Carbon Monoxide in the Biosphere:
CO Emission by Fresh-Water Algae
FINAL REPORT TO:
Coordinating Research Council
Thirty Rockefeller Plaza
New York, New York 10020
and
Air Pollution Control Office, EPA
Principal Investigators;
H. L Crespi and J. J. Katz
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Carbon Monoxide in the Biosphere: CO Emission
by Fresh-Water Algae*
H. L. Crespi, Doris Huff, H. F. DaBoll and J. J. Katz
Chemistry Division
Argonne National Laboratory
Argonne, Illinois 6C439
..
This work supported by Coordinating Research Council--U.S.
Environmental Protection Agency and under the auspices of the
U.S. Atomic Energy Commission. Work performed during the
year 1971. Final report, October, 1972.
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SUMMARY
The chemical processes associated with the biosynthesis
and degradation of the photosynthetic pigments in algae produce
large amounts of CO. Senescent cells are found to produce CO
in excess of that that can be accounted for only from degrada-
tion of chlorophyll indicating the existence of additional plant
sources of CO. Our results indicate that plants may be the
o
source of 10 tons or more of CO per year.
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An accurate assessment of the role of carbon monoxide in
the atmosphere requires that all large natural sources of car-
bon monoxide be detected and the magnitude of their CO contri-
butions to the atmosphere be determined. The discovery of
large and previously unknown natural sources of carbon monoxide
would obviously have considerable impact on the generally held
opinion that the carbon monoxide of the atmosphere is largely
man-made (1). We believe, on the basis of experimental work
described here, that the biogenesis, and, particulary the degra-
dation, of photosynthetic pigments are such large and previously
unsuspected contributors to CO in the atmosphere. The older lit-
erature contains intimations to this effect. Thus, there are
reports that higher plants (2) and marine algae (3) evolve CO.
Ocean waters appear to act as a natural source of CO (4,5).
An atypical fresh-water blue-green alga has also been shown to
produce CO (6). The relationship between these observations and
the generality of the phenomenon have, however, not been made
explicit.
There are at least two pathways by which CO can be produced
by algae and higher plants. Blue-green algae have two character-
istic photosynthetic pigments called phycocyanin and phycoerythrin,
whose prosthetic groups, phycocyanobilin and phycoerythrobilin,
result from the oxidation of a tetrapyrrole macronucleus such as
chlorophyll (6). (See Figure 1 for the structural formula of
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these tetrapyrrole compounds. ) The chemistry of this process
probably is similar to the conversion of the heme of hemoglobin
in animals to the bile pigments, a process which, as is well
known (7), produces one molecule of CO for each molecule of heme
transformed to bile pigment. The macrocycle ring is opened by
oxidation of the carbon atom at the a bridge position to yield
a linear tetrapyrrole and a molecule of carbon monoxide. The
chemical relationship between chlorophyll and algal bilins is
very similar to that of heme and bile pigment, and each molecule
of tetrapyrrole converted to bilin would produce one molecule of
CO (6). Thus, as the blue-green algae grow, they evolve CO.
It would be expected that as all blue-green algae contain phyco-
cyanin and/or phycoerythrin, all would produce CO. However,
under our culture conditions, production of CO by blue-green
algae by this process turns out to be strongly species specific,
and for no very obvious reason.
The degradation of chlorophyll in dying plant material is
a second way for.,plants to produce CO, for the degradation of
chlorophyll might also follow a course analogous to the degrada-
tion of heme. All photosynthetic organisms that fix carbon
dioxide and produce oxygen, without exception, contain chlorophyll
a. Thus, it might be supposed that the degradation of chlorophyll
in algae and in higher green plants could produce large amounts
of CO.
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-5--
Th e work described here shows that the catabolism of
chlorophyll in algae does indeed make a large contribution to
the carbon monoxide content of the atmosphere. This is a
previously unrecognized large natural source of CO that will
have to be taken into account in describing the inventory of CO
in the atmosphere. Our observations furnish an initial basis
for the interpretation of the CO isotope studies of Stevens (8),
which appear to require the existence of a large, unknown nat-
ural source of CO.
Techniques of Algae Culture
Both green and blue-green algae were cultured by the
techniques described by DaBoll et al. (9) and Crespi et al. (10).
