest population
of methane-oxidizing bacteria and the sudden drop of
methane concentration at the boundary of the aerobic

and anaerobic zones in a stratified reservoir. A

68

similar distribution of methane was also observed by
Tiedje (unpub.) during the summers 1970 and 1971 in
Wintergreen Lake. Methane bacteria (see reviews by
Stadtman, 1967 and Toerien and Hattingh, 1969) may also
be important CO2 fixers in the anaerobic zone.

There was no significant correlation between dark
CO2 uptake from 0 to 5 m with any single or any combi-
nation of the following factors tested: available light,
temperature, oxygen, alkalinity, depth, net light CO2
uptake, and pH.

Excretions of the 14C assimilated are shown in
Table 11. For convenience, excretion in the transparent
bottles is called light excretion and that in the dark
bottles is called dark excretion. Light excretion rates
were highest in June and lowest in September-October
within the study period. Light excretion rates averaged
60.63 mg C per m2 per day or about 3.5% of that of the
total net CO2 uptake in the transparent bottles.

Most of the available information concerning
excretion of newly fixed carbon came from the works of
Fogg (1952), Wetzel and his coworkers (1969, 1971).

Data collected by Fogg (1952) from pure cultures of
Anabaena showed that extracellular non-nitrogenous
organic compounds were almost equal to extracellular
nitrogenous compounds. These nitrogenous substances

were composed mainly of peptides and amides. In his

659

 

 

 

 

 

 

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70

study, young cultures excreted the highest per cent of
nitrogen fixed (50%). The per cent excreted decreased
toward the end of the exponential phase then increased
to a lesser extent in stationary phase.

The growth of the Anabaena bloom in Wintergreen
Lake followed, more or less, the classic growth curve
(Fig. 15). However, the organic carbon excretion pattern
ig_§itg_shown in Figure 16 and Table lla was different
from that observed by Fogg (1952) in pure cultures of
Anabaena. This difference may be due to a number of
reasons: (1) Fogg (1952) measured in detail only the
excretion of nitrogenous substances, the present study
measured total substances excreted. The excretion of
Microcystis which were also present in large numbers may
cause this different pattern. (2) Extra-cellular organic
carbon measured in sign could represent the amount left
behind after intense metabolic activities of epiphytic
bacteria (Odum, 1957; Wetzel, 1969; Allen, 1971; also
cf. Wetzel, 1968), free-living bacteria (Wright and
Hobbie, 1965, 1966; Hobbie and Wright, 1965; Wetzel,
1967) or by algae themselves (Khoja and Whitton, 1971;
also cf. Danforth, 1962).

The total amount of carbon excreted (3.5% of the
total net CO2 fixed) in Wintergreen Lake was higher than
the 1.5% observed by Fogg (1958, cited by Fogg, 1962) in

a Swedish lake when Gloeotrichia and Aphanizomenon

 

 

Figure 15.

71

The density of Aphanizomenon plus Anabaena
populations (filaments/l x 105, O ) and
total heterocysts (numbers/l x 105, O ) at
0 m (A), l m (B), 2 m (C), and 3 m (D)
during the summer of 1971.

POPULATION

ALGAL

72

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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75

were the predominant plankton. It is reasonable that the
percentage of newly fixed carbon that was excreted also
varies with the type of algal bloom, lake environment,
and analytical procedure. The excretion of newly fixed
carbon also has been shown to be influenced by environ-
mental conditions (Fogg, 1952). Fogg and Nalewajko
(unpub. cited by Fogg, 1962) found that the liberation
of extracellular products amounted to 90% of the total
carbon fixed in Lake Windermere, English Lake District,
in which diatoms were the most abundant planktonic algae.
Miller (1971) reported that the mean annual secretion of
dissolved organic carbon by phytoplankton in a near-by
marl lake (Lawrence Lake) was 5.8% of net production.
However, this lake did not have blue-green algal blooms
similar to those in Wintergreen Lake.

Extracellular products measured from 3-5 m probably
resulted from the activity of both anaerobic and aerobic
bacteria in this zone. Total light excretion (in trans-
parent bottles) sometimes exceeded net primary productivity
(Tables 11 and 10). The excretion of organic carbon by
bacteria has been reviewed by Wilkinson (1958). Extra-
cellular polysaccharides may exceed cellular dry weight
in certain conditions. The mean dark excretion by plank-
tonic organisms was 28.4 mg C per 1112 per day (8.8% of
total carbon fixed in the dark and 46.8% of total extra—

cellular products in transparent bottles). Thus, overall,

76

dark excretion and light excretion were almost equal.
However, the vertical distribution of these excreted
products was ununiform (Table 11). Furthermore the
excretion in transparent bottles minus that in dark
bottles (9.92 mg C/mZ/day) in the epilimnion

(l-2 m) was higher than dark excretion (4.78 mg C per

m2 per day). The excretion in transparent bottles minus
that in dark bottles (0.67 mg C per m2 per day) in the
lower region (3-5 m) was, however, lower than dark
excretion (4.68 mg C/mz/day).

Results of statistical analysis of excretion data
are shown in Table 12 which indicates that the excretion
of newly fixed carbon in transparent bottles (0-2 m) was
highly correlated with the combination of oxygen,
available light, pH,depth, and net primary productivity.
Among these factors, pH was inversely related to excretion'
at certain high pH values. The correlation between
available light and total excretion in transparent
bottles was also not linear. This excretion was, how-

ever, highly correlated with net primary productivity.

There was no significant correlation between total

I

excretion in transparent bottles in the 3-5 m zone and
any one or combination of the tested factors such as net
primary productivity, oxygen concentration, total alka-

linity, available light, temperature, pH, and depth.

77

*
Table 12. Multivariate statistical analysis

 

Dependent variable: Excretion in transparent bottles
(0-2 m)
y = 4813.105 + 2.143 x1 + 0.035 x2 — 1135.703 x3 + 66.548x§
+ 7.378 X4 + 0.020 X5

 

(a) Overall Analysis

 

 

Sum of df Mean of F Sig.
Squares Squares
Regression
(about mean) 2942.164 6 490.360 13.96 <0.0005
Error 702.491 20 35.124
Total
(about mean) 3644.655 26

 

(b) Multiple Correlation Coefficients

 

 

 

Mean = 15.883 mg C/m3/day R2 = 0.8073 R = 0.8985
. Partial
Reg. Sig. Corr. R2
Coef. Level Coef. Deletes
Constant 481.105 0.008
Oxygen (X ) 2.143 0.015 0.513 0.738
Light (X 7 0.035 0.012 0.527 0.732
pH2(X3) -ll35.703 0.007 -0.554 0.721
(X3) 66.548 0.007 0.558 0.719
Depth (X4) 7.378 0.029 0.466 0.753
Light CO2 Fixed (X5) 0.020 <0.0005 0.749 0.560

 

*
Factors tested: Light net CO uptake, oxygen,
alkalinity, available light, tempera ure, pH, and depth.

 

78

It is concluded that the production of planktonic
organisms in Wintergreen Lake during the period 22/6 to

5/10, 1971, had the following characteristics:

1. Net primary productivity of planktonic communi—
ties was high, ranging from 300.0 to 2,762.5 mg C per m2
per day and was mostly due to the activity of blue-green
algae in the epilimnion (0-2 m).

2. Net primary productivity in the l-2 m region
was correlated significantly with the combination of

available light and chlorophyll a concentration. The net

primary productivity of the 3-5 m region was correlated
with a set of factors: available light, total alkalinity,

temperature, and oxygen concentration.

3. Dark CO2 fixation averaged 322.40 mg C per m2
per day or 21.40% of the net primary productivity (net
CO2 fixation). There was no correlation between dark
fixation and physical, chemical, and biological para-

meters measured at all depths.

4. Total excretion averaged 60.60 mg C perm2
per day or 3.5% of total net carbon fixed. Excretion is
believed to be from algae and bacteria, however, algae
were probably the most important contributors because

of their higher biomass.

5. Total net productivity including net primary

productivity, dark fixation, and excretion in Wintergreen

79

Lake was 1,890.00 mg C per m2 per day or about 150 tons
of organic carbon produced during the period of 22/6 to

5/10, 1971, in the lake.

VI. gg SITU NITROGEN FIXATION

A. Introduction

 

Blue-green algae (planktonic, benthic, and epiphy—

tic) together with bacteria, e.g. Clostridium, Azotobac-

 

Egr, photosynthetic bacteria, etc., are the most likely
organisms to contribute to the nitrogen income of the
aquatic environment. Estimation of the rates of atmos-
pheric nitrogen fixation by these organisms in situ is
difficult. The measurements based on total nitrogen
change (Bremner, 1965; Nishigaki and Shioiri, 1959), or
on 15nitrogen uptake (Dugdale 35 31., 1959; Dugdale and
Dugdale, 1962; Dugdale 35 31., 1964; Goering and Neess,
1964; Stewart, 1970a; Horne and Fogg, 1970), have certain
limitations. The first technique is not sufficiently
sensitive and is indirect, thus incorporating many
serious errors. The 15nitrogen technique is time con-
suming, expensive, and requires SOphisticated equipment
(mass spectrometer). In the last five years, the study
of biological nitrogen fixation both i§_§i£2 and ig_!£££g
has progressed rapidly following the discovery by
Scholhorn and Burris (1966, 1967) and by Dilworth (1966)

that nitrogenase also reduced acetylene to ethylene.

80

81

Hence, the measurement of the rate of reduction of
acetylene may be used as an index for the rate of
nitrogen fixation. This method has been used widely

by biologists, oceanographers, soil scientists, limnolo-
gists etc. to monitor biOIOgical nitrogen fixation
(Stewart gt_al., 1967, 1968; Hardy et 31., 1968; Brezonik
and Harper, 1969; Jewell and Kulasooriya, 1970; Elleway
g; 31., 1971: Stewart 23 $1., 1971; Macgregor and Johnson,
1971). The advantages of this technique are that it is

lSN, 106 times

sensitive (103 times more sensitive than
more sensitive than Kjeldahl analysis), cheap and rapid
(Hardy st 31., 1968). The main disadvantage, however,

is that it is indirect. However, previous studies com-

paring the 15

N and acetylene reduction methods showed a
consistent correlation, thus supporting the validity of

the latter.

B. Materials and Methods In Situ

 

The technique used for this study was adopted from
that of Hardy, Holsten, Jackson, and Burns (1968). The
samples obtained for acetylene reduction were taken at
the same time as those for limnological and productivity
studies (Chapters III, V). Sporadic checks indicated
that water without phytOplankton and from below 3 m,

did not reduce acetylene in both the summer of 1970 and
1971. Therefore, the routine acetylene reduction assay

was limited to the depths 0, 1, 2, and 3 m. The major

82

steps for preparation of the sample for the acetylene
reduction assay are summarized in Figure 17. Water was
collected at station A (Fig. 1) with a 3-1iter Van Dorn
sampler (l). A 1 liter sample (0.5 liter if phytoplankton
density was high) was filtered through a flour sifter
(top 2) in which the agitating part was removed and the
bottom screen was lined with a nylon netting of 20 0
square pore size (Nitex, Tobler, Ernst Traber, Inc.,
New York, N.Y.). The filter retained more than 95% of
the algal population as revealed by microscopic exami-
nation. The reason for not using a finer netting was to
avoid lengthy filtration periods and thus prolonged
exposure of algae to the surface environment. Algae
retained on the filter were carefully poured into a
50-ml beaker (3) and the volume was brought to 40 ml
with filtrate from the same depth. A lO—ml sample (4)
was transferred to each of four 26-ml serum bottles

(A. H. Thomas Co., Philadelphia, Pa.). In the summer
of 1970, the samples were not flushed with a nitrogen-
free gas mixture, however, in the summer of 1971, they
were flushed with a gas mixture containing 0.04% C02,
22% 02, and 78% argon (Matheson Gas Products, Joliet,
Ill.) (6). Immediately after flushing the bottles were
capped with rubber serum stoppers (A. H. Thomas Co.,
Philadelphia, Pa.); excess pressure was released by a

quick puncture with a needle. All of the bottles

83

Figure 17. Procedure for in situ acetylene reduction
assay in the lake.

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

85

prepared in this manner were stored in a dark box while
preparing ones from other depths. A portion of the head
space gas was taken out (1 or 2 ml) prior to addition of
1 ml (1970) or 2 m1 (1971) of acetylene (Matheson Gas
Products, Joliet, Ill.) (7). The algae in one of the
four bottles from each depth were killed immediately
after addition of acetylene by injecting 0.2 ml of 2%
HgClZ. This bottle served as the control. The sample
preparation for the four depths took about 40 min.
Sample bottles were incubated at ll-12 AM at their
original depth (8). The reduction reaction was stopped

by injecting 0.2 ml of 2% HgCl after one hour of incu-

2
bation in the lake (9). Samples were kept in an ice
chest during transportation from the lake to the
laboratory. Samples were allowed to equilibrate at

room temperature and 0.5 ml of the headspace gas was
injected into a gas chromatograph (10) equipped with an
Hz-flame ionization detector (Model 600-D, Varian Aero-
graph, walnut Creek, Calif.) and interfaced with a
Varian recorder (Model 20). The flow rates were:
nitrogen carrier gas (Hi-pure), 25 ml/min; H2,25 ml/min;
and compressed air, 300 ml/min. All of these gases were
purchased from General Dynamics (Detroit, Mi.). The
column was packed with Porapak N (100-120 mesh, Waters

Assoc. Inc., Framingham, Mass.). The length of the

columns varied, depending on the oven temperature

86

(3mm x 1.3 m at 85 C in 1970 or 3mm x 1.0 m at 45 C in
1971). It was found that good separation of peaks could
be achieved at low temperatures with the shorter column
even when the samples contained large quantities of
methane gas. The quantity of ethylene produced in the
sample was determined from a standard curve relating
peak height to millimicromoles ethylene. Certified
standards of 47 ppm, 830 ppm ethylene in nitrogen were
obtained (Matheson, Co., Joliet, Ill.) to prepare the
standard curve. Standards were run at each analysis
period to correct for changes in detector sensitivity.

The effect of changes of light intensity and tem-
perature on the rate of acetylene reduction by natural
populations of algae was done as follows: a water sample
was taken from 1 m at midnight (18/7/71), brought to the
laboratory, and the acetylene reduction assay was
carried out immediately. The samples were incubated
in a constant temperature aquarium described in Chap-
ter VIII at 10, 20, 30, and 35 C under 100, 300, 600,
and 1,000 ft—c, which was provided by one fluorescent
tube and four tungsten flood-light bulbs.

The effect of different light quality on the rate
of acetylene reduction was done using a unialgal culture

of Aphanizomenon (Durham strain received from Dr. J. H.

 

Gentile, National Marine Water Laboratory, West Kingston,

R.I.) grown in nitrogen-free ASM-l medium (Gorham 33 al.,

87

1964) under fluorescent light (about 250 ft-c). Sub-
samples of this culture were incubated with acetylene
(20%) in 26-ml bottles covered with white, green, and
red cellophane papers. Triplicate samples were incu-
bated at 25 C on a multi-purpose rotator (Model 150V,
Scientific Industries Inc., Queens Village, N.Y.) to
ensure even distribution of light.

