Ultraviolet effects of physiological activities of blud [sic]-green algae : final report

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aQK757

.N49

1979

FINAL REPORT

ULTRAVIOLET EFFECTS OF PHYSIOLOGICAL
ACTIVITIES OF BLU^-GREEN ALGAE

J. W. Newton
D. D. Tyler
M. E„ Slodki

Northern Regional Research Center
Agricultural Research
Science and Education Administration
U.S. Department of Agriculture
Peoria, Illinois 61604

Project Officer:

R. J. McCracken

Agricultural Research, Science and Education Administration
U.S. Department of Agriculture
Washington, D.C. 20250

Prepared for

Environmental Protection Agency
BACER Program
Washington, D.C. 20460

United States
Department of
Agriculture

II I . I . ■

National Agricultural Library

Introduction

The blue- green algae (Cyanobacteria) are found widespread in
nature, in soil, water, and in association with a variety of plant and
marine life (2). Various species can tolerate a variety of climatic
conditions and are found even in hot springs and arctic regions. These
cells lack differentiated chloroplasts and contain chlorophyll in
membranous structures; consequently, they have recently been classified
as blue-green bacteria, analogous to photosynthetic bacteria. The
cyanobacteria carry out a typical plant- type photosynthesis, however,
with water photolysis and oxygen evolution as major features. Consequently,
these ubiquitous organisms constitute a particularly useful microbial
system for monitoring worldwide environmental effects on plants as might
result from enhanced solar UV-B (280-320 nm) irradiation due to depletion
of stratospheric ozone (10) .

We have evaluated both Anabaena flos- aquae and the water fern
Azolla as laboratory test systems for environmental studies. Azolla is
an aquatic nitrogen- fixing plant which contains a symbiotic cyanobacterium,
Anabaena, within its leaf cavity (4). This fern is also found worldwide,
but is particularly important for its use as a green manure in rice
paddies in the Orient. Many species of cyanobacteria fix atmospheric
nitrogen and contribute to nitrogen input into soils in a variety of
ways . Both systems appear to be particularly important contributors of
nitrogen to rice culture.

2

.

Our studies show that the nitrogen- fixing enzyme system in cyanobacteria
is particularly sensitive to UV-B damage. Furthermore, inhibition of
nitrogenase activity (measured as acetylene reduction) takes place in
the absence of any nucleic acid damage or lethal effects on the cells.

These studies indicate, therefore, that measurement of acetylene reduction
activity in nitrogen- fixing systems may provide a simple biochemical
assay for assessing the effects of UV-B on plants.

Materials and Methods

Azolla caroliniana, a nitrogen- fixing water fern, was obtained from
Dr. S. A. Peters, C. F. Kettering Foundation Laboratories, Yellow
Springs, Ohio, and was grown on modified Hoaglands salts as described by
Peters and Mayne (6). Anabaena flos- aquae (Lyngle.) Breb. ATCC 22664
was grown on nitrogen- free BG-11 medium C8)* Cultures of plants and
cyanobacteria were grown at 25°C in light chambers under cool white
fluorescent lamps at light intensity of 10-20 watts/M2. Measurements of
total light intensity were made with a Yellow Springs Instrument Co.

(Yellow Springs, Ohio) model 65A Radiometer equipped with a 6551 Radiometer
probe having a constant wavelength response from 0.28 to 2.6 microns
(reduced to 65% at 0.21 microns).

UV-B irradiation of samples was obtained using a bank of six 8-watt
RPR 3000 A Rayonet photochemical reactor lamps (Southern New England
Ultraviolet Co., 954 Newfield St., Middletown, Conn.) placed above
cyanobacterial and plant material at 25°C in flat dishes covered with
5 mil cellulose acetate films. The unfiltered RPR 3000A lamp has, in
addition to UV-B, a strong emission in the short wavelength region
(Amax ^254 nm). Such lamps were used either singly or in multiples to
increase irradiation.

3

(We are grateful to Drs. K. Eskins and H. J. Dutton of this Center
for suggesting the use of these lamps as a source of UV-B radiation.)

The lamps were aged 100 hours and did not significantly decrease in
irradiance levels during prolonged use thereafter. As recommended by
the Agricultural Equipment Laboratory of the Beltsville Agricultural
Research Center (BARC) , 5 or 10 mil cellulose acetate (CA) film was used
to filter out low wavelength UV radiation from the lamps (5) . The CA
was pre- irradiated 6 hours and discarded after 30-40 hours of use.

Since we have no knowledge of the actual targets involved, other than to
exclude DNA, our data are reported as total incident UV-B light over the
range indicated and does not assume any biological effectiveness of a
particular wavelength.