The nutrient solutions contain a standard mix of salts (9) and
carbon dioxide is fed to the cultures as a mixture of 5$ COp-95^
Np. The feed gas is passed over Hopcalite (a manganese-copper
oxide catalyst) to remove all traces of CO. The light intensity
is controlled by means of neutral density screens. As a culture
grows from a small inoculum, the light intensity is increased in
steps of from 12.5 to 25 to 50 to 100 percent of the maximum
light intensity. All cultures are thermostated for temperature
control. Figure 2 is a photographic view of the "algae farm".
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Analysis of GO, Chi, Bilin
An P and M Model 700 gas chromatograph with a flame ioni-
zation detector was modified for the detection of low concen-
trations of carbon monoxide (11). A gas sampling system was
added so that the sample can be swept directly from a collection
tube into the chromatograph gas carrier stream. A catalyst
tube with furnace and hydrogen supply was placed bewtween the
chromatographic column and the detector to convert CO to methane
(CO may be determined directly with a thermal conductivity
detector,, but the sensitivity is low). A nickel catalyst is used
for this conversion. Carbon dioxide is removed by a COg absor-
ant positioned before the sample collecting tube, and Linde 5A
molecular sieve is used for the GC column packing to retain any
residual trace of carbon dioxide and to separate methane, if any,
from CO. An absolute sensitivity of 0.005 microliters (1 ppm
CO in a 5 ml sample) is readily attained, with a precision of
ħ 10$. Our standard gas mixtures (Matheson) have also been
analyzed manometrically. Chlorophyll was extracted and analyzed
according to the methods of Strain and Svec (12) and phycocyano-
bilin was determined as phycocyanin (13).
Results and Discussion
The results of a survey for CO evolution of algae under
routine culture in our laboratory are shown in Table 1.
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Table 1. CO Production by Various Algae
Species Type Co Evolved
Phormidium luridum Blue-green +
Fremyella diplosiphon Blue-green
Synechococcus lividus Blue-green
Chlorella vulgaris Green +
Scenedesmus obliquus Green +
The blue-green alga Phormidium luridum produces CO at the
rate of 100-300 |_ig per gram of new growth, a CO production rate
that correlates with the rate of biosynthesis of the photosyn-
thetic pigment phycocyanin. However, the blue-green algae
Syne choc oc cus_ lividus and Fremyella diplosiphon evolve CO at very
much lower levels or perhaps not even at all. As all these blue-
green algae contain phycocyanin, it would appear that either
different biosynthetic pathways are used for the production of
phycocyanin, or that the algae differ from each other in their
ability to utilize endogenously produced CO. It is entirely pos-
sible that CO in these organisms is being fixed, as recent
reports (14) indicated that many soil organisms are able to fix
CO. The environmental impact of blue-green algae may thus involve
both large-scale emission and fixation of CO.
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Figure 3 is a plot that shows CO evolution by a culture of
the blue-green algae Phormidium luridum. CO was evolved
throughout the culture cycle at a rate dependent on light in-
tensity and at a level consistent with the amount of CO expected
from the extent of phycocyanobilin biosynthesis. An average of
about 50 x 10~3 p,l/min of CO were evolved during the approxi-
mately 160-hour lifetime of the culture. Integration over this
time period gives a total of ^80 jj,l or 600 u.g total CO evolved
by this culture. At harvest, our Phormidium cultures contain
6.0 grams of newly formed algae of which about 8 percent is
phycocyanin. Since phycocyanin is 4 percent bilin by weight,
the culture will have generated 19.2 milligrams of bilin from
which 925 |_ig of CO is expected to be evolved. This number is
somewhat higher than the measured value of 600 |j,g, but the CO
that was evolved is probably due to bilin biosynthesis, as the
rate of CO evolution appears related to light intensity is shown
by Figure 3, and therefore to biosynthetic activity.
Figures 4 and 5 illustrate the growth and CO emission of
Phormidium luridum under conditions similar to the cultures shown
in Figure 3, but over a longer time period. As the culture be-
came very dense, the rate of CO evolution increased to levels
much higher than those expected from bilin biosynthesis only.
-S
A total of 7-10 x 10 -" moles of CO were emitted during the course
_c
of each of these experiments. About 2-3 x 10 J moles of CO is
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_
expected from bilin synthesis, which leaves an excess of 5-7 x 10 J
moles of CO. While this number corresponds clearly to the total
amount of chlorophyll in these cultures, there is no evidence
of chlorophyll degradation during the course of the experiment.