To check the effect of phosphate on the rate of
acetylene reduction, various amounts of KZHPO4 were
added to 10 ml of concentrated algae samples from 2 m
to have final amended concentrations of 0.0, 10, 50,
100, and 1000 09/1. The incubation was 1 hr at 2 m.

The experiment was repeated twice on 3/7 and 16/7/71

(Anabaena was dominant).

C. Results and Discussion

 

 

The annual nitrogen income of Wintergreen Lake con-

sists of combined nitrogen and gaseous nitrogen.

1. Combined Nitrogen Sources: These sources are

 

migrating Canada geese and ducks, local geese and ducks,
effluent from a dairy feed-lot lagoon, wastes from the
near-by school, fertilizer and soil nitrogen run-off
from the surrounding cropped lands, underground water,
and aerial precipitation (rain and snow).

The contribution of nitrogen by local and migrat-

ing waterfowl to Wintergreen Lake is being studied by

88

Manny and Johnson. The nitrogen input by waterfowl
during winter alone was estimated to be about 5.0 mg
N03, 36.1 mg organic nitrogen per 102 (Manny, 1971).

There were about 300 resident waterfowl (Manny,
1971) and several thousand Canada geese and ducks which
spent a few days before flying south during late fall
and early winter. Fresh droppings of local waterfowl
contained on the average of 1.6 — 2.0% (dry weight)
nitrogen (microneldahl analysis). Such a high nitrogen
content of the excreta compared to the 0.15% reported
by Sylvester and Anderson (1964, cited by Manny, 1971),
might be explained by the nitrogen rich diet provided
by the sanctuary personnel and visitors to the waterfowl.

The concentrations of total nitrogen in the effluent
water from the dairy lagoon and the school lagoon (1.6 -
4.9 mg/l) were always higher than those in open water
(1.5 - 2.2 mg/l) in Wintergreen Lake during March-April
periods in 1970 (Manny, 1971).

The contribution of nitrogen by fertilizer run-off
or by underground water could be important particularly
in the spring. The downward movement of nitrate becomes
significant under irrigated conditions and during the
rainy season (Murphy and Gosch, 1970) especially in
porous soils (Linville and Smith, 1971) like those
which surround Wintergreen Lake.

Aerial precipitation is also an important source of

nitrate and ammonia (Delwiche, 1970). Manny (1971)

89

estimated that freshly fallen snow from Wintergreen Lake
contained (ug/l) 1124 nitrate, 16.3 nitrite, and 724
ammonia. The nitrogen content of rain water received

in Wintergreen Lake was, however, not measured.

2. Fixation gf_Atmospheric Nitrogen: Measurable

 

 

acetylene reduction in Wintergreen Lake water was con-
fined to the euphotic zone (0-3 m, Fig. 18 and 19). The
acetylene reduction by photosynthetic bacteria and other
indigenous flora in the hypolimnion was undetectable

(see Chapter IX for details). The reduction of acetylene
by mud flora was, however, not studied.

The acetylene reduction of Wintergreen Lake water
was significant only during the period of the blue-green
algal bloom in both 1970 and 1971. The fixation was
maximum in July then decreased gradually to undetectable

levels in mid-August. A dense Aphanizomenon population

 

returned in late November and early December after the

fall turnover (1970). This later bloom of Aphanizomenon

 

was more dense (approx. 5 times more filaments/1) than
that in the summer. The late algal bloom usually
accumulated in a surface layer approximately 5 m wide
and about 0.5 - 1.0 cm thick, near the southwest end
of the lake. The trichomes appeared old and clumped
together forming visible bundles. This phenomenon was

also observed by Horne and Fogg (1970) in Windermere

90

Figure 18. Rates of acetylene reduction at 0, l, 2, and
3 m in Wintergreen Lake in 1970 when
Aphanizomenon was dominant.

 

15

10

H4 FORMED (mumole/I /hr)

C:2

91

 

 

l

15

   

July

30
August
SUMMER 1970

30

 

92

 

.mH musmflm

93

K00 mmEZDm

 

 

 

 

$393 230. 0:2.
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94

(England) in 1966 where the 15N2 fixation per volume

was the highest observed in the lake. The acetylene
reduction rate by the windblown bloom in Wintergreen
was, however, undetectable. Microscopic observation
showed very few heterocycts but a large number of
gonidia.

The rate of acetylene reduction by Wintergreen
Lake water during the summer bloom of 1971 was almost
5 times that in 1970, although the number of filaments

and heterocysts of Aphanizomenon (Fig. 20) per volume

 

(1970) was almost 10 times greater than that of
Anabaena (Fig. 15) in 1971. The lower fixation rate

in 1970 may have been due to the presence of nitrogen
gas in the incubation bottles, but Stewart 35 31. (1971)

observed that N2 did not measurably affect the acetylene

 

reduction by Aphanizomenon in Wisconsin lakes because N2
is less soluble in water than acetylene. This ineffi-

ciency of Aphanizomenon in reducing acetylene (Stewart

 

3E El‘! 1967) and in N fixation (Stewart gt_al., 1968)

2
was observed in both unialgal cultures and in algal
samples taken from Lake Monona, Mendota, and Kegonsa,
Wisconsin.

The multivariate statistical anslysis (Table 13)
indicates that the weekly rate of acetylene reduction
(1971) was significantly correlated with the combination

of light, oxygen, alkalinity, depth, filament density,

heterocyst density, and pH.

Figure 20.

95

Density (numbers/l x 105) of the

Aphanizomenon filaments ( Q ) and hetero-
cysts (ief)_in 1970 at 0 m (A), l m (B),
2 m (C), and 3 m (D). The data for 5/8
is from the diurnal sampling.

POPULATION

ALGAL

96

 

 

 

 

 

 

 

 

 

 

  

 

 

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JULY AUG. SEPT. OCT.

97

Table 13.

*

Multivariate statistical analysis

 

Dependent variable:
in the summer of 1971

Rate of acetylene reduction (0-2 m)

 

 

 

 

 

 

 

y = -1290.98 + 52.87xl - 2.88x1 + 243.97x2 - 15.43x3
+ 8.03X + 99.54X - 0.13x + 0.34X - 9.15X
3 4 5 6 7
(a) Overall Analysis

Sum of Mean of .

Squares df Squares F 819'
Regression

(about mean) 79706.83 9 8856.31 15.03 <0.0005
Error 6582.75 11 589.34
Total
(about mean) 86189.58 20
(b) Multiple Correlation Coefficients
Mean = 68.21 mumole C2H4/l/hr 02 0.92 0.96
. Partial 2
Sig. R
Reg. Coef. Corr.
Level Coef. Deletes

Constant -1290.98 0.055
Light (X1) 52.87 0.001 0.79 0.80
X1 -2.88 0.001 -0.88 0.79
O ygen (X2) 243.97 0.001 0.88 0.79
X? -15.43 0.002 -0.77 0.81
A kalinity (X3) 8.03 0.050 0.55 0.89
Depth (X4) 99.54 0.003 0.74 0.83
Filaments (X ) -0.13 0.004 -0.74 0.83
Heterocysts 7X6) 0.34 <0.0005 0.89 0.63
pH (x%) -9.15 0.003 -0.75 0.82

 

*
Factors tested:

 

Available light, oxygen, alka-

linity, net primary productivity, temperature, pH, depth:

heterocyst and heterocyst-bearing algae.

98

(a) Heterocyst and Filament Densities. The

 

reduction of acetylene in Wintergreen Lake, as in the
Wisconsin Lakes (Goering and Neess, 1964; Stewart 33 31.,
1967; Stewart 35 31., 1968; Stewart 35 31., 1971), the
English Lake district (Horne and Fogg, 1970), Santuary
Lake, Pennsylvania (Dugdale and Dugdale, 1962), Sargasso
and Arabian Sea (Dugdale 33 31., 1964), Yellowstone hot
spring (Stewart, 1970a), algal crust in the Arizona
desert (Macgregor and Johnson, 1971), and flooded rice
soils (Nishigaki and Shiori, 1959), was associated with
the presence of heterocyst-bearing blue-green algae
(Fig. 11). Heterocysts are speculated to be the site
of nitrogen fixation (Stewart 25 31., 1968; Ogawa and
Carr, 1969; Wolk, 1970; Jewell and Kulasooriya, 1970;
Nelson 33 31., 1971; Wolk and Wojciuch, 1971). In
Wintergreen Lake, however, the efficiency (acetylene
reduction/heterocyst) varied considerably with depth,
month, and time (Table 14) although a positive cor-
relation (R = 0.89) between the weekly rates of acetylene
reduction and the frequency of heterocysts was found.
More direct evidence that heterocyst-bearing

Aphanizomenon and Anabaena were the agents catalyzing

 

the reduction and not epiphytic bacteria associated with
the algae, nor the planktonic bacteria, is that the
filtrate going through 20-u netting did not reduce

acetylene and the reduction of acetylene by an algal

99

 

 

 

 

 

 

 

 

 

 

000.0 0000 000.0 mmu0
00m.0 mmu0 0nm.0 0m00
mmm.0 mmu0 000.0 0mum
mom.0 mmum 0n>.0 Em mm0~0
000.0 Ed omum0 mm0.0 0N000
000.0 mmuo0 00m.0 0005
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ammooumumm\0c00mcum 0809 00>000000m\0c00>£um 0509
s 0 00 0n\m\m~nnm so 000
mm~.0 m0m.0 m0m.0 In m0m.0 In 000.0 000.0 m
500.0 0mm.0 000.0 mmm.0 0mm.0 000.0 000.0 m00.0 N
000.0 000.0 m00.0 000.0 Nb0.0 000.0 000.0 000.0 0
000.0 mmm.0 000.0 mnm.0 0m0.0 m0m.0 NN0.0 000.0 0
24 mmum 20 0000 Em 000m Ed 0000 Ed 000m 2m m0um0 2m 0005 Sm 0000
0n\>\0|m no Amv
an 000.0 000.0 0N0.0 m00.0 II II m
000.0 m00.0 000.0 000.0 000.0 0m0.0 000.0 N
000.0 000.0 In 000.0 000.0 000.0 000.0 0
000.0 000.0 000.0 000.0 0N0.0 0N0.0 000.0 0
20 0000 :0 0000 24 mmuo0 20 Noam 20 0010 20 omu00 20 0000 00000
0500\0\ma0 so 000
ammoonmumm\0c00>£um

 

Amno0 x un\ummoouwumn\00os 1&0

 

ammoou0u0£ 00m 00050009 0c00m£00 00 0000 mo £0000000> 00:0500 .00 00908

100

sample which was forced through 25 G x 5/8" hypodermic
needle (to break algal cells) was undetectable. This
is in agreement with data reported by Stewart gt El‘
(1971) that the concentration of algal sample from
Lake Mendota up to 4,000 times by filtering did not
affect the rate of acetylene reduction per water sample
unit. Also, non-heterocystous blue-green algae, for

example Oscillatoria (Horne and Fogg, 1970) or Cladophora

 

 

and Microcystis (Stewart gE_§l., 1967), presumably

 

loaded with epiphytic bacteria taken from the same

water at the same time with nitrogen-fixing Aphanizomenon

and Anabaena, did not fix 15N2 (Horne and Fogg, 1970)

 

or reduce acetylene (Stewart gt al., 1967). The negative
correlation between the rate of acetylene reduction and
filaments (trichomes) density was not expected, though
it is consistent with available information that fila-

ments without heterocysts do not reduce acetylene.

(b) Available Light. The correlation between

 

available light and weekly rate of acetylene reduction
was not linear (Table 13) apparently because high
available light became partially inhibitive to the

rate of acetylene reduction. The pattern of this
relationship may be seen in Figure 21, where strong

light intensity on the water surface apparently inhibited
acetylene reduction, but at 3 m the acetylene reduction

was negligible due to the low light intensity. The

101

Figure 21. Distribution with depth and date in 1971
of the following:

(A) Heterocystous algae (filaments/1 x 105)

(B) Heterocysts (number/l x 105)

(C) Net primary productivity (mg C/l/day)

(D) Rate of acetylene reduction (mu mole/
l/hr x 102)

 

ethyl.
O I ?
O
1
2
3
D

14
c 02

 

 

102

het. filam.

 

 

 

 

d

 

 

 

 

 

 

 

DEPTH (m)
U N -i 0

v w

 

 

 

 

wn—oo
Fl ll
d v
t

 

FW—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

103

response of the natural population of algae taken from
Wintergreen at midnight (18/7/71, 23.1 C) to various
temperatures and light intensities was mixed (Table 15a).
At 10 C, the reduction rate was highest at 100 ft-c,

but at 35 C, the highest reduction rate was obtained

at 1,000 ft-c.

In general, the reduction rate at a given light
intensity increased with the increasing temperature.

The reason for sampling at night was to eliminate as
much as possible the available carbon and light-induced
ATP. Samples were kept dark and aerobic until assay
which lasted 30 min.

The transmittance of light of different wavelengths
was not the same through Wintergreen water. As shown
in Figure 5, less blue light than red or green light
was present in the water below the surface. Red and
green lights are not optimum for all pigments of blue-
green algae (cf. Brody and Brody, 1962; Bogorad, 1962;
O'heocha, 1962), thus probably limiting photosynthesis
as well as nitrogen fixation.

In the present study, the relative rates of
acetylene reduction by Anabaena under different quality
lights were: white, 154; red, 141; and green, 138.5
(mm ethylene peak per sample per hr). Red and green
light were less effective than white; but the difference

was small.

104

Table 15a. Effect of light, temperature on rate of
acetylene reduction by a natural population
of blue-green algae obtained at midnight.
Data expressed in mumoles of ethylene pro-

duced per 1 per hr

 

 

 

 

Ft-c\\¥Temp. (C) 10 20 3O 35
100 12.30 31.00 32.60 29.40
300 5.70 30.50 23.50 42.80
600 8.80 27.00 37.50 44.00
1000 9.10 27.30 39.60 44.40
Table 15b. Effect of added phosphorous on rate of
acetylene reduction
Exp. 1 (3/7/71) Exp. 2 (16/7/71)

 

 

Added Phosphate

Ethylene produced Ethylene produced

 

(“g/1) (mm peak height/ (mm peak height/
l/hr) l/hr)
0 44.70 47
10 82.80 36
50 101.75 31-6

100 75.50 29.2
1000 --- 33.0

 

105

(c) Phosphorous. The ecological importance of

 

this element in the development of algal blooms has been
discussed briefly in Chapters IV, V. Stewart 33 El.
(1970) reported that additions of phosphate would stimu-
late the rate of acetylene reduction by laboratory grown
blue-green alga and that this technique might be used to
measure available phoSphate in natural waters. However,
in this study the effect of added phOSphorous on the rate
of acetylene reduction by natural population was incon-
clusive (Table 15b). In the first experiment, the rate
of acetylene reduction increased with increasing KZHPO4.
But in the second experiment the rate of acetylene
reduction in the presence of added phosphate was lower
than that of the control. It should be noted that the
concentration of phosphorus during the first experiment
was higher than that during the second experiment

(Fig 10A). Added phosphorous may change the delicate
ecological balance of the community through a number of
ways, (1) change of pH of the water (see review by Kuhl,
1962) (2) precipitation of iron as ferric phosphate
(Hutchinson, 1970), which is essential for algal growth
and known to be present at very low concentrations during
the growing season (McMahon, 1969), (3) stimulation of

growth of dominant Microcystis and their associated

 

bacteria (Shapiro gt_§l., 1969), causing other elements

to become limiting to the nitrogen-fixing algae,

106

(4) inhibitive effect of added phosphorous to photo-
synthesis of algae such as that which occurred in Cayuga
Lake, New York (Hamilton, 1969). The change of water pH
by added phosphate is unlikely because the pH of all
bottles including the control did not change after incu-
bation.