2

UV-B irradiance levels in W/m were measured with an Optronics
Laboratories, Inc. Model 725 UV-B Radiometer (7). We calibrated this
instrument against a Rayonet lamp which had been scanned at distances of

13 and 20 cm [5 mil CA filter) with the Instrument Research Laboratory,

2

BARC, spectroradiometer over the 250-400 nm region. Integrated W/m

over the range of 280-320 nm at these distances were taken as reference

2

points CO . 44 and 0.82 W/m , respectively) and linearly extrapolated to
provide estimates of higher UV-B irradiances.

Cyanobacterial suspensions of 40 ml were stirred during irradiation.
Aliquots were removed, rapidly agitated to separate clumped cells,
plated on BG-11 (N free) medium, and assayed for nitrogenase, fixation
of C1402 and hydrogen evolution. The data reported are typical examples
selected from many experiments which all gave consistent results.

4

Acetylene reduction and hydrogen evolution were measured gas
chromatographically on cyanobacterial and fern preparations incubated in
light in screw-capped vials containing argon-acetylene or argon atmospheres.
Samples of the gas phase were periodically withdrawn with gas -sampling
syringes. The ethylene formed from acetylene was separated on columns
of Poropak R (9) and hydrogen measured using a molecular sieve 5A column

Cl).

14

C C>2 fixation was measured on aliquots of either A. flos- aquae or

14

fern fronds in growth media containing Na2HC 0^. Samples were collected
on glass fiber papers, rinsed with 6N HC1, and the incorporated C^
determined in a liquid scintillation counter using a water-miscible
scintillation fluid.

Concentrations of A. flos -aquae in irradiated suspensions, determined
by measurement of optical densities at 650 nm, were correlated with
protein content (3) . With our cultures, an optical density of 1.0 at
650 nm corresponded to approximately 200 ygrams algal protein per milliliter.
Results

Because of their extensive pigment system, cyanobacteria are known
to be fairly resistant to short wavelength UV irradiation and to possess
an active photoreactivation system (11). In our early studies, we
confirmed both of these effects and determined killing curves for our
strains using an unfiltered Rayonet UV lamp (Figure 1). Comparison of

Fig. 1

5

killing curves obtained by plating cell aliquots on plates which were
immediately incubated in the light with those allowed to incubate in the
dark 24 hours before illumination showed an active photoreactivation of
UV killing.

Figure 2 shows that when CA is used as a filter to remove short
Fig. 2

wavelength UV, the killing effect is virtually eliminated, even though

the measured UV-B radiation intensity has now been increased fivefold to

2

approximately 2.1 W/m . Note also that although the time scale has
changed from minutes to hours of irradiation, no lethal effect can be
observed.

We attempted to increase the UV-B irradiation by using a curved
bank of six lamps with a reflector to impinge the light more directly on
the reaction vessel. Figure 3 illustrates the results of such an

Fig. 3

experiment in which the UV-B intensity has been approximately doubled to
2

5.2 W/m . These data indicate some killing; however, there was only a
slow decline in the population of viable cells which suggests that only
a fraction of the cells may be sensitive to high intensity UV-B. It
would be of interest to use this approach as a means of selecting strains
with either enhanced resistance or sensitivity to UV-B.

6

Two biosynthetic activities of A. flos- aquae were examined after
exposure to sub-lethal doses of UV-B: fixation of and nitrogen

fixation (measured by acetylene reduction and hydrogen evolution) .

Table 1 lists the effects of total UV irradiation and UV-B on acetylene

Table 1

reduction by Anabaena and indicates a decline in activity of algae
irradiated with UV-B in the absence of a lethal effect. For physiological
studies, concentrations of suspensions of A. flos -aquae were increased
tenfold. Plate counts of these suspensions indicated that, over the
range of 6-80 yg protein/ml, identical survival curves were obtained
allowing direct comparison of the results of viable cell count and
physiological activity of the suspensions.

Data in Table 2 show that, under similar conditions of irradiation,

Table 2

effects of UV-B on CC>2 fixation were slight. From these results, it
appears that the nitrogenase system is a more specific and sensitive
target for UV-B damage in A. flos -aquae.

Experiments were performed to gain some insight into the nature of
the nitrogenase inhibition by UV-B. Since nitrogenase is a multienzyme
complex which can be assayed for in a variety of ways, we have also
measured the effect of UV-B on the ability of the complex to photoevolve

7

molecular hydrogen. As can be seen in Table 3, the effect of UV-B on

Table 3

nitrogenase is negligible when this assay is used. Apparently, the

activity of nitrogenase measured specifically by the acetylene reduction

assay is the most sensitive indicator of UV-B damage.

of

Visible photobleaching/suspensions occurred after 6 hours irradiation
with UV-B. However, no destruction of a specific pigment could be
detected by examination of difference spectra of acetone extracts from
irradiated and unirradiated cells.