These results indicate a source of CO other than bilin synthesis
or chlorophyll degradation. It is possible that Ph. luridum, a
filamentous organism, some form of cell senescence occurs
throughout the growth cycle. It has been observed in this Lab-
oratory (J. Norris, personal communication) that Ph. luridum
cells often generate a very weak electron spin resonance photo-
signal, an indication of possible cellular degradation. We have
also found that the "typical" results depicted in Figures 3-5 are
not consistently reproducible, so that one must postulate processes
involving carbon monoxide other than those associated with the
simple scheme outlined for tetrapyrrole metabolism.
Figures 6, 7, and 8 show the results of monitoring the CO
evolution by cultures of the green alga, Scenedesmus obliquus.
Because green algae contain no bilinoid pigments, no CO is ex-
pected from this source, and no CO evolution is, in fact, ob-
served until after about 450 hours of growth, when a dramatic
output of CO begins. CO emission begins as soon as the algae go
into the stationary phase of growth and begin to die. Table 2
shows the results of analysis of various portions of the curves
shown in Figures 6, 7, and 8. During the initial phase of CO
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Table 2. Correlation of CO Emission with Chlorophyll
Disappearance in S. obliquus
Experiment
Culture
Days
Total CO
Emitted
(moles x 10
5
Decrease in
Chlorophyll
(moles x 105)
Fig. 5
Fig. 6
Fig. 6
Fig. 7
Fig. 7
3
(Days 23-26)
6
(Days 23-29)
7
(Days 29-36)
2
(Days 23-25)
3
(Days 32-35)
5.4
11.0
2.3
5-5
9-9
13.7
6.7
10.0
9.0
evolution and chlorophyll disappearance, the amount of CO evolved
is equal to or lower than the amount of chlorophyll that has
disappeared (on a molar basis). In the later stages of growth,
however, considerably more CO is evolved than can be accounted
for by chlorophyll degradation. The data of Figure 8 indicate
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that CO evolution from S_. obliquus is related to plant senescence.
At day 26 the entire algal culture was centrifuged, washed and
resuspended in fresh nutrient medium. Total chlorophyll content
then increased and CO evolution decreased as the cells became
more viable and at day 32 CO evolution abruptly increased as
chlorophyll content went sharply down.
Figure 9 shows the CO evolved from a culture of Chlorella
vulgaris as a function of growth and chlorophyll content. Unlike
the other green alga, S_. obliquus, the Chlorella culture emitted
CO at a low level through most of its life cycle and there is no
rapid emission of CO as chlorophyll content drops at senescence.
These data are consistent with the following interpretation:
(1) the biosynthesis of bilinoid pigments in blue-green algae
yields CO; (2) the degradation of chlorophyll in both blue-green
and green algae yields COj (3) an additional unknown source of
CO exists in plants. It is possible that additional CO is evolved
from the oxidation of phenolic types of compounds (15,16) as it
has been shown that flavonoid degradation by molds yields CO (17).
Figure 10 summarizes our results. The fact that some of the algae
examined here do not emit CO may be due to the fact that CO can
be fixed by many organisms (14). Thus, the algae may fix the CO
that they generate, or the particular heterotrophic flora
supported by these cultures may fix CO. Our results complement
those of C. M. Stevens and co-workers (8) who have observed CO
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emissions from trees. These workers have observed, in the
northern hemisphere, an autumnal burst of CO that corresponds
o
to an emission of the order of 5 x 10 tons of CO over a 1.5
month period. Fogg (18) estimates that the total annual world
yield of photosynthesis to be 25 x 1010 tons of organic matter.
If it is assumed that 0.5$ of this organic matter is chlorophyll,
chlorophyll degradation could yield 6 x 10^ tons of CO per year.
Bilin biosynthesis could account for about the same amount of
o
CO per year, so these two sources could emit a total of 1 x 10
tons of CO per year. The data of Stevens e_t al. (8) indicate
natural sources of CO in addition to bilin biosynthesis and
chlorophyll degradation, and our data indicate that algae and
other plants are contributing in further unknown ways to the
large natural CO emissions.
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REFERENCES
1. L. S. Jaffe, Ann. N. Y. Acad. Sci. 174, 76 (1970)-
2. S. Wilks, Science 129, 964 (1959).
3- C. C. Delwiche, Ann. N. Y. Acad. Sci. 174, 116 (1970).
4. J. W. Swinnerton, V. J. Linnenbom and R. A. Lamontagne,
Science 167, 984 (1970).
5- D. F. Wilson, J. W. Swinnerton and R. A. Lamontagne,
Science 168, 1577 (1970).
6. R. F. Troxler, A. Brown, R. Lester and P. White, Science
167, 192 (1970).