The effect of added phosphorus on the activity of
natural population deserves more discussion. Similar
inhibition of added phosphorus on CO2 uptake was also
observed by Wetzel (1966); Goldman and Armstrong (1969);
Kalff (1971); Sakamoto (1971) in short-term experiments.
However, Goldman and Armstrong (1969) and Sakamoto (1971)
found that CO2 uptake responded to the increasing phos-
phorus added if the incubation of samples with phosphorus
lasted longer than two days. It seems that the uncertain
response of algae to added phosphorus related the phos-
phorus pool in cells. Algae can accumulate phosphorus
more than they actually need and use it later during
environmental stress (cf. O'Kelley, 1968). When phos-
phorus was supplied to phosphorus starved algae, it may
be used for synthesis of the nucleic acids necessary for
the reproduction, therefore reducing CO2 uptake or
acetylene reduction. The increase in CO2 uptake in
long-term experiments was probably due to a demand for

carbon skeleton by the newly formed cells. Cell division

107

stimulated by a moderate amount of phosphate was
observed by Goldman and Armstrong (1969) in long-term
experiments in Lake Tahoe.

Thus the resistance of the biological system
against temporary environmental stress may pose some
problems in interpretation of nutrient-limiting experi-
ments, thus may be misleading. Three approaches may be
used to monitor limiting factors which affect the develop—
ment of algae and their productivity. (1) Changing a
factor in question by adding nutrients or manipulating
physical factors, then monitoring the response of a
natural population or pure culture. This method has
been used widely by limnologists (Gerloff and Skoog,
1954; 1957; Goldman, 1960, 1964; Wetzel, 1966; Goldman
and Armstrong, 1969; Stewart 33 31., 1970; Kalff, 1971;
Sakamoto, 1971). (2) Measurement of chemical, physical,
and biological parameters then analysis by the multi-
variate statistical method to determine limiting factor(s).
(3) Analysis of cellular composition and then compared
with critical composition determined under laboratory
conditions (Gerloff and Skoof, 1954).

The last approach is difficult and time consuming
hence it is not practical. The first method is con-
venient and fast but has some inherent uncertainties
due to the resistance of the populations to change as

discussed above. The second method requires long-range

108

study and a great number of sampling times and also has
an inherent limitation in that the values measured by
available technology may not be the true value due to
the mobility of elements. According to Pomeroy (1960):
Measurement of the concentration of dissolved
phosphate in natural waters gives a very limited
indication of phosphate availability. Much or
virtually all of phosphate in the system may be
inside organism at any given time, yet it may be
overturning every hour with the result that there
will be a constant supply of phosphate for organisms
able to concentrate it from a very dilute solution.
The advantage of this method is that one is able to
analyze a large number of factors at the same time.
The use of any one of the above approaches to study
a complex ecosystem such as Wintergreen Lake would be
shortcoming due to the inherited uncertainties discussed

above. The combination of the first two approaches

would be desirable.

(d) Oxygen. It is well known that oxygen inhibits
nitrogenase activity of whole cells (Stewart, 1969;
Stewart and Lex, 1970; Stewart and Pearson, 1970; Drozd
gt 31., 1970). In the present study, the in_§itu data
indicate that low levels of oxygen did not appear to
inhibit the rate of acetylene reduction (Table 12),

though higher levels may.

(e) Combined Nitrogen. The periods of measurable

 

acetylene reduction and large numbers of heterocysts in

109

1970—1971 coincided with the lowest concentrations of
nitrate and ammonium (Fig. 10, B, C) in Wintergreen Lake
water. This inverse correlation between nitrogen fix-
ation or number of heterocysts and combined nitrogen
under laboratory and in situ_conditions is well docu-
mented. Ogawa and Carr (1969) reported that ammonium
has the strongest suppressive effect on heterocyst form-
ation and that heterocyst production was evident 24 hr

after transferring Anabaena variabilis from nitrate con-

 

taining medium to a nitrogen-free medium. According to
Horne and Fogg (1970), 15N2 fixation maxima in surface
water (0-5 m) in Windermere and Esthwaite Water (England)
coincided with the lowest values of nitrate-N concen-
tration (200 09/1). The concentration of ammonium-N
rarely exceeded 20 ug/l, but it rose sharply after the
fixation peak; probably due to algal excretions or lysis
(Fogg, 1952; Fogg and Westlake, 1955). The concen-
trations of nitrate at the fixation maxima were even
much lower (1 09/1) in Lake Mendota (Stewart gt_§l.,
1971) and undetectable in Sanctuary Lake, Pennsylvania
(Dugdale and Dugdale, 1962). The observation that com-
bined nitrogen inhibits nitrogen fixation by living
cells is well accepted but the mechanism of the inhi-
bition is not certain. Stewart gt El' (1968) found that

ammonium and nitrate did not have an immediate effect

on acetylene reduction of several pure, unialgal cultures,

110

or natural populations of blue-green algae. They
speculated that combined nitrogen did not inhibit
nitrogenase activity but suppressed nitrogenase syn-
thesis. This argument is in line with the findings of
Hardy EE.E£° (1968) that the nitrogenase preparation of

Azotobacter was not sensitive to low levels of ammonium

 

but rather was inhibited by high concentrations of

NH: ions as well as that of Na+ ions. Jewell and
Kulasooriya (1970) found that the rate of acetylene
reduction by blue-green algae grown on a nitrogen-free
medium declined after a maximum value until most of the
newly fixed nitrogen in the cellular pool was used for
cellular synthesis, after which the rate of reduction
increased again. These authors concluded that the
complex pattern of nitrogen fixation observed, depended
on the internal nitrogen pool, age, and possibly newly
excreted organic nitrogen. Nelson and his coworkers
(1971) also found that the internal nitrogen pool could
regulate nitrogenase activity; furthermore, they found
that upon transferring from a nitrate-containing medium
to a nitrogen-free medium, Anabaena g2. produced more
heterocysts and nitrogenase under an argon-CO2 atmosphere
than under a Nz-CO2 atmOSphere. Thus, it seems that N2
does not directly control nitrogenase synthesis nor
does combined nitrogen control its activity (gig

classical allostery). Rather, combined nitrogen in

111

the medium and/or the cellular pool as well as other
physiological parameters of the cell seem to turn on

or off the nitrogenase synthesizing activity. When
nitrogen becomes available from newly fixed nitrogen

or from an external source, the cell may, in addition

to stopping new nitrogenase synthesis, shut off fixation
by diverting energy and the limited reducing power from
nitrogenase. The delayed inhibition (60 min) of
acetylene reduction by combined nitrogen observed by
Stewart and his coworkers (1968) on Nostoc is consistent
with the above explanation since the heterocysts could
carry out fixation with ATP provided by photosystem I

in the heterocyst (Fay 35 31., 1968) until available
reducing power and/or carbon skeleton already pumped

in from vegetative cells is exhausted, hence, nitrogen
fixation ceases.

The relatively smooth curves for acetylene
reduction for 1970 and 1971 (Fig. 18, 19), as com-
pared to Figure 1 of Jewell and Kulasooriya (1970),
could be due to a number of factors, (1) the long
spacing of time between samplings in this study,
therefore the turning points were missing, (2) the
algal population in Wintergreen Lake water was in a
steady state in which sinking filaments were replaced

by young active filaments, (3) the excreted organic

112

nitrogen was utilized immediately by the non-nitrogen—
fixing organisms, (4) any combination of the above

factors.

(f) Other Factors. The positive correlation between

 

acetylene reduction and depth is understandable since
only data from 0-2 m were analyzed and, as Figure 11
indicated, higher rates of acetylene reduction occurred
at 1 and 2 m. There was a relationship between total
alkalinity and acetylene reduction; however, this cor-
relation was not very significant. The non-linear
negative correlation between pH and acetylene reduction
indicates that most of the time, high pH was inversely
related to acetylene reduction. The lack of correlation
between acetylene reduction and CO2 uptake or chlorophyll

a was probably due to the interference of the Microcystis.

 

(g) Diurnal Acetylene Reduction. The diurnal

 

acetylene reduction measurement was done once in 1970
and twice in 1971 during the period of high acetylene

reduction. Both the bloom of Aphanizomenon (1970) and

 

Anabaena (1971) had the capacity to reduce acetylene at
night although the pattern and the significance of the
reduction by the two blooms were different. Figures 22
and 23 show the variation of acetylene reduction rate,
algal population, and heterocysts at each depth during

a 24-hr period on 4-5/8/70. The reduction rates, at

113

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TIME OF DAY

TIME OF DAY

Figure 23.

115

Densities of Aphanizomenon heterocysts
during the diurnal sampling period of
4-5/8/70.

 

NO. OF HETEROCYSTS /L|TER x 106

.00
O

N
O

8

8PM

116

 

 

 

TIME OF DAY

117

all depths measured, except at 3 m, were higher during
the night than during the day. The reduction rate at

3 m was zero at night, reached the maximum value at 5 PM
then decreased to zero again at sunset. The highest
reduction rate was recorded at sunrise at 2 m which also
correSponded to high algal and heterocyst pOpulations.
The most significant observation from this experiment is
the apparent inhibition of acetylene reduction during

the day (5.20 x 10'6

mumoles ethylene/filament produced
at 5:00 AM compared to 1.13 X 10.6 mumoles ethylene/
filament produced at 10:50 AM). Figures 22 and 23 also
show the vertical diurnal distribution of the blue-green
algae population as is discussed in Chapter V.

Figure 24 shows the acetylene reduction pattern
during the 36-hr period of 5-6/7/71. The top curve
shows the relative distribution of light received. The
reduction rate, however, was highest during the day and
lowest at night. The lag in the maximum rate of reduction
during the first day was correlated to a cloudy sky
(storm) which cleared at 6 PM. On the following clear
day (6/7/71), the reduction rates reached a maximum in
the early morning (8 AM) then decreased while the algal
population did not change. This low acetylene reduction
(0-2 m) may have been due to the high light intensity
and/or excess oxygen from photosynthesis (Stewart and

Pearson, 1970).

118

Figure 24. Diurnal solar radiation received (relative
units, top), rates of acetylene reduction,
Aphanizomenon plus Anabaena population
densitiés, and total heterocyst densities
during the diurnal sampling period of
5-6/7/71.

 

9H4 FORMED (mu

3

FILAMENTS/ L X10

NO OF HE TEROCYSTSILX'O:3

A

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119

K 1 A A A L A A A A A A

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I-IAAIAJIAIAIIAAIIL

  

 

 

 

 

 

 

120

Another 28-hour sampling period (27-28/7/71) at
l m was designed to follow more closely the diurnal
variation of acetylene reduction. The sky was clear
except for clouds at 11 AM (27/7/71) and at 9 AM
(28/7/71). The lag in the reduction maximum (about
2 PM), Figure 25, was probably related to the high
turbidity of the water; only 35% of the surface light
reached 1 m depth and the secchi disc transparency was
near the lowest observed value (0.9 m). The reduction
rate was lowest at 10 PM then increased rapidly after

sunrise. Here again, Anabaena and Microcystis apparently

 

moved up during the daytime and sank at night (Fig. 26).

The double peaks (10 AM and 5-6 PM) of the rate
of acetylene reduction by Anabaena blooms reported by
Stewart st 31. (1971) in Green Bay were not observed
in any diurnal measurement at Wintergreen Lake.

The capacity of blue-green algae to grow hetero-
trophically in the dark in nitrogen-free medium has
been reported by Watanabe (1967) and Fay (1965), but
it has not been studied in the natural environment. The
dark fixation of 15N2 reported by Horne and Fogg (1970)
in Esthwaite and Windermere by natural populations of

Aphanizomenon and Anabaena or the acetylene reduction

 

in the dark detected by Stewart and his coworkers (1967)
in Lake Mendota were limited to dark incubations for a

short period after light. The light energy stored as

121

Figure 25. Diurnal rates of acetylene reduction at 1
during the diurnal sampling period of
27-28/7/71 when Anabaena was dominant.

 

 
 

 

 

 

 

5 O

AL£\_\0_OE1F5 Owl—210“.

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5

0:

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DAY

 

 

TIME OF DAY

123

Figure 26. Densities of Anabaena filaments (l),
Anabaena heterocysts (2), and Microcystis

 

colonies (3) at l m during theriurnaT
sampling period of 27-28/7/71.

ALGAL COUNTS PER LITERX'IO

124

 

 

 

 

 

TIME OF DAY

125

ATP could be sufficient to allow algae to fix nitrogen
or to reduce acetylene in such a short period. The
dark acetylene reduction reported here was carried out

by long-term light starved cells of Aphanizomenon and

 

Anabaena in which light-induced ATP was probably
exhausted. The argument that epiphytic bacteria were
responsible for the acetylene reduction does not appear
to be true because, as mentioned earlier, the algal
sample forced through 25 G 5/8" hypodermic needle
reduced insignificant amounts of acetylene. Acetylene
reduction by planktonic bacteria was also unlikely;

therefore, Aphanizomenon and Anabaena were the organisms

 

responsible for acetylene reduction during the night.
The energy and reductant needed by the blue-green algae
for dark acetylene reduction was probably provided
through several respiratory and fermentive pathways
(Leach and Carr, 1970, also see review by Gibbs, 1962).
The relative importance of the contribution of extra-
cellular organic carbon and internally accumulated
photosynthetic products, however, was not known. Khoja
and Whittman (1971) recently reported a list of 10
species of blue-green algae including Anabaena sp.,
which can grow heterotrophically in dark in nitrogen-
free medium.

Aphanizomenon seems more efficient in acetylene

 

reduction at night and at low light intensity (sunrise)

126

than under bright day light (Fig. 22). The acetylene
reduction at sunrise was almost 400% that at full light.
This hypothesis was supported by a laboratory study in

which a unialgal culture of Aphanizomenon was grown

 

and assayed for acetylene reduction at a light intensity

of either 300 or 1,200 ft—c. Aphanizomenon reduced

 

more acetylene at the low light intensity than at the
high light intensity (140 mm ethylene at 1,200 ft-c
versus 166 mm at 300 ft-c).

The reverse situation was, however, obtained with

the Anabaena bloom which reduced more acetylene in the

 

day than at night. The acetylene reduction at night
by the bloom of Anabaena accounted for approximately
20% of that reduced in the day (Fig. 25).