Discussion

From a practical standpoint, it is obvious that assessment of the

environmental effects of enhanced UV-B irradiation on biological material

is going to require development of simple assay procedures with wide

applicability. Our studies have consistently revealed a surprising

sensitivity of the nitrogenase complex to UV-B irradiation. The UV-B

2

irradiation level (ca. 3 W/m ), which we find inhibitory to nitrogenase,
is approximately the same as that of noon sunlight in the 280-330 nm
region. The main drawback to this approach to this means of assessment
of environmental damage is that it requires the use of those limited
systems which possess nitrogenase activity.

It should be emphasized that, by performing direct microbiological
plate counts on a large population of irradiated cells, we have ruled
out the possibility that the UV-B effect observed on nitrogenase is due
to nucleic acid damage. This finding suggests that the cellular target

8

may be another pigment associated with the nitrogenase complex or its

electron transport system. Further studies on the action spectrum of *

this effect may help to reveal the cellular component involved as UV-B

receptor.

The Azolla system provides an opportunity to examine the effect of

UV-B on a plant and, simultaneously, its symbiont. Since nitrogenase

activity (acetylene reduction) is exclusively a property of the symbiont,

this specific physiological activity can be measured after irradiation

14

of the fern. Measurement of fixation of C 02 by the symbiosis serves

as a general index of the physiological activity of the system. Data in

14

Table 4 summarize such an experiment, in which C 02 fixation and
Table 4

acetylene reduction are measured in UV-B- irradiated plants. Although
there was a slow decline in general physiological activity of the plants
as the culture aged, the nitrogenase activity of irradiated plants
showed a significant decrease over control plants.

Information now available (12) on the effects of short wavelength
UV irradiation on biological material has come virtually exclusively
from studies of microorganisms. It seems likely, therefore, that
microorganisms may again prove to be the material of choice to study
biological UV-B effects. Nitrogen fixation consumes a substantial
fraction of the energy of a cell in which it occurs; consequently, it is
possible that a minor physiological disturbance would be expressed more
readily in such a system. Furthermore, this assay (acetylene reduction)
is readily adaptable to field studies and could serve as a convenient
assay for a variety of environmental studies.

9

There seems little doubt that the green and blue- green algae will
be organism of choice to study large populations of plant material under
controlled conditions. Furthermore, since algal nitrogen fixation is
confined to blue-green algae (cyanobacteria) , we seem to have selected
an ideal class of microorganism for evaluation of UV-B effects on plant
material. Worldwide distribution of these organisms suggests that they
might, in this way, serve as a convenient indicator of the extent of
stratospheric ozone depletion.

Abstract

The effect of UV-B (280-320 nm) irradiation on physiological
activities of Anabaena flos- aquae and the water fern Azolla carol iniana
has been studied where lethal effects of irradiation are known to be
absent. Nitrogenase activity specifically declined at low levels of UV-
B, under conditions which had little effect on general physiological
activity of the irradiated cells. These findings indicate that measurement
of acetylene reduction (nitrogenase assay) may serve as a simple biochemical
assay to assess environmental UV-B damage to plants due to depletions of
stratospheric ozone.

10

References

1. Benemaim, J. R. , Berenson, J. A., Kaplan, N. 0., and Kamen, M. D.
1973. Hydrogen Evolution by a Chloroplast-Ferredoxin-Hydrogenase
System. Proc. Natl. Acad. Sci. U.S. 70, 2317-2320.

2. Fogg, G. E. , Stewart, W. D. P., Fay, P., and Walsby, A. E. 1973.

The Blue Green Algae, Academic Press, New York and London.

3. Layne, E. 1957. Spectrophotometric and Turbidimetric Methods for
Measuring Proteins, In Methods in Enzymology, S. Colowick and N. 0.
Kaplan (eds.), Academic Press, New York 3_> 447-454.

4. Moore, A. W. 1969. Azolla: Biology and Agronomic Significance.
Bot. Rev. 35_, 17-34.

5. Rowan, J. D. , and Norris, K. H. Instrumentation for Measuring
Irradiance in the UV-B Region. U.S. Environmental Protection
Agency. Annual Report, 1977, Interagency Program on Biological and
Climatic Effects Research, Washington, D.C.

6. Peters, G. A., and Mayne, B. C. 1974. The Azolla, Anabaena Azollae
Relationship. I. Initial Characterization of the Association.

Plant Physiol. S3, 813-819.