7. T. Sjostran, Ann. N. Y. Acad. Sci. 174, 5 (1970).
8. C. M. Stevens, L. Krout, D. Walling, A. Venters,
A. Engelkemeir and L. E. Ross, Earth and Planetary
Sciences Letters, In press.
9. H. F. DaBoll, H. L. Crespi and J. J. Katz, Biotech. Bioen^
;4, 281 (1962).
10. H. L. Crespi, H. F. DaBoll and J. J. Katz, Biochem.
Biophys. Acta 200, 26 (1970).
11. J. W. Swinnerton and V. J. Linnenbom, J. Gas Chromatog.
5, 570 (1967).
12. H. H. Strain and W. A. Svec, in The Chlorophylls, L. P.
Vernon and G. R. Seely, Eds., Academic Press, N. Y., 1966,
13. H. L. Crespi, U. Smith and J. J. Katz, Biochemistry "]_,
2232 (1968).
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14. R. E. Inman, R. B. Ingersoll and E. A. Levy, Science
172, 1229 (1971).
15. S. Miyahara and H. Takahashi, J. Biochem. 69, 231 (1971).
16. T. Matsuura, H. Matsushima and H. Sakamoto, J. Am. Chem.
Soc. 89, 6370 (1967).
17. D. W. S. Westlake, J. M. Roxburgh and G. Talbot,
Nature 189, 510 (1961).
18. G. E. Fogg, Photosynthesis, The English Universities
Press, Ltd., London, 1968.
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FIGURE LEGENDS
Figure 1 -- Structural formulas of heme, bilirubin, chloro-
phyll a,, phycocyerobilin, and phycoerythrobilin.
Figure 2 -- View of the algae farm showing Lucite rocking
trays and associated equipment.
Figure 3 -- The production of carbon monoxide by the blue-
green alga Phormidium luridum. Culture conditions
are as described in the text, with 5$ COp-95^ N~
being fed at a rate of 18 ml/min. As the light
is stepped up in intensity, CO emission increases,
a reflection of an increased rate of bilin synthesis,
At about 95 hours, the lights were turned off, and
at 100 hours, turned on again.
Figure 4 Growth and CO production by the blue-green alga
Phormidium luridum under standard culture condi-
tions, but with a 5$ CO^-^fo N2 feed rate of 9
ml/min. As compared to the culture described by
Figure 1, there is a large CO output as the cul-
ture ages .
Figure 5 -- Growth, CO production, chlorophyll and bilin levels
during the course of culture of Ph. luridum
under culture conditions as for the culture of
Figure 3- There are large CO emissions without
concommitant disappearance of chlorophyll.
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Figure 6 -- Growth, CO and chlorophyll production by the green
alga Scenedesmus obliquus. CO production corre-
lates with cessation of growth and a decrease in
chlorophyll content of the cells.
Figure 7 Growth, CO and chlorophyll production by S_.
obliquus followed far into the stationary phase
of growth. Even after disappearance of most of
the chlorophyll, CO is being emitted at a rapid
rate.
Figure 8 Growth, CO and chlorophyll production by a culture
of S_. obliquus thas was centrifuged, washed, and
resuspended in fresh nutrient medium at day 26.
Although the culture was in the stationary phase
of growth at the time of resuspension, cell den-
sity increased further in the fresh medium, as did
the chlorophyll content, and CO emission dropped.
After a few days, the normal processes of
senescence resumed.
Figure 9 -- Growth, CO and chlorophyll production by the green
alga Chlorella vulgaris. Small amounts of CO are
emitted throughout the life cycle.
Figure 10 - A schematic picture of CO emission by algae.
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CH=CH2
H2C = CH
Heme
CH 3
CH3
CH2
I
R02C-CH2 CH3 0
Chlorophyll
CH
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Bilirubin
Phycocyanobilin
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FIGURE 2
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c
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160
140
120
100
80
60
40
20
0
Light Intensity (% of maximum)
12 25 50 100 0100
0
T
feed gas at 18 m l/min.
harvest
2 4
Days of Growth
CO Production by Phoridium luridum
FIGURE 3
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Light Intensity (% of maximum)
12 25 50 100
0
6 8 10 12
Days of Growth
14
16
Growth and CO Production by Phormidium luridum
FIGURE 4
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Growth, CO and Chlorophyll Production by Scenedesmus obliquus
FIGURE 8
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FIGURE 9
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Chlorophyll
Precursors
Senescence
Chlorophyll * CO
t
Growth
Green and
Blue-green Algae
Blue-green
Algae
Bilins and CO
FIGURE 10
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