The recovery of light-starved Aphanizomenon and

 

Anabaena was rapid. When a unialgal Aphanizomenon

 

culture was assayed at the end of a lO-hr dark-growing
cycle, the reduction rate was saturated within two hr
after the light was turned on (Fig. 27). A similar

recovery in a sample of Anabaena, taken from Wintergreen

 

Lake (7/19/71) and kept in dark for about 17 hr, was
obtained within 2 hr (Fig. 28). These results may
explain the early daily maximum of the rate of

acetylene reduction at sunrise by Aphanizomenon and by

 

Anabaena at 8 AM.

127

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131

Thus the diurnal data indicate that acetylene
reduction at night was significant. How light-starved
cells acquired the ATP required for acetylene reduction
remains puzzling. It could be provided by respiration
or fermentation of photosynthetic reserves.

The combination of laboratory and in situ data

 

over the two-year period leads to the following con-

clusions:

(l) Heterocyst bearing Aphanizomenon (1970) and

 

Anabaena (1971) were the main organisms responsible for

the acetylene reduction measured. In general, acetylene
reduction rates were correlated with heterocyst number.

There is no evidence that planktonic or epiphytic

bacteria associated with phytoplankton reduced acetylene.

(2) The period of acetylene reduction and presence
of heterocyst bearing blue-green algae coincided with
the period when the concentration of combined nitrogen

(nitrate and ammonium) was at low levels.

(3) The acetylene reduction was limited to the
euphotic zone (0-3 m). The effects of light on acetylene
reduction rate were related to depth and water trans-

parency.

(4) Both Aphanizomenon and Anabaena in Wintergreen

 

Lake were capable of reducing acetylene both at day

and night. Total acetylene reduction averaged 3,083.5

132

umole/mZ/day assuming a lZ-hr reduction period. Using
the theoretical conversion factor of 1.5 (ethylene pro-
duced: ammonia formed) the total nitrogen fixed by
planktonic algae in Wintergreen Lake was estimated about
315 Kg ammonia per season or 21 Kg ammonia per hectare

per season.

VII. ACETYLENE REDUCTION BY NITROGEN-
FIXING ORGANISMS ASSOCIATED

WITH DUCKWEEDS

A. Introduction

 

Duckweeds or Lemnaceae are the smallest aquatic
flowering plants. Their distribution is world wide
(Daubs, 1965). Rao (1953) reported that thick duckweed
communities presumably modified the thsical and chemical
properties of the water underneath favoring the develop-
ment of blue-green algae over other algae, although any
direct effect of duckweeds on the growth of these blue-
green algae has not been elucidated. On the other hand
Bottomley (1919) reported that exudates of nitrogen-
fixing organisms and other bacteria promoted growth of
Lemna. In this part of the study, the composition and
the distribution of the duckweed community in Wintergreen
Lake and in the small lagoons and shallow ponds connected
with it were conducted. The nitrogenase activity of
organisms associated with duckweeds and the morphologi-

cal aspect of this interaction were also investigated.

133

134

B. Materials and Methods

 

Duckweeds were identified using Daubs' Monograph
of Lemnaceae (Daubs, 1965) as a guide. For the acetylene
reduction assay, water containing duckweeds was collected
at station B (see Fig. l) and passed through a flour
Sifter with a metal screen of 1.5-mm square openings.

The sifter retained Lemna and Spirodela. The filtrate

 

which contained Wolffia was passed through a second
modified sifter that was equipped with nylon netting

of 35-u pore size (Nitex, Tobler, Ernst Traber, Inc.,
New York), which retained all Wolffia. Water passing
this filter was called the last filtrate. Since it was

too time consuming to separate Lemna from Spirodela they

 

were always assayed together for acetylene reduction.

Lemna and Spirodela or Wolffia were removed from the

 

Sifter and dried briefly by pressing them gently between
towel paper. One gram of each category was immediately
weighed and transferred to a 26-ml serum bottle. The
last filtrate was used to bring the total volume in

each bottle to 16 ml. The details of preparation and
analysis for acetylene reduction were identical to those
in Chapter VI. Triplicate samples of the last filtrate,

W01ffia and the mixture of Lemna and Spirodela, were

 

incubated with 10% acetylene for one hour at the lake
water surface. The reduction was stopped by injecting

0.5 m1 of 2% HgCl2 into the bottles. One bottle from

135

each category which had HgCl2 added at the beginning
of the incubation served as a control.

The effect of light intensities and temperatures
on the rate of acetylene reduction by duckweeds was
studied as follows: samples of duckweeds collected from
Wintergreen Lake were prepared as described above and
incubated in the constant-temperature aquarium under
light of 100, 300, 600, and 1,000 ft-c intensities.
Temperatures tested were 10, 25, and 35 C.

Axenic cultures of Lemna perpusilla Torrey and

 

Lemna gibba Limnaeus obtained from Dr. J. A. D. Zee-

 

vaart (Plant Biology Laboratory, Michigan State Uni-
versity) were also tested for acetylene reduction.
They were grown in Hutner's medium with or without
combined nitrogen. The carbon source was either 1%
sucrose (w/v) or 0.1% NaHCO3. They were grown in a
temperature-controlled growth chamber (Model CEL 25-7,
Sherer-Gillett Co., Marshal, Michigan) under 600 ft-c
(when supplied with sucrose) or 1,200 ft-c (when CO2
was carbon source) of fluorescent and tungsten light.
Growth temperature was 20-22 C. The acetylene assay
was performed with 5- or 7-day-old cultures. The
details of the preparation for acetylene reduction
were the same as those described above.

Fresh duckweeds were examined under a stereo-

microscope at lOOX magnification to locate "algal

136

colonies." The colonized leaves were sliced vertically
into thin sections by hand using razor blades and
examined immediately under the Leitz phase microscope.
Leaves were also pressed to release the blue-green algal

colonies from duckweed.

C. Results

1. Composition of Duckweeds. The community of

 

duckweeds was composed exclusively of Lemna, Spirodela

 

polyrhiza, and Wolffia columbia, although the percentage

 

 

of each species varied with time. Species of Lemna were
not identified. During the early summer (June), Lemna

and Spirodela dominated the population (ratio between

 

them was 1:1) with very few Wolffia observed. Wolffia
became more prevalent in late July and finally overgrew
the others in late September (the ratio between Lemna

and Spirodela was about 1:2 at that time). Lemna and

 

Spirodela appeared to be struggling for survival; their

 

leaves were smaller and turion formation was obvious.

2. Distribution of Duckweeds. Duckweeds, at their

 

 

maximum development in July and August, formed a dense
layer covering the shallow lagoons completely, the dairy
lagoon, and the Duck pond. In Wintergreen Lake itself,
duckweeds formed a broad irregular marginal band 5-15 m

wide along the shores. They were entangled with emerged

137

Ceratophyllum, Myriophyllum, Potagometon, Najas, and

 

Nuphar along the west and south-west sides. Duckweeds
were also piled up in a thick layer 3-8 m wide by the
prevailing south to north wind at the north end during
early July. Their distribution was reduced to the very
edges of the lake at the end of the summer. The remain-
ing duckweeds were eaten by ducks, and other birds migrat-
ing from Canada (October-December) or died when the water

froze.

3. Nitrogen Fixation. Wolffia, the water surround-

 

ing the duckweed or axenic Lemna did not reduce acetylene.
Egmna and Spirodela, with associated microflora, from the
lake did. Table 16a shows the quantities of ethylene
produced by duckweeds collected in the late summer;
turions were present in all samples. Photoreduction of

acetylene by Lemna and Spirodela in situ was five times

 

 

as much as dark reduction (Table 16b). The acetylene
reducing activity seemed to be associated with the leaves
rather than the roots (Table 16b). The higher rate of
activity of the root-free plants than the whole plants
may be explained as follows: the root-free leaves may
release some organic materials which enhanced the
activity of the associated nitrogen-fixing organisms.
Also, the higher availability of light may have been
reSponsible for this discrepancy since only 0.5 g of

wet weight of root-free leaves was assayed. To determine

138

Table 16a. In situ rate of acetylene reduction by whole
plants of Lemna and Spirodela collected from
Wintergreen Lake in 1971

 

 

 

C H Formed

 

 

Date 2 4
(mumole/gwet wt/hr)
24/8 93.08
1/9 46.80
7/9 90.00
21/9 27.20

 

Table 16b. Effect of plant part and light on in situ
acetylene reduction by plants of Lemna and

Spirodela (samples taken from Wintergreen
Lake on 1/9/71).

 

 

 

C H Formed

 

 

Conditions 2 4
(mumole/gwet wt/hr)
Whole plants (light) 46.80
Excised roots (light) 4.20
Leaves only (light) 92.16
Shaken water (light) 2.13
Water (light) 0.00

Whole plants (dark) 10.08

 

139

how firmly nitrogen-fixing organisms were attached to
duckweeds, water containing the Lemnaceae was shaken
by hand for five minutes and Wolffia in the first filtrate
was picked up and discarded. The filtrate prepared in
this way (without Wolffia) reduced very little acetylene
(Table 16b).

The reduction of acetylene by freshly collected

Lemna and Spirodela from Wintergreen Lake which were

 

incubated in a temperature controlled aquarium increased
with increasing temperature and light intensity (Fig. 29).
The low acetylene reduction in the laboratory study may
have been due to the prolonged darkness during transpor-
tation from the lake to the laboratory.

Axenic cultures of Lemna perpusilla or Lemna gibba

 

 

did not reduce acetylene. Leaves of Lemna in all nitrogen-
free cultures turned yellowish within 10 days due to
nitrogen deficiency. Upon introduction of a growing

unialgal culture of Aphanizomenon, the dying Lemna

 

recovered within a week and reproduced normally for 6
months; the other cultures that did not receive Aphani-
zomenon eventually died. The surviving Lemna depended

completely on blue-green algae for its nitrogen.

4. Anatomy Study. The anatomy of Lemna and Spiro-

 

dela leaves (fronds) was similar to that of the floating
leaves of more complex aquatic flowering plants. The

leaf was made up mainly by spongeneous meSOphyll with

Figure 29.

140

Effect of light and temperature on the

rate of acetylene reduction by natural
samples of Lemna and Spirodela incubated

in the laboratory. Samples were taken from
Wintergreen Lake on 4/9/71.

 

141

 

 

 

 

 

 

 

 

O 5 ‘ 0
t5 t5 MoguxmfiElEvomeOu vaU

142

large lacunae (Jacob, 1947). The upper epidermis had a
thick cuticune and the lower was bare but protected by a
thin mucilagenous layer. Each leaf had two pockets in
which flowers, fruits, or turions were formed. Three
species of heterocyst bearing blue-green algae were

found to attach to Lemna and Spirodela leaves. Two

 

species had a tapering filamentous form and belonged to

the genera Calothrix and Dichothrix (Fig. 30). Both had

 

 

sheaths and base heterocysts. The other species was
fragile, formed irregular colonies, and belonged to the

genus Anabaena (Fig. 30). Calothrix, Dicothrix, and

 

 

 

Anabaena were found mostly inside the pockets of mature

 

Lemna and Spirodela plants or in the lower mesophyll of

 

older leaves which had turned yellow.

D. Discussion

 

From the above observations and experimental data,
it may be concluded that heterocyst bearing blue-green

algae tightly associated with Lemna and Spirodela plants

 

were capable of reducing acetylene. The seasonal suc-
cession of species in the natural environment may be
restated to emphasize the role of available nitrogen and
other nutrients. The symptoms of nitrogen deficiency

described by Jacobs (1947) were observed on Lemna and

 

Spirodela, but not on Wolffia, when available nitrogen

 

concentration in the water was lowest. Wolffia, which

143

Figure 30. Algae associated with Lemna and Spirodela.

 

 

(1) Anabaena colony which developed on
lower mesophyll of Spirodela polyrhiza.
The figure also shows heterocysts (arrow).

(2) Small colony of Calothrix (arrow)
entangled with organic matter inside
Spirodela polyrhiza pocket.

(3) Blue-green algae separated from lower

mesophyll of Lemna showing heterocysts
(arrow).

Scale: Bars represent 10 u.

 

 

144

 

 

 

 

 

145

apparently could not compete well with other duckweeds
for nutrients in the early summer, outgrew Lemna and

Spirodela when nitrogen, phosphate, and possibly other

 

elements were depleted.

There are several possibilities for the nature of
the interaction between the duckweeds and the algae.
Symbiosis is unlikely for the following reasons: Lemna

and Spirodela showed a nitrogen deficiency while associ-

 

ated blue-green algae fixed nitrogen actively. Blue-green
algae were reported to secrete nitrogenous substances
which were made up mainly of ring structured polypeptides
which may not be usable by duckweeds (Fogg, 1952, 1966;
and Bottomley, 1919). These observations and experimental
results are not contrary to the laboratory data. Nitrogen

starved Lemna grown with Aphanizomenon, showed signs of

 

recovery when Aphanizomenon became old, and presumably

 

some filaments lysed; also the number of bacteria origi-
nally associated with this alga was high at this time

(about 106

cells per ml). Thus it is possible that Lemna
utilized nitrogenous compounds resulting from bacterial
degradation of excreted polypeptides. Thus duckweed
might not behave like a partner of symbiosis with blue-
green algae but rather assimilate nitrogen from lysed
cells or from byproducts of bacterial degradation. The
nitrogen deficiency of duckweeds indicated that they

were not obtaining sufficient nitrogen from the associated

blue-green algae.

146

Commensalism is probably more descriptive of this
association. Blue-green algae as well as other micro-
organisms may find duckweed leaves a good place for
physical support, protection against direct sun light,
and an excellent source of carbohydrates and growth
factors. No heterocyst bearing blue-green algae were
ever detected attached to the upper leaf surface or the
root system. Wetzel and Manny (1972, in press)
reported that about 0.02-0.07% of CO2 uptake by

Lemna perpusilla was excreted under laboratory conditions.

 

Masses of blue-green algae entangled with mucilagenous
substances in the pockets (Fig. 30, 2) and the lower
mesophyll of duckweeds were observed under the microscope
by using India ink. These substances were possibly
secreted by both the algae and duckweeds.

Thus heterocyst bearing blue-green algae associated

with Lemna and Spirodela appeared to be epiphytes and

 

active nitrogen fixers in the natural environment when
available nitrogen was limited. The contribution of this
association to the nitrogen income of Wintergreen Lake
and its associated lagoons was estimated at 37 kg NH3
per season. Thus total nitrogen fixed by planktonic
algae and duckweed—algae association during nitrogen

fixation season was 352 kg NH The economic implication

3.
of this loose association and the food quality of Lemna-

ceae in cattle feeding are very important.

147

The present finding of the association between
duckweeds and blue-green algae which fix atmospheric
nitrogen add to the possible economic potential of these
plants. It was an old practice of the Vietnamese farmers
to fertilize their small backyard vegetable garden with
Azolla and duckweeds to improve yield. Since duckweeds
have a great capacity to tolerate pollution (Coler and
Gunner, 1970), they can be grown in carbon-enriched
ponds or lakes for mass production. Muzafarov gt 31.