7. Norris, K. H. , and Rowan, J. D. Instrumentation for Measuring
Irradiance in the UV-B Region. Rev. Sci. Instrum. In preparation ,
1978.

8. Stanier, R. Y. , Kunisawa, R. , Mandel, M. , and Cohen-Bazire, G.

1971. Purification and Properties of Unicellular Blue-Green Algae
f Order Chroococcalesl . Bact. Rev. 35, 171-205.

11

9.

Stewart, W. D. P. , Fitzgerald, G. P. , and Burris, R. H. 1967. In
Situ Studies on ^ Fixation Using the Acetylene Reduction Technique.
Proc. Natl. Acad. Aci. U.S. 58_, 2071-2078.

10. U.S. Congress. Senate Committee on Aeronautical and Space Sciences
Subcommittee on the Upper Atmosphere. 1975. Stratospheric Ozone
Depletion: Hearings Part 1 § 2, 1-1060. Washington, D.C., Government
Printing Office.

11. Van Baalen, C. , and O’Donnell. R. 1972. Action Spectra for
Ultraviolet Killing and Photoreactivation in the Blue Green Alga
Agmenellum quadruplicatum. Photochem. Photobiol. L5_, 269-274.

12. Witken, E. M. 1976. Ultraviolet Mutagenesis and Inducible DMA
Repair in Escherichia coli. Bact. Rev. 4£, 869-907.

12

Table 1

Effect of UV-B on nitrogenase activity

of A. flos- aquae

Irradiation

a

time

h

Acetylene reduction^

Control

UV-B

uvc

nmol/h/mg protein

0

1,490

1,490

945

0.5

—

—

370

1

—

—

105

2

—

—

10

3

1,300

840

—

6

1,350

340

—

aUV-B, 2.1 W/m2, cell suspension, 40 ml; protein
65 yg/ml.

■L

Aliquots, 5 ml of suspensions incubated in light
in atmosphere of argon- 90%, acetylene 10 % for
assay.

cRayonet lamps without cellulose acetate filter,

2

10 W/m separate experiment, 40 ug/ml algal protein.

13

Table 2

Effect of UV-B on fixation of ^CO ^ by
A. flos-aquaea

Irradiation

time3,

h

fixed

Control

UV-B

uvb

cpm/mg protein/min in light

0

9,300

9,200

8,800

2

9,800

7,400

70

4

9,200

5,700

6

7,800

5,500

a

Cell suspension, 37 ml; protein, 50 yg/ml.

UV-B, 2.1 W/m2.

■L

DRayonet lamps without cellulose acetate filter,
10 W/m2.

14

Table 3

Effect of UV-B on photoevolution of H2 by

A. flos- aquae

Irradiation

timea

h

H2 evolution*3

Control

UV-B

nmol/h/mg protein

0

460

460

3

350

343

6

265

215

o

Cell suspensions, 40 ml, 80 yg protein/ml,

exposed to 2.1 W/m^ UV-B.

u Aliquots, 5 ml, of suspension incubated

2

anaerobically (argon atm.); 30 W/m white
light for assay.

15

Table 4

14

Effect of enhanced irradiation with UV-B on C02 fixation
and acetylene reduction by Azolla

Irradiation

Control

UV-B

enhanced

* • a
time

14C°2

Acetylene

14

°°2

Acetylene

days

fixed*3

reduced0

fixe?

reduced0

1

24,000

450

20,200

300

2

17,800

380

15,200

100

4

7,200

320

5,900

130

6

4,350

350

4,700

100

Visible light

, 10 W/m2,

supplemented with UV-B,

2 W/m2.

^cpm/g plants

(wet) /min

in visible light

, 30 W/m2

t

c 2

nmol/g plants (wet)/h; argon atm., visible light, 30 W/m .

16

Figure Legends

Fig. 1. UV killing and photoreactivation of A. flos- aquae. Single,

unfiltered, 8W Rayonet lamps, 15 cm from surface of stirred cell suspension.

2

Algal protein, 6 yg/ml; total light, 2.7 W/m .

Fig. 2. UV-B irradiation of A. flos -aquae. Six Rayonet lamps in flat
bank array held 17 cm from surface of stirred cell suspension (40 ml,

6 yg protein/ml] . Total light, 5 W/m^; UV-B, 2.1 W/m^. Cellulose
acetate filter (CA) , 10 mil.

Fig. 3. Effect of higher UV-B intensity on A. flos -aquae. Six Rayonet

lamps in curved reflector fixture held 17 cm from surface of stirred

2

cell suspension (40 ml, 7.6 yg protein/ml). Total light, 12.5 W/m ; UV-
2

B, 5.2 W/m . Cellulose acetate filter, 10 mil.

17

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