Commercially available air lift fermentors were used to simultaneously monitor biomass production, N2-fixation, photosynthesis, respiration, and sensitivity to oxidative damage during growth under various nutritional and light regimes, to establish a data base for the integration of these organisms into a Closed Ecological Life Support System (CELSS) program. Certain cyanobacterial species have the unique ability to reduce atmospheric N2 to organic nitrogen. These organisms combine the ease of cultivation characteristics of prokaryotes with the fully developed photosynthetic apparatus of higher plants. This, along with their ability to adapt to changes in their environment by modulation of certain biochemical pathways, make them attractive candidates for incorporation into the CELSS program

Belkin, S.

Fry, I.

Packer, L.

English

NASA Technical Reports Server

Application of Photosynthetic  fixing Cyano- 
bacteria to the CELSS Program 
by Lester Packer, Ian Fry, and Shimshon Belkin, 
Membrane Bioenergetics Group, Applied Science 
Division, Lawrence Berkeley Laboratory, University 
of California, Berkeley, CA 94720 
Abstract 
Certain cyanobacterial species have the unique 
ability to reduce atmospheric N2 to organic nitrogen. 
These organisms combine the ease of cultivation charac- 
teristic of prokaryotes with the fully developed photo- 
synthetic apparatus of higher plants. This, along with 
their ability to adapt to changes in their environment by 
modulation of certain biochemical pathways, make them 
attractive candidates for incorporation into the CELSS 
program. 
A high percentage of the cyanobacterial biomass is in 
the form of protein, much of which exists in discrete high 
molecular weight granules (phycobilisomes and carboxy- 
somes). We have shown that with proper manipulation of 
the osmotic environment, high amounts of granular glycogen 
can also be produced. These particular fractions, along 
with the ability of certain species to aggregate, may 
negate future need for conventional harvesting and frac- 
tionation techniques, such as high speed centrifugation. 
We are undertaking a study, using commercially 
https://ntrs.nasa.gov/search.jsp?R=19860010458 2020-03-20T15:10:51+00:00Z
,.,. - ? 
; . .  ;: F -*F ;, . , ;. : 
..-, ?,#. '-9 &/ 3 .  
available air lift fermentors, to simultaneously monitor 
biomass production, N2-fixation, photosynthesis, respira- 
tion, and sensitivity to oxidative damage during growth 
under various nutritional and light regimes, to establish 
a data base for the integration of these organisms into a 
CELSS program. 
Introduction 
The use of biological components in the CELSS program 
as subsystems for the revitalizaton of air, waste pro- 
cessing, and for the production of food has been proposed 
for long-term space flight('). To this end, many systems 
of biomass (protein, carbohydrate, lipid) production are 
under consideration by NASA., Employment of photosynthetic 
systems (higher plants, green algae and cyanobacteria) 
allow biomass production from relatively simple components 
which are readily recycled in a CELSS system, namely C02, 
minerals (NO;, PO;, k ,  Na+, etc. ) and micronutrients. 
The production of plant material in a closed system 
presents several problems, two of which we shall address 
here. A primary consideration is that of the energy 
requirement for continuous operation, including illumina- 
tion, temperature control and various maintenance mech- 
anisms. One way in which the energy demands can be 
lessened is by modifying the quality of the light used. 
Illumination of photosynthetic systems with white light 
340 
(300-700nm range) is wasteful, since photosynthetic 
organisms utilize only selected regions of the visible 
, * 
spectrum. Tailoring the emission profile of the light '.. 
source to match that of the action spectrum of photosyn- 
thesis, or to select an I1emission bandw in a region of 
maximal absorption by some of the photosynthetic pigments 
would present an energy saving, especially if the selected 
I1emission bandw was in the blue region, when using fluor- 
escent light sources. 
A second concern is the probability of nitrogen loss 
from the closed system by th& action of contaminating 
denitrifying bacteria, which degrade NO; to N2 gas, which 
is then lost from the biological system(2). Denitrifica- 
tion is most likely to occur irrespective of the method of 
waste processing due to the storage of human waste and 
non-food biomass (cellulose), which would result in an 
irreversible loss of biologically essential nitrogen. 
To address the above problems, we have proposed the 
use of cyanobacteria (blue-green algae) as the photosyn- 
thetic organism of choice. 
Cyanobacteria o c c ~ , ~ ~ ~  a unique position in the 
hierarchy of plant form and structure. Like higher 
plants, they carry out