(1968) found that Lemna minor L. contained 30-32% of

 

albumins, 30-35% of starch plus vitamins. Recently
duckweeds have been used to feed cattle since they are
nutritious, easy to grow in mass, and fast growing
(Abdulaev, 1969). Poultry has been shown to prefer
Lgm23_and both yield and quality of meat increased when
poultry was fed with Lemna (Muzafarov 35 31., 1968).

These workers found that the amount of vitamin A and
carotene in the liver and yolk of tested poultry increased
2-2.3 times over the controls.

The duckweed-algae association also offers some
potential for solving problems of recycling elements in
closed environments such as long-term space travel, if
higher plants have to be used as proposed by Whatley
(1971). The advantage of this association is that it

can provide oxygen and human food; it needs a very small

148

space for growth, and can grow on liquid waste, and is
apparently more palatable than many algae.

More study of physical, physiological relationship
between duckweeds and their associated communities as
well as the contribution of duckweed—blue-green algae

association is needed.

VII I . PHOTOSYNTHETIC BACTERIA

A. Introduction

 

The photosynthetic bacteria are among the most
primitive, currently living forms (Losada et_gl., 1960)
which are widespread in nature. They are comprised of
green sulfur, purple sulfur, and purple non-sulfur
bacteria. The photosynthetic bacteria are characterized
by their ability to grow under anaerobic conditions
when exposed to light and in the presence of an oxidizable
substrate, the nature of which varies with the species
(van Niel, 1931-1971). The photometabolism of C02,
organic acids, alcohols, etc., which are believed to be
the major fermentative products, by photosynthetic bac-
teria has been studied extensively in several laboratories
(van Niel, 1931-1971; Muller, 1933; Foster, 1940; Gest
EE.E£°' 1950; Glover 22 31., 1952; Eisenberg, 1953; Korn-
berg and Lascelles, 1960; Losada gt‘al., 1960; Stanier
gt 31., 1959; Hurlbert and Lascelles, 1963; Nesterov
§E_§1,, 1965; Anderson and Fuller, 1967; Evans g£_§l.,
1966; Stokes and Hoare, 1969; Schick, 1971c, also cf.

Wiessner, 1970). The metabolism of CO and simple

2

organic acids by photosynthetic bacteria grown

149

150

anaerobically in the light could occur through several
alternative pathways; the reductive pentose phosphate
pathway (Losada gt 31" 1960), the reductive tricarboxylic
acid cycle (Evans 33 31., 1966), the forward tricarboxylic
acid cycle, and the glyoxylate cycle (Losada 35 31., 1960),
etc. The importance of each cycle for any one organism
is dictated by its nutritional history, the composition
of the carbon source, and the incubation conditions of
the experiment. It has been shown that photosynthetic
bacteria prefer to assimilate simple organic acids over
CO2 (Anderson and Fuller, 1967). In most cases, the
simultaneous assimilation of CO2 and organic acids
results in a decrease or complete suppression of fixation
of the exogenous C02. Several species of photosynthetic
bacteria have also been shown to have the ability to fix
atmospheric nitrogen when grown in nitrogen-free media,
in the light, and under anaerobic conditions (Kamen and
Gest, 1949; Lindstrom 33 31., 1950; Lindstrom gt 31.,
1951; Gest 3E 31., 1950; Wall §E_§l,, 1952; Newton and
Wilson, 1953; Okuda and his coworkers, 1960-1961; Arnon
35 31., 1961; Winter and Arnon, 1970; Evans and Smith,
1971; Schick, 1971a, 1971b; also cf. Stewart, 1966).

The purpose of this research was to isolate photo-
synthetic bacteria from Wintergreen Lake and to study
their capacity to reduce acetylene and the various factors

which affect this activity.

151

B. Materials and Methods

 

1. van Niel's Modified Medium. For the enrichment

 

cultures a standard modification of van Niel's mineral
medium (1944) was used consisting of 0.5 g (NH4)ZSO4,

0.5 9 K HPO4, 0.2 g M9804, 0.2 9 NaCl, 3.0 g NaHCO

2 3,
1.0 g yeast extract in 900 ml distilled water. The

medium was adjusted to pH 6.8 with 5% H3PO4. Three grams
of sodium acetate was dissolved in 100 ml of distilled
water. The salt and acetate solutions were autoclaved
separately and mixed together when they cooled to room

temperature.

2. Medium "98." Van Niel's modified medium gave
low cell yields when yeast extract was omitted. Unless
otherwise noted, the following medium "98" was used in
all experiments with pure cultures of photosynthetic
bacteria. Solution 1: l l of deionized water with

40.0 g KH P04 and 60.0 g K2HP04. Solution 2: l 1 of

2

deionized water with 2.0 g MgSO 7H 0, and 0.5 9 CaCl

4' 2 2'
Solution 3: (Pfennig and Lippert, 1966) 500 mg of EDTA
(ethylene diamine tetraacetic acid) was dissolved in

100 m1 deionized water. The solution was adjusted to

pH 6.8 with 10% NaOH. EDTA was then mixed with other
salt solutions consisting of 800 m1 deionized water with

200 mg FeSO4.7H20, 10 mg ZnSO4.7H20, 3 mg MnC12.4H20,

152

2 mg NiCl .6H20, and 3 mg Na MoO 2H 0. This solution

2 2 4' 2
was diluted to l l with deionized water.

The final mineral salt solution was prepared by
adding 15 m1 of solution 1, 100 ml of solution 2, and
10 ml of solution 3 to 500 ml deionized water, mixed.
This solution was diluted to 900 ml with deionized water.
The pH of the final mineral solution was 6.7-6.9. The
organic substrate and 2.0 mg of yeast extract were
dissolved in 100 ml of deionized water. The final
mineral salt and organic substrate solutions were auto-

claved separately, cooled to room temperature under

repurified nitrogen gas then mixed together.

3. Isolation. All isolations of photosynthetic

 

bacteria were done in the summer of 1970. Sediments or
partly degraded algal mats which floated to the surface

of the lake were collected into 120 ml screw-caped bottles;
the bottles were then filled with surface lake water.

The algal mats were decaying residue from the lake bottom
which had migrated to the surface. These mats were often
dotted with red spots of colonies of Rhodopseudomonas,

Rhodospirillum on the surface layer, and pink colonies

 

of Thiopedia underneath. Portions of lake water from

 

the anaerobic zone were also collected into other bottles
which were then filled with van Niel's modified medium.
All bottles were incubated under a 60-watt tungsten bulb

at room temperature. Visual development of purple color

153

occurred within 4-7 days in the bottles containing the
algal mat, 15-20 days in those containing sediment, and
5-10 days in those containing hypolimnion water. This
primary enrichment was subcultured several times in the
above medium. Cell suspensions from these secondary
enrichments were diluted in sterile deionized water

from which a loopful was transferred to one of a series
of test tubes (15 X 150 mm) containing 15 ml of nitrogen-
free medium "98" with 1.7% agar, kept at 45 C in a water
bath. Each inoculated tube was shaken using a test tube
mixer (Fisher Mini-Shaker Model 58, Fisher Scientific Co.,
Chicago, 111.). A loop of agar from this tube was then
transferred to the second molten agar tube, mixed and

so on, to give a total of four dilutions of agar shake
tubes. Tubes were flushed with nitrogen gas (Hi pure,
99.9998% pure) and capped with rubber stoppers. All
tubes were incubated at room temperature under 60-watt

incandescent light. Irregular colonies of Rhodomicrobium

 

began to develop within 10—15 days, in the bottom half

of the test tubes. Rhodopseudomonas developed earlier

 

(5-7 days) and formed spindle colonies throughout the
agar column. The tubes and then the agar column were
broken and the individual colonies exposed at the agar
break were picked and suSpended in oxygen-free sterile
deionized water to repeat the shake tube isolation.

Rhodopseudomonas cells were usually trapped inside

 

154

colonies of Rhodomicrobium by the branched network of

 

the latter. Therefore, shake tube isolation and sub-
culturing in liquid medium were repeated until pure
cultures were obtained.

Rhodopseudomonas strains were isolated using either

 

the above shake-tube technique or by streaking a cell
suspension on agar slants that were prepared in wide-
mouth bottles under a nitrogen atmosphere (Hi pure).

Rhodopseudomonas developed faster than Rhodomicrobium;

 

 

therefore they could be isolated easily using the latter

technique.

4. Preparations for Electron Microscope Studies.

 

For morphological studies, cell suspensions were diluted
in deionized water to about 103 cell/ml. A drOp of this
suspension was transferred to an electron microsc0pe grid
(200 mesh) which had been previously coated with a thin
layer of 0.05% collodion (for negative stain) or 0.25%
formvar (for carbon shadow). The grid with the cell
suspension was allowed to sit for about 2 min, then it
was drained and air dried. Air dried cells on grids
were coated with gold-palladium #46 at 35 or 45°. The
negative stain was done with 0.5% of phosphotunsgtic
acid in water. The prepared grids were examined with a
Hitachi electron microscope (HU-ll, Hitachi, Ltd.,
Tokyo) or a Philips EM-300 electron microscope (T.M.N.V.

Philips of Holland).

155

5. Anaerobic Gas Flushing System. Unless other-

 

wise noted, all work on photosyntehtic bacteria, such

as transferring, preparation of media, nitrogen fixation
assay, etc., was done under repurified nitrogen or argon.
Traces of oxygen in these gases were removed by the Hun-
gate technique (Hungate, 1950) as modified by Bryant and
Robinson (1961). The anaerobic flushing system (Fig. 31)
composed of a vertical furnace (Sargent-Welch Scientific
Co., Detroit, Mi.), maximum temperature of about 500 C,
which contained a quartz tube (homemade, 2.5 X 3.0 cm)
filled to two-thirds of its length with cupric oxide wire
(CuO, Fisher Scientific Co., Fair Lawn, N.J.). The tube
was plugged with a rubber stopper. The gas source was
connected to a one-quarter-inch copper tube which
extended through the stopper and cupric oxide and

almost reached the bottom of the quartz tube. The
scrubbed gas exited through a similar c0pper tube which
was connected to four rubber tubing lines. Each line
was attached to a glass syringe (S-ml) filled with cotton
and equipped with a large stainless steele needle (B-D
Yale Luer-lok, 18 gauge 31/2," Becton, Dickinson and Co.,
Rutherford, N.J.). The syringe could be taken off the
line and autoclaved. The needle was bent to make a hook
of about 45 degrees. During aseptic work, the needle
was sterilized by heating over a bunsen flame prior to

inserting into the container mouth. The flow of gas

156

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157

 

 

 

 

 

 

 

 

 

158

was adjusted by the tank regulator. Before use, the
cuprix oxide wire in the tube was heated to about 500 C,
then reduced by flushing with hydrogen gas until the
wire turned pink. It was necessary to keep the furnace
heating all the time and to regenerate the cupric oxide

once a week.

6. Photometabolism g£_Rhodopseudomonas and Rhodo-

 

 

microbium. Tests for growth of the isolated photosynthetic
bacteria on organic substrates were conducted in 17 X 150 mm
test tubes which contained 9'ml nitrogen-free mineral medium
"98" plus 0.2% (unless otherwise noted) of one of a

variety of organic substrates. During media preparation

and inoculation the test tubes were continuously flushed
with repurified nitrogen. One ml from an early stationary
phase culture (cells were grown in nitrogen-free medium

"98" containing 0.3% sodium acetate) was pipetted into

each test tube and capped with a rubber stopper. One

tube without substrate served as the control. Tests were
carried out in duplicate and the experiment was repeated
twice. Growth was estimated from the measurements of the
optical density at 680 mu in l-cm cuvettes by using a
Spectronic 20 colorimeter. After 10 days of incubation

the optical density of the control was subtracted from

that of the tubes containing the tested organic sub-

strates to obtain the final growth measurement. The

tubes were supported by a circular wood frame which held

159

the tubes equidistant from a lOO-watt tungsten bulb.
The incubation temperature was about 27 C and the light

intensity was 300 ft—c at the tubes.

7. Acetylene Reduction Assay. Rhodomicrobium was

 

 

used to study the ability of this genus to reduce

acetylene. For convenience, Rhodomicrobium was grown in

 

a continuous culture apparatus which was constructed
from a 1.5 1 erlenmeyer flask (Fig. 31). The culture
was stirred continuously by a magnetic stirrer and
illuminated with a 60-watt tungsten bulb (about 150 ft-c
at the surface).

The culture could be kept at a late log phase by
this system. Cells used for preparations of all experi-
ments described below were taken from this continuous
culture.

Acetylene reduction assays were done 18 to 20 hours
(log phase) after subculturing to broth medium from the
continuous culture. Unless otherwise indicated, all
cells used for acetylene reduction were grown in
nitrogen-free medium "98" containing 0.0002% yeast
extract, 0.3% acetate, stirred by magnetic stirrer and
incubated under an atmosphere of repurified nitrogen,
and in the presence of continuous tungsten light.

To determine the optimum acetate concentration,
time of incubation and quantity of acetylene for the

assay, cells were grown at about 30 C, 300 ft-c, harvested

160

in log phase (for optimum acetate concentration experi—
ment, culture was centrifuged at 3,000 x g for ten
minutes, resuSpended in fresh medium) and assayed for
acetylene reduction under a repurified argon atmosphere.
For studies on the effect of light intensities and
temperatures, cells were grown in side-arm flasks (Nephelo
culture flask, screw cap, 500 ml, Belco Glass, Inc.,
Vineland, N.J.) placed in an aquarium (0.31 X 0.32 x
0.77 m) in which the water temperature could be con-
trolled, at four different light intensities (100, 200,
300, 400 ft-c) which were obtained by varying the length
of the light path in the aquarium. The water was main-
tained at desired temperatures of 25, 30, 37 C by a con-
stant temperature circulator (Model 20 CTC, Bronwill
Scientific Inc., Rochester, N.Y.). Cultures were con-
stantly stirred by magnetic stirrers placed underneath
the aquarium. Continuous light was provided by a row
of four tungsten flood-light bulbs (75-150 watts).
Light received at the walls of the individual flasks
was measured using Tri-Lux-Footcandle meter (Kling Photo
Corp., New York, N.Y.). Growth was followed by measuring
optical density at 680 mu. Acetylene assays were done
with 16 ml of suspensions of cells taken directly from
the culture flasks without resuspending in new medium.

The cells were incubated under 10% acetylene in argon

161

in 26-ml serum bottles at the same temperature and
light intensity at which they were grown.

The effect of various combined nitrogenous com-
pounds on acetylene reduction was also studied. In
this study, cells were grown under 300 ft-c at about
30 C in 500-ml side-arm flasks containing 400 ml medium
"98," stirred continuously by a magnetic stirrer. The
acetylene reduction assay was done at log phase without
resuspending cells in new medium. Ten ml of the culture
was pipetted into each of a series of 26-m1 serum bottles
into which 1 m1 of solutions of various concentrations of
yeast extract, ammonium chloride, or sodium nitrate was
added to have the final concentrations indicated. Cells
then were incubated under 10% acetylene in a repurified
argon athSphere at 30 C and 300 ft-c. A half ml of
head space gas in the bottle was injected into the gas
chromatograph at the indicated times. A control (no
combined nitrogen added) was always used.

Induction of acetylene reduction activity was
measured using cells grown in medium "98" with 0.1%
NH4C1 and 0.0002% yeast extract in a nitrogen atmosphere.
The cells were harvested at log phase, centrifuged at
3,000 x g for 10 min and resuspended in nitrogen-free
medium "98" in 500-ml flasks and the flasks were flushed
continuously with repurified nitrogen, argon, or helium

(about 100 ml/min). Samples for the acetylene reduction

162

assay (triplicate) were done at the times indicated in
26-ml serum bottles. Bottles were incubated for 1 hr

on the disc of a multi-purpose rotator (Model 150 V,
Scientific Products, Inc., Queens Village, N.Y.) to
insure uniform light distribution (300 ft-c, 28 C). The
experiment for each type of gas was repeated twice.

Since the quality of light affects bacterial photo-
synthesis (Pfennig, 1967), it should similarly affect
nitrogen fixation which derives its necessary energy
from photosynthesis. This implies that when some wave
lengths of light are selectively eliminated, the acetylene
reduction rate could be affected. This was done by wrap-
ping white, red, yellow (amber), and blue transparent
cellophane paper around the cultures. The absorption
spectrum of each color of paper was determined on paper
which had been wrapped around a l-cm cuvette and placed
in a scanning Beckman grating spectrophotometer (Model
DB-G, Beckman Instruments, Inc., Fullerton, Calif.). The
transmission of white cellophane was practically unchanged.
The red cellophane absorbed all wavelengths up to 570 mu
but began to transmit light which reached maximum trans-
mittance from 625 to 750 mu then the percent transmittance
decreased again. With the yellow cellophane, maximum
transmittance was obtained with the wavelengths greater
than 550 mu and to lesser extent with wavelengths shorter

than 400 mu; maximum absorption occurred in the range of

163

400-500 mu. Finally, the light blue cellophane trans-
mitted maximally in the regions of 375-475 mu and 700-
750 mu. In summary, white cellophane supplied all of

the tungsten light wavelengths. The yellow cellophane
and to a lesser degree the blue cellophane supplied most
of the wavelengths which are absorbed by both carotenoids
and bacterial chlorophyll. Finally the red cellophane
transmitted wavelengths longer than 600 mu.

In the first experiment, cells were grown under
tungsten light in 500-ml side-arm flasks; samples were
wrapped with transparent colored cellophane paper (as
indicated) when incubated with acetylene. In the second
experiment, cells were grown in flasks wrapped with white,
red, yellow, and light blue cellophane papers. These
flasks were placed at an equal distance from a lOO-watt
tungsten bulb (about 300 ft-c at flask wall). Samples
for acetylene assay were also wrapped with the same
colored cellophane paper as grown and incubated at 300 ft-C.

A standard curve (Fig. 32) relating optical density
at 680 mu to dry weight was prepared as follows. Cells
were grown in nitrogen-free medium under nitrogen gas
at 300 ft-c and 30 C, harvested at log phase, centrifuged,
'resuspended, and diluted to various optical densities
(Spectronic 20, at 680 mu) using the original supernatant.
Triplicate cell suspensions (10 ml) were dried in an

oven at 80 C for 24 hr, then weighed. A correction was

164

 

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mo :6 omo um >uamcmp Havaumo mcflumamu w>uso Unmpcmum

.Nm muswflm

165

3;: Emmi; Ea

 

 

 

 

 

166

was made for the weight of salts in the suspensions.
In all experiments this curve was used to obtain dry

weight values.

C. Results and Discussion

 

l. Enrichment and Isolation. The frequency of the

 

development of photosynthetic bacteria in the enrichment
cultures varied depending on the inoculum source. Visible
color development occurred in all bottles inoculated with
algal mats, in 50-70% of the bottles inoculated with mud
and in 80-100% of the bottles inoculated with water. The
composition of photosynthetic bacterial community also
depended on inoculum source. Water samples taken from

3-5 m always supported the development of Thiopedia.

 

Thiopedia usually appeared first followed by Chromatium

 

 

domination after 10 to 20 days. The intense decomposition

could have produced a high concentration of H28 which

is thought to inhibit Thiopedia and favor Chromatium.

 

 

It is worth noting that Thiopedia was able to grow on

 

the wall of bottles containing sediment taken from deep
water which were kept in the refrigerator at 10 C in the
dark. Visible development occurred only above the sedi-
ment. The fermentation of organic material at this
temperature is expected to be extremely slow, hence

H25 released was also low. The energy source in this

particular condition is not known; it is probable that

167

light was admitted by the occasional opening of the

refrigerator. Several attempts to isolate Thiopedia and

 

Chromatium by using various media containing bicarbonate,

or various organic carbon sources with added H S, how-

2

ever, failed.

Thiopedia and Rhodospirillum developed first in

 

 

samples containing the algal-mat, however, Rhodospirillum

 

rapidly overgrew Thiopedia. Rhodospirillum occasionally

 

 

constituted 98% of the total bacterial population in the
enrichment cultures. Upon sitting for some time or

transferring to medium "98," Rhodopseudomonas became

 

dominant and Rhodospirillum eventually disappeared.

 

Rhodomicrobium usually appeared in an old culture when

 

Rhodospirillum had almost disappeared; the ratio between

 

Rhodomicrobium and Rhodopseudomonas was about 2:1 after

 

 

the disappearance of Rhodospirillum.

 

Enrichment cultures containing mud taken from
shallow water, which appeared to have less freshly
deposited organic material, usually supported the growth

of Rhodopseudomonas first, then later Rhodomicrobium.

 

 

All attempts to isolate Rhodospirillum also failed.

 

Rhodomicrobium and ten strains of Rhodopseudomonas were

 

 

isolated; these genera were apparently less sensitive
to oxygen than the others. The isolates were all budding
bacteria containing bacterial chlorophyll, and grew on

acetate as the sole carbon source in a nitrogen-free

168

medium under anaerobic conditions and light. Some

strains, presumably belonging to the species Rhodopseu-

 

domonas palustris, required p-aminobenzoic acid (Hutner,

 

1946) for maximum growth. All isolates grew well when
supplied with yeast extract as low as 0.0002% (w/v) in

nitrOgen-free medium.

2. Some Characteristics 2£_the Isolated Rhodomi-

 

 

 

crobium. This organism was isolated for the first time
by Duchow and Douglas (1949) and was classified in the

family Hyphomicrobiaceae under the name of Rhodomicrobium

 

vannielii (Duchow and Douglas, 1949; Douglas, 1957).

 

This organism reproduces by budding and has morphology

similar to Hyphomicrobium, however, it is an anaerobic,

 

photosynthetic, non-sulfur bacterium. Mature cells of
the isolate had an oval to rod shape (l-l.2 x 1.5-2.5 u);
cells grown in nitrogen-free medium were smaller. Round
young cells were formed at the tips of "stalks." These
newly formed cells were motile when separated from motor
cells (Fig. 33, 3). Each motor cell can bear as much as
4-5 stalks which originated from its body and grew in
different directions. Two to four stalks sometimes
appeared to join without a mature cell at the joining
point. Mature cells in cultures about 1-2 weeks old
supplied with acetate had a refractile granule near

each end (Fig. 33, 4). These granules were probably

poly-B-hydroxy butyrate which was previously reported

169

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170

 

 

   

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171

by Hoare and Gibson (1964) in Rhodomicrobium vannielii

 

grown with acetate as the carbon source. These granules
were, however, not observed when cultures were supplied
with other organic carbon such as mannitol, starch, etc.

Rapid and extensive growth of Rhodomicrobium was

 

achieved with medium "98" in the presence or absence of
combined nitrogen under light. Exogenous CO2 was not
required when organic carbon served as the carbon source.
This result seems contrary to the earlier observation by
Duchow and Douglas (1949) and Trentini and Starr (1967)
that Rhodomicrobium required exogenous CO for anaerobic

2
heterotrOphic growth. However, in the present study

 

significant CO2 was produced during photoheterotrophic
growth. Increasing quantities of CO2 in the head space
of an acetate grown culture was observed by head space
analysis with a Carle Basic gas chromatograph equipped
with a thermoconductivity detector (Model 8000, Carle

Instrument, Inc., Fullerton, Calif.) or by trapping 14CO
14

2

when uniformly labeled C acetate was the substrate.
Photodegradation of organic acids has been shown to

supply both sufficient reducing power and CO2 for hetero-
trophically growing cultures of other purple photosynthetic
bacteria (Muller, 1933; Gest 35 31., 1950; Glover 33 31.,
1952; Losada 32 21., 1960).

Attempts to grow Rhodomicrobium under the light in

 

nitrogen-free medium containing CO as the sole carbon

2

172

source and hydrogen as the reductant has failed so far
although Pfennig (1969) and Hoare and Hoare (1969)
succeeded in growing this organism autotrophically in
the presence of hydrogen and combined nitrogen.

Old cultures of Rhodomicrobium isolated from Winter-

 

green Lake formed a cyst-like structure (0.5-1.0 x

0.8-1.0 n) which had an angular shape under the phase
microsc0pe (Fig. 33, 4 and 5). Negatively stained
preparations examined by electron microscope (Fig. 33,

2 and 6) also showed an irregular angular shape; some

had the appearance of a corn grain, some had an angular
egg shape. All were nonmotile. These peculiar structures
were observed by Murray and Douglas (1950) in old cultures
but were ignored until Gorlenko (1969) who isolated a
budding photoheterotrophic bacterium, physiologically

and morphologically similar to Rhodomicrobium, from

 

Lake Bolshoi Kichier, reported that the angular-shaped
structure had characteristics of a true spore. He called
these structures "spores" because they stained positive
with specific spore stains and they survived heating at
100 C for 30 min. The tests for heat survival of the

angular structures from the present Rhodomicrobium

 

isolated was, however, inconclusive. Therefore, these
structures are subsequently referred to as "cysts."
The nitrogen or carbon source did not affect cyst

formation. Old colonies in agar shake tubes contained

173

about 50% or more cysts which appeared in pairs thus
giving an appearance of dividing cells but without
visible connecting stalk between the two cells.
Gorlenko (1969) reported that he observed some
germinating "spores" which stained like vegetative cells
and formed stalks before producing young cells at the
stalk tips. Some of his pictures showing germinating
"spores" were, however, seemingly old vegetative cells
which were split away from the branched network by
mechanical factors as usually observed in the present
study. Nonetheless, observations in the present study
are in agreement with Gorlenko in that the angular
structures were produced in old cultures and these
structures produced vegetative cells under optimum
nutritional conditions. Information accumulated so far
does not permit any speculation on.the physiological

nature or the ecological role of the cysts.

3. Photometabolism of Organic Compounds. The

 

 

growth of Rhodomicrobium on a variety of organic sub-

 

strates in nitrogen-free medium "98" supplied with 0.0002%

yeast extract (as well as RhodOpseudomonas) is presented

 

in Table l7.‘ In general, Rhodomicrobium was capable of

 

using a wider range of organic substrates than Rhodopseu-

 

domonas. Rhodomicrobium metabolized all sugars tested

 

except ribose. This result is contrary to the report

of Pfennig (1969) that Rhodomicrobium vannielii did not

 

174

u s .o .m .4 .m .wmo.o u N.H

.wHo.o u m .wNo.o

"How ammoxm wN.o mumz mCOHumuuchcoo mumnumnsm HH<

mmB cpsoum on» H «m.o can» uwummnm u m .m.o on H.o n N .H.o 0» mo.o

«*

.pmanHnCH
.mmmmuucH uHcd

 

 

 

 

 

 

 

 

 

 

 

.Q.o o u o “cocoa mumuumnsm on can 30Hn3 Honucoo map Hm>o .a.o CH mmmmuocH #02«

o N hHocmmoum

N H mHocmusm
I H mummHsmOHna H N mHocmnum
o o mcHGMHNIHQ H H «Hocmnumz
o o mcHosHo m m mHoauaamz
o H mchumhu o H NHouHmocH
m N mcHosmH H o mcmsHoa
o m chmummm< N N HoumohHU
o H mumuummmm o H mcHHMHmw
N H mumconoum o m CHHDGH
o H muMCOHSHUMHMO o m :HanO
H H mumoNcmm H N HHocHouomwm
H H mumHsoHHmm o m noumum
o H mumumusHo o H HoanHom
m N mumumom Hanna 0 o 0mocHnmu¢
m m mumHmz H o mmonHm
o m wumfimusHo o N wmouomHmula
m m mum>su>m o H mmosfimnm
o o mumHuHU o m mmonuxmp ouomm
o H mHMCHoosm o m mmouosm
N H mummmfidm o m meHQOHHmOIH+Vp
m m mumuomq o m HouHGGMZIAIVp
N m mumumo< o N mmouomqla+vp

mmcoE ESHW mmsoa EdHn
nonsmeOUOSm IouoHEopoam «amumuumnsm Ionawmmmuonm IOHUHEOUonm «4mumnumn9m
0Hcmmuo 0Hcmmuo
«suzouo umz «nu3ouo umz
mccsomfioo

UHGmmuo mo mumHHm> m :0 mucoEOpsmmmmpocm can EsHbouoHEoconm Ho nusouw

 

 

.hH OHQMB

175

utilize glucose, fructose, and mannitol. Table 17

also indicates that Rhodomicrobium can metabolize

 

carbohydrate polymers such as starch, chitin, and
inulin. This surprising metabolic capacity to utilize
sugar may be explained by the fact that acetate left
over in the inoculum may have facilitated or initiated
the metabolism.
This explanation is in line with the observation
of van Niel (1931) that the development of representatives
of Athiorhodaceae in media containing sugars was greatly

enhanced by contamination with lactate. Rhodomicrobium

 

and Rhodopseudomonas did not grow well when alcohol

 

served as the carbon source. This was probably due to
lack of CO2 (Qadri and Hoare, 1968) which was probably
for alcohol metabolism.

Rhodomicrobium and Rhodopseudomonas utilized.most

 

 

of the simple organic acids tested particularly those of

the tricarboxylic cycle with the exception of citrate

which may be impermeable to the cell (Eisenberg, 1953).
It is necessary to emphasize again that CO2 and

reducing power were not provided by external sources in

this experiment therefore they must have originated

from the degradation of the organic compounds or from

the acetate contamination (Muller, 1933; Ormerod, 1956).

4. Acetylene Reduction. Rhodomicrobium was

 

 

chosen for studies on acetylene reduction because:

176

(l) nitrogen fixation in this organism has not been
studied in detail since Lindstrom et 21. (1951) showed
that this organism can grow in nitrogen-free medium and
fix 15N2, (2) the effect of polymorphism observed by
Douglas and Wolfe (1959) and Gorlenko (1969) on the
efficiency of nitrogen fixation by this organism could
be pronounced, and (3) the growth of this organism in

the absence of exogenous CO has not been studied.

2
The maximum acetylene reduction rate was obtained
with 0.3% (w/v) or more of acetate. This concentration
of acetate also supported maximum growth of this organism.
The maximum rate of acetylene reduction was achieved with
10% acetylene in an argon atmosphere (Fig. 34, left).
The ethylene formation leveled off after one hour of
incubation (Fig. 34, right). The initial lag in
acetylene reduction shown in Figure 34, right, was
presumably due to the low solubility of acetylene in
water (Morrisson and Boyd, 1966, p. 239) or to the
effect of transfer (Hardy 35 31., 1968). Both growth
and acetylene reduction were optimum in a pH range of
6.5-7.1.
Ethylene production and growth in a nitrogen-free

medium by Rhodomicrobium were light dependent (Fig. 35)

 

as is the case for other purple photosynthetic bacteria
(Kamen and Gest, 1949; Newton and Wilson, 1953; Arnon

§£_§£., 1961; Evans and Smith, 1971; Schick, 1971a,

177

.mM@ mommmupmmc ucmcH80p mnu

mm comma cuH3 mmHuuon momma :H nuon .AuzmHuv GOHHOSU
loud mamHmcum mo wmusoo wfiwp .AummHv coHumnucmocoo
owqumom mamum> ESHQOHUHEoposm ma coHuospmu mcmHhuwoe

.vm musmHm

(ww) lHOEJH >013de

178

8:9

 

 

/
0
2‘0

4‘0 6 0

TIME (min)

 

 

 

 

 

 

g a g a:
m 8 v- V
0
5t
03
”—3,
z
O
0
03%
° 2
—(\l
\ I
(\J
(NU
J 1 1 1 1 I O
O O O O O
3 o «0 (\I

(Ju/Uuw) 1H9|3H wad t7H Z:3

179

 

.EDHQOHOHEOUOHE an A DC 1,330.3 new A .v
coHuospwu wcmeumom Ho mums mcu co poHHmm xumc m mo pummmm

.mm musmHm

 

 

l l

J J

Fri]?

1‘

 

TIME (hr)

 

 

 

1
Q 00 (0 v4 7N2
(EOLXUM/fiw/GIOWW) oawaos H :>

V

32 36

28

24

181

1971b). The continuation of ethylene production at a
lower rate, after the light was turned off as is shown
in Figure 35 (arrow) indicates that acetylene reduction

in Rhodomicrobium was a dark reaction and not related to

 

the light-dependent process of ATP production and mobili-
zation of reducing power (Arnon et 21,, 1961) which were
necessary for acetylene reduction. Dark nitrogen fixation

by light-grown cells of Rhodospirillum rubrum (Schick,

 

1971a) and Chloropseudomonas ethylicum (Evans and Smith,

 

1971) has been reported. Schick (1971a) found that
nitrogen fixation continued but at a lower rate for
20 min after the light was turned off. The low but
significant rate of acetylene reduction in the dark
reported here probably was due to the accumulation of
ATP, reducing power, and early products of carbon metab-
olism while in the light but which were exhausted after
the cells were subjected to darkness for one hour
(Fig. 35). Acetylene reduction resumed again when
the light was turned on, however, the rates of reduction
were much lower than those before the light was turned
off. This appears to be due not to the dark period (4 hr)
but to the older age of the culture. This explanation is
supported by the growth and acetylene reduction data
shown in Figure 36.

The effect of polymorphism on the acetylene

reduction was studied in a culture inoculated with cells

182

HOV

 

.musuHso EdHnouoHfioponm m CH pm>ummno
30:30.3 can A C v COHuospmu mcmHmpmom Ho mmumm

.mm musmHm

 

 

2‘0

 

 

CD

28

TIME (hr)

12

184

harvested by centrifuging from a stationary-phase culture
previously grown with 0.10% of ammonium chloride in the
medium. The culture was continuously flushed with
repurified nitrogen gas. Samples assayed for acetylene
reduction were taken at the times indicated. The growth
was slower in this experiment when the flushing rate
exceeded lOOml/min, presumably because of the flushing
out of newly formed CO2 which is necessary for acetate
photometabolism (Stanier et 31., 1959). The acetylene
reduction curve in Figure 36 indicates three distinct
~stages. The first stage (0-10 hr) which was the initial
lag in acetylene reduction. This could be due to a
number of factors: (1) classical growth lag in new
medium (Lamanna and Mallette, 1965), (2) effect of
dissolved oxygen in newly prepared medium, (3) large
endogenous nitrogen pool in inoculum which suppressed
nitrogenase synthesis, (4) or the composition of the
polymorphic pOpulation (relative percentages of cysts,
motile, and non-motile vegetative cells). The first
three factors are unlikely. Growth in new media

(Fig. 35) and induction of acetylene reduction using a
109 phase inoculum (Fig. 41) occurred only in a short
time after transfer. The preparation of medium was done
under a strictly anaerobic atmosphere. However, micro-
scopic observation supported explanation (4) since the

culture contained about 55% non-motile branched cells,

185

45% cysts and very small number of motile vegetative cells
in stage one (0-10 hr). When the acetylene reduction

rate became more significant in stage 2 (10-20 hr),

the motile cells and young branched network (which had
vegetative cells without PHB granules) became more
dominant and the cyst population diminished. At the

end of stage two (approximately at 20 hr) the pOpulation
of the culture was comprised of approximately 70%

branched cells and 30% motile cells with a small number

of cysts.

In stage 3 (> 20 hr), the percentage of motile cells
decreased and cyst forms increased. Angular structures
could readily be seen at the end of phase 3. Branched
cells began to accumulate visible granules of PHB at the
beginning of the stationary phase. Thus, acetylene
reduction activity appeared to correlate with the
presence of motile cells and young network and it
diminished when cells were preparing to form cysts.

The small fluctuation of acetylene reduction rates in
phase 3 appears to be due to experimental errors. How-
ever, the acetylene reducing system may be suppressed by
an increase of nitrogen in the internal nitrogen pool

as was observed in blue-green algae by Jewell and
Kulasooriya (1970) or by the extracellular organic
nitrogen excreted or released by lysing cells (Okuda

EE,E£°' 1961a) or a combination of these three.

186

Literature concerning nitrogen fixation or acetylene

reduction by spores of Clostridium or cysts of Azotobacter

 

 

is nil. Recently Hitchins (personal communication) found

that cysts of A, vinelandii reduced only acetylene upon

 

germination. It is expected that this observation also

holds for cysts of Rhodomicrobium even though the con-

 

ditions for cyst formation in the two organisms are

quite different. Cyst formation in Azotobacter is stimu-

 

lated by B-hydroxybutyrate, crotonate, or butanol (Lin
and Sadoff, 1968; Hitchin and Sadoff, 1970) while cyst

formation in Rhodomicrobium was apparently triggered

 

by nutrient exhaustion.

The optimum light intensity for Rhodomicrobium

 

growth was 300-400 ft-c, regardless of the temperature
tested as shown in Table 18. The growth rate was the
same regardless of whether ammonium or atmospheric
nitrogen was the nitrogen source since the generation
time of a culture supplied with 0.1% (w/v) of ammonium
chloride was 10.50 hr as compared to 10.66-12.56 hr for
cultures in nitrogen-free medium (Table 18, column 4).
A similar generation time of 10.50 hr was reported by

Trentini (1967) who grew Rhodomicrobium vannielii in a

 

medium containing bicarbonate, lactate, and ammonium
sulfate under 700 ft-c light. However, the best growth
rate (5.10 hr) was obtained when he supplied cultures

with a mixture of 10 vitamins and aspartic acid.

187

mmmmm COHuUSOmu QOHmumom ou HOHHQ mHmumemEEH OOHumm mcHHSU cmHMHsono mmB w

 

 

"muoz
mEHu coHumumcww
.i
0.0H O.m mm.mm m0.0 OOH
m.HO O.vm ON.mH hm.H OON mm
O.mvH 0.0mN mm.NH mm.H oom
0.0NH n.vON >O.HH OO.H oov
O.mOH 0.00 mH.Hv mm.O OOH
v.vNN O.NON mv.om O0.0 OON om
O.mnm 0.0vm mO.HH O0.0 oom
O.va 0.000 Om.NH mm.H OOO
m.mNH O.NNH NH.mm hm.H OOH
0.00N 0.00m NO.MN mn.H OON mN
0.00m 0.0mm mN.HH NN.H OOm
O.mmN 0.0mm O0.0H mm.H oov
us\uanms sue us
lune loss pamflms lonumc loo
OHMMMOEJE \mHmemmWHosnfi *0 Hum Hmch uanH musumnmmswe

 

 

EdHnouoHEoponm an
GOHuospmu mcmeumom Ho mpmu cam cpzoum co musumnmmfimu cam uanH Ho uommmm .mH mHQMB

188

The rates of acetylene reduction per cell dry
weight are shown in Table 18 and Figure 37. It appears
that under these conditions, the rate of acetylene
reduction was highest under 300 ft-c at 30 C. Acetylene
reduction did not correlate with final cell yield
(Table 18, column 5 and column 3). At 37 C under 400 ft-c
fast growth and a high dry weight did not result in a
high acetylene reduction rate (120 mpmole C2H4 per mg
dry wt per hr at 37 C and 400 ft-c as compared to
206 mumole C2H4 per mg dry wt per hr at 25 C under
200 ft-c. The self-shadow regulation is unlikely when
all final yield and reduction rates are considered
(Table 18, columns 3 and 6). Larger filaments were
observed at 37 C under 300-400 ft-c (as compared to
those under the same light conditions at lower temper-
atures) which could account for the low rate of reduction
since Conti and Hirsch (1965) and Morita and Conti (1963)

observed that the stalks of Rhodomicrobium had little

 

or no bacterial chlorophyll. It may follow that stalks
were not active in acetylene reduction hence they could
cause the inconsistency between dry weight and acetylene
reduction. The age of the culture at the time the assay
was done could also be important because of the narrow
Optimum for acetylene reduction as shown in Figure 36.
The formation of abnormal stalks which were nearly as

wide as the mature cells reported by Trentini and Starr

189

Figure 37. Effect of light and temperature on the
rate of acetylene reduction by
Rhodomicrobium.

 

190

 

 

 

 

 

 

 

 

 

400 ,-

.
0
o
2

79353335323

300-
lOO-

191

(1967) at 37 C was not found in the present study;
however, filaments at 37 C were a little larger than
those formed at lower temperatures.

All previously studied nitrogen-fixing organisms
use N2 only if there is no other utilizable nitrogen
source present (Kamen and Gest, 1949; Gest et 21., 1950;
Ormerod et 31., 1961; Sorger, 1969; and Schick, 1971a).
The effect of combined nitrogen on acetylene reduction by

Rhodomicrobium is shown in Figures 38, 39, and 40. The

 

reduction was suppressed by as little as 0.0005% (w/v)
of combined nitrogen; however, the effective suppression
time was different depending upon the nitrogen source
and the cell c0ncentration: (a) Yeast extract (Fig. 38):
0.0005% (w/v) of yeast extract did not suppress the
acetylene reduction activity of the whole cells. The
initial high quantity of ethylene produced in samples
containing 0.005% and 0.05% yeast extract was probably

a lag effect due to stimulation by the yeast extract.

(b) Sodium nitrate (Fig. 39): 0.0005% of sodium nitrate
suppressed nitrogenase activity for only 20 min before
the reduction rate returned to values similar to the
control. The addition of 0.05% of nitrate inactivated
nitrogenase system only for 60 min. (c) Ammonium
chloride (Fig. 40): 0.0005% of ammonium chloride sup-
pressed acetylene reduction for only 5-10 min. However,
concentrations of 0.005% and 0.05% of ammonium chloride

inhibited reduction for 80 and 140 min respectively.

192

Figure 38. Effect of different concentrations of
yeast extract on the rate of acetylene
reduction by Rhodomicrobium.

 

2
(GLC peak, mm) x10
0)

CH
0024

193

 

 

 

QC]-

0.0005%

0. O O 5%"

005%

 

1'0

2b 3b 30 5b 610
TIME(min)

 

194

Figure 39. Effect of different concentrations of
sodium nitrate on the rate of acetylene
reduction by Rhodomicrobium.

 

m
0

ETHYLENE (GLC peak,mm)x10

[\J
h

00 R3 0)

Dr

195

 

 

Iéfi/

 

o 21014lOl6'OJ 80'

TIME (min)

 

1001 12b

196

Figure 40. Effect of different concentrations of
ammonium chloride on the rate of
acetylene reduction by Rhodomicrobium.

 

C2H4 (GLC peak,mm)

400

300

200

100

197

 

 

TIME (min)

 

 

198

All concentrations of combined nitrogen tested
have failed to stop completely the activity of nitro—
genase system. These results further support the idea
(see Chapter VI) that combined nitrogen did not act
directly (allosteric control) on the nitrogenase system
but rather the cells divert the limited energy and
reducing power to the other processes such as ammonium
assimilation which require lesser quantities of energy
and reductant for synthesis of cellular materials. The
shift of metabolic process concerning nitrogen metabolism
would not stop completely the flow of available electrons
and energy to the already synthesized nitrogenase and
nitrogen fixation would still proceed, but at a lesser
rate until nitrogenase was diluted by cell division.

The immediate suppression of acetylene reduction
by nitrate as is shown in Figure 39 seems contrary to
the observations by Taniguchi and Kamen (1963) in which

Rhodospirillum rubrum and Rhodopseudomonas spheroides did

 

 

not use nitrate as a nitrogen source until after being
grown in the presence of nitrate as the only available
nitrogen source. Since the metabolism of nitrate by

Rhodomicrobium was not studied in detail, any conclusion

 

drawn from this limited information would be premature.
Figure 41 shows the induction of acetylene
reducing activity when cells grown on combined nitrogen

were transferred to nitrogen-free medium and incubated

Figure 41.

199

The induction of nitrogenase synthesis by
argon or nitrogenase in Rhodomicrobium
(Ethylene produced by Rhodomicrobium cells
following transfer from a combined-nitrogen
medium to a nitrogen-free medium-grown cells
of Rhodomicrobium was incubated under an
argon or nitrogen atmosphere).

(GLC peak ,mm/ hr)

c2 H4

200

 

400-

300—

 

Argon —->

<— Nitrogen

 

 

 

TIME (hr)

 

201

under nitrogen or argon gas. The rate of acetylene
reduction in an argon atmosphere was much higher than
that in the sample in the nitrogen atmosphere. Each
point was the average of three samples. When the experi-
ment was repeated using a helium atmosphere, the results
were similar to data for the argon atmosphere. The high
nitrogenase synthesis under argon and helium was also
observed by Nelson et_al. (1971) with Anabaena sp. which
did not grow during the experimental period because of
the lack of nitrogen (argon gas contained less than 5 ppm
N2 by volume). The specifications for the argon used in
this experiment indicated no more than 10 ppm N2 was

present. Thus it is unlikely that N was the specific

2
inducer for nitrogenase synthesis, however, the cell
apparently develops a greater nitrogen reducing ability
under conditions of nitrogen starvation.

Table 19 shows the effects of light quality on the

acetylene reduction by Rhodomicrobium grown under tungsten

 

white light but assayed under colored light. Since the
yellow cellophane transmitted most of the wavelengths
which are selectively absorbed by bacterial photo-
synthetic pigments, its high rate of acetylene reduction
is predictable. The effect was pronounced, however,
with red and blue light, which absorbed, respectively,
all and a portion of wavelengths available to the exist-

ing carotenoids. However, when Rhodomicrobium was grown

 

202

Table 19. Effect of light quality on acetylene
reduction by Rhodomicrobium*

 

 

C2H4 Formed/
Sample/hr
(GLC peak , mm)

Light Conditions

 

White 115.70
Red 94.00
Yellow (amber) 123.50
Light blue 94.25

 

*Culture grown at 30 C under 300 ft-c continuous
white tungsten light. Samples were wrapped with cor-
responding colored cellophane papers when incubated with
acetylene under the same light and temperature as grown.
Each value is the average of duplicates.

203

in cellophane-wrapped cultures, yellow light supported
growth as well as did white light, but the highest
yield was obtained under blue and red light (Table 20,
column 2).

It is necessary to note that the results of these
experiments only indicate the qualitative effects of

light quality on growth and acetylene reduction by

 

Rhodomicrobium, because the amount of light received
by the cultures was not the same after going through

colored cellophane papers.

5. Conclusion. From information collected in this

 

part of the study, it can be concluded that: (1) Several

isolates of Rhodomicrobium and Rhodopseudomonas can grow

 

 

on numerous organic carbon compounds as the sole carbon
source in the presence or absence of combined nitrogen.
(2) The acetylene reduction activity of whole cells was
suppressed by utilizable combined nitrogen. (3) Rhgdg-

microbium had the ability to synthesize nitrogenase in

 

the absence of combined nitrogen under N2, argon, or
helium atmosphere. (4) Elimination of certain light
wavelengths affected the rate of acetylene reduction.

(5) Rhodomicrobium appeared to undergo a change of form

 

(life cycle) that under the laboratory conditions, can
be depicted as follows (page 205). This "life cycle"
was based only on microscopic observation and yet to be

proved by further studies.

204

Table 20. Effect of wavelength on growth
reduction by Rhodomicrobium*

 

and acetylene

 

 

Color Paper Final(g;y Weight (mfimgieiggmggy
weight/hr)
White 1.90 134.20
Red 1.90 192.20
Yellow (amber) 1.85 113.80
Light blue 1.70 200.00

 

*
Cultures were started at the same

dry weight per

volume, incubated at 28 C under 300 ft-c continuous white

tungsten light and correspondent colored
Acetylene reduction assay was done under

cellophane papers.
the same con-

ditions as grown. Each value is the average of duplicates.

205

Non-motile

cell Motile

"Cyst"

Figure 42. Suggested life cycle of Rhodomicrobium.

 

IX. PHOTOMETABOLISM BY NATURAL
POPULATIONS OF PHOTOSYNTHETIC

BACTERIA

A. Introduction

 

The function of natural populations of photo-
synthetic bacteria in the environment has been ignored,
although they were discovered in the nineteenth century
(Kondrat'eva, 1963). Some of the reasons for this lack
of progress are: (1) they are difficult to work with
in §i£2_due to the lack of portable instrumentation to
provide a thoroughly anaerobic system, (2) the most
prevalent of these organisms in nature have not been
grown in pure culture.

The purpose of this study was to gain some insight
about the activity of natural populations of photo-

synthetic bacteria.

B. Materials and Methods

 

Samples for the photosynthetic pigment study were
taken at station A (10/7/71) with a 3-1 Van Dorn sampler,
stored in l-l brown polyethylene bottles and brought

to the laboratory immediately. The qualitative

206

207

extraction of pigments was done as follows. Approximately
100 ml water sample from 2 m and 50 m1 from 3 m, 4 m, and
5 m were collected on Millipore filters (GS 0.22 0) under
low vacuum (about 1/3 atm). The filters were then soni-
cated for 10 sec at maximum power (3/8" diameter tip,
Biosonik III, Bronwill Scientific, Inc., Rochester, N.Y.)
in a 7:2 (v/v) acetone methanol mixture (Cohen-Bazire
gE_§1,, 1957). Sonicated samples were centrifuged at
10,000 xg for 10 min and the supernatant was scanned
in a l-cm cuvette with a scanning spectrophotometer
(Model DB-G, Beckman Instruments, Inc., Fullerton,
Calif.). The extraction lasted about 20 min.

The in_§itu assimilation of acetate was measured
as follows. Water samples from various depths were col-
lected by a 3-1 Van Dorn sampler and transferred to a
500-ml flask which was continuously flushed with argon.
Ten ml of the sample was pipetted into each of two 26-m1
clear serum bottles which were continuously flushed with
argon (Fig. 17, step 6). A one-half ml aliquot of a

stock solution of 14

Cl-acetate (made to specific activity
of 1 uc per 100 pg per ml—-New England Nuclear, Boston,
Mass.) was injected into each bottle. The bottles were
capped and incubated at the original depth for 0.5 hr.
Metabolic activity was stopped by adding 0.2 ml of

Lugol's solution. Samples were filtered onto Millipore

filters (GS 0.22 0) under low vacuum. Adsorption was

208

corrected for by subtracting the radioactivity found in
filters which were from samples which had Lugol's solution
added immediately after the acetate addition. Filters
were dried under low vacuum in a desiccator overnight
then dissolved in 19 ml of Bray's cocktail containing
4% cab-o-sil. (Bray's modified cocktail contained 60 g
Naphthalene, 4 g PPO [2,5-Diphenyloxazole], 200 mg
Dimethyl POPOP 1,4 bis-[2-(4-methyl-5-phenyloxazolyl)J-
Benzen, 100 ml Methanol, 20 ml Ethylene glycol and.made
to l l with p-dioxane). The samples were counted in a
Tri-carb liquid scintillation spectrometer (Model 3310,
Packard Instrument, Inc., Downers Grove, 111.).
Laboratory studies of photodegradation of acetate
by the natural population of photosynthetic bacteria was
done as follows. Water was pumped at night into a 5-
gallon bottle which was flushed with excess lake water
to displace residual oxygen, capped, and brought to the
laboratory. Ten ml duplicates of the samples were
incubated with 100 ug of uniformly labeled plus unlabeled
acetate (New England Nuclear, Boston, Mass.). The
samples were incubated for 1 hr under light intensities
and temperatures indicated, in a constant temperature
aquarium described in Chapter VIII. Duplicate bottles
were incubated in the dark at each temperature as the
controls. At the end of the incubation, the bacteria were

killed with 0.2 ml of 2% HgClz. The pH of the sample was

209

adjusted to 11 with l N NaOH to trap 14CO2 in solution.
After two hours the samples were transferred to 125-ml
flasks which were stoppered with rubber stoppers through
which a 0.8 X 12 cm glass tubes penetrated. The outside
end of the glass tubes was capped with a serum cap; a
two-m1 plastic cup containing 1 ml of 1 N NaOH was hung
near the other end of the glass tube which was inside
the flask and above the liquid level. One ml of l N HC1
was injected into the flask to lower the pH of the sample
to below 3. The flasks were gently shaken at room temper-
ature for 10 hr. One-half ml of the NaOH from the plastic
cup was transferred to 19 ml of Bray's solution containing
4% Cab-O-Sil and counted. Errors due to photodegradation
by high light intensity or volitalization of acetate
accounted for less than 0.5% of the radioactivity trapped.
In situ acetylene reduction by anaerobic water was
assayed by filling 120-ml clear serum bottles with water
taken from 4 and 5 m by a Van Dorn sampler. Excess
water was flushed through the bottle to assure the
removal of oxygen. Each bottle was capped with serum
stoppers which were pierced by a 18 G 1 1/2" needle
to release the small amount of air trapped in the inner
rim of the stopper. This needle was then connected to
a lO-ml plastic syringe, and a second needle which was
connected to an argon source was inserted. Ten ml of

water was withdrawn by the syringe with the volume being

210

immediately filled with argon. Both needles were then
removed. One ml of argon from the prepared bottles was
taken out and replaced by 1 ml of acetylene. Bottles
were suSpended at the original depths for 2 hr after
which the metabolic activity was stopped by adding

0.2 ml of 2% HgClZ.

C. Results and Discussion

 

During the stratification period, Wintergreen Lake
water was divided into three different regions: aerobic
zone 0-3 m, intermediate zone 3 m, and anaerobic zone

4 m-bottom. Thiopedia occupied the oxidized-reduced

 

intermediate metalimnion (3-3.5 m) and the green sulfur
bacteria were found from 3.0 m to near the bottom.

The population of photosynthetic bacteria was
estimated in the summer of 1970 by using the inverted
microscope technique. The population of Thiopedia was

about 105-107 plates/ml, and that of green sulfur

6

 

bacteria between 104-10 cells/ml. The green sulfur

bacteria (Chlathrochloris sp. and Prosthecochloris sp.,

 

 

but not Chromatium) developed in small clumps; therefore

 

the above figure for the green sulfur bacterial popu-
lation was only a rough estimation. Caldwell (1971
unpub. MSU) estimated that the green sulfur bacteria
accounted for 90% of the total bacterial population in

the region between 4-5 m.

211

Figure 43 shows the absorption spectra of pigments
extracted by the acetone-methanol mixture. The maximum
absorption peak at 660 mu was due to the chlorophyll a
of blue-green algae. The maximum absorption peak at
650 mu was characteristic of chlorobium chlorOphyll
(Clayton, 1963). The absorption peak at 765 mu cor-
responded to that of bacteriochlorophyll (Clayton, 1963)

probably from Thi0pedia.

 

The low absorption peak of the 2 m sample was
probably due to the short duration of the extraction.

The amount of bacteriochlorophyll extracted was
surprisingly low (Fig. 43, 3 m sample) although the

pOpulation of Thiopedia was large enough to give the

 

water from 3 m a pink color. Perhaps ThiOpedia relied

 

on their carotenoids which are photosynthetically active
(Pfennig, 1967) as their principal light trapping pig-
ments. No attempt, however, was made to extract the
carotenoids.

The amount of chlorobium chlorophyll (660 mu peak)
appeared high although its presence at 3 m was visually

masked by the pink color of Thiopedia. Figure 44 shows

 

the relative acetate uptake by natural bacterial popu-
lations assayed in situ. The maximum uptake at 3 m
corresponded with the presence of a dominate population

of Thiopedia. The acetate uptake by dark and light

 

bottles was similar. The possible explanation for the

212

.Hn\n\OH co cmxmu mum3 mmHmEmm .nmmnmuomn

MDHHSm cmmum .18 ommv Hco ESHQOHOHBU paw AMHmeOHcB

.18 momv Hnom mo mmocu new AmmmHm cmmumlman Ho :5 OOOO

m Hco mo mEmeE COHHQHOmnm mzonm mnsmHH one .A>\> .Nubv
Hocmcuma cam 0c0pmow mo musuxHS m an pmuomuuxm mCOHHMHsmom
Hmusumc EOHH mucmEmHm OHHmnucwmouonm mo muuommm GOHHQHOmnd

 

.mv mnsmHm

213

CON

3 .

 

 

0mm

00m
A

®

 

THE :10an m><>>

H

ooh)

 

 

q

Omum

 

d

005

005

 

 

0mm

H

00m
H

 

OR

000

1

CI 00..

 

33NV11IWSNV81°Io

214

l4Cl-Acetate assimilated in 30 min by 10 ml

of the natural microbial population (105 cpm =
approx. 1.6 pg acetate). The oxygen and
temperature profiles on the day of the experi-
ment (3/7/71) are also shown.

Figure 44.

215

 

 

 

 

 

3 I
0 /
..... m
C
m 2..
C 0 T.
e
k
01 .m
5 W
e
t
hm/o
OleT n.v e
4 m M
e
18- .IK
2
molar AU./ A
O
O
0
0 I I
214 /
'12"
mIZH r
[‘7
1|. 0V,
j” 1 1
I 2 3

 

 

 

AEOIHamO

216

absence of a light effect are: (l) photosynthetic

bacteria assimilated acetate in the dark (Uffen and Wolfe,
1970), (2) organisms other than photosynthetic bacteria were
responsible for the uptake, (3) reserve ATP pools were
sufficient for a short dark assimilation of acetate
(incubation was 0.5 hr). No attempt was made in_§itu_

to verify any of these hypotheses. However, experimental
results shown in Figure 45 indicate that mineralization

of assimilated acetate by a natural population (dominated

by Thiopedia) assayed in the laboratory was light depen-

 

dent. Figure 45 shows the rate of photodegradation of

acetate to CO by 10 m1 of photosynthetic bacteria per hr.

2
The highest degradation occurred at 100-600 ft-c and

at 29 C where trapped CO represented 2.4% of the added

2
acetate. Dark degradation at best accounted for about
40% of the total degradation. The considerable dark
CO2 evolution could be due to the activity of methane-
forming bacteria which also metabolize the fermentative
end-products to CO2 (cf. Toerien and Hattingh, 1969).
Various representatives of photosynthetic bacteria
have been shown to grow effectively utilizing light and
fatty acids or a variety of other fermentative end-
products (Van Niel, 1931-1971; Muller, 1933; Stanier
gt 31., 1959; Thiele, 1968; Pfenning, 1969; also see

Chapter VIII) under laboratory conditions. Certain

organic compounds were converted completely into cellular

Figure 45.

217

Effect of light and temperature on
mineralization of added acetate by a
natural population of photosynthetic
bacteria taken from 3.5 m in Wintergreen
Lake on 10/8/71 (Hg acetate degraded/ 10 ml
sample/hr). The population was dominated
by Thiopedia and green sulfur bacteria.

 

218

 

 

 

 

UmNzoLmEE 8.308 .38.

219

material (Muller, 1933) while very little exogenous CO2
was consumed (Losada et 31., 1960). There is no reason
why this photosynthetic mode of life did not occur in the
hypolimnion of Wintergreen Lake where light was available
(Fig. 4). The high methane concentration in the hypo-
limnion (Tiedje, unpub.) indicates that the fermentative
end-products, which are substrates for the methane-forming
bacteria and photosynthetic bacteria, were abundant in
this region.

The processes of methane production in lakes are
poorly understood although they are very important in
the recycling of carbon. Information on these processes
has accumulated mostly through studies in rumen (Hungate,
1966) and sewage digesters and with pure cultures (Siebert
and Hattingh, 1967; Hattingh et_§l., 1967; Toerien and
Siebert, 1967; Toerien et_al., 1968; Siebert et_al.,
1968; Toerien and Hattingh, 1969; Toerien,.l970).
Methanogenic bacteria appear to utilize either H2 and
CO2 or organic fermentative end-products, primarily
acetate. Photosynthetic bacteria are known to utilize
similar substrates as well as more complex organics. The
present studies on the assimilation of organic compounds
and mineralization of acetate suggest that the photo-
synthetic bacteria may be important in all steps of

recycle of the organic carbon in natural water.

220

Several attempts to measure acetylene reduction
by samples from 4-5 m gave negative results. This could
be due to: (l) suppression of nitrogenase by high con-
centrations of combined nitrOgen present in the hypo-
limnion, (2) inability to reproduce lake conditions in

the assay bottle (particularly anaerobiosis).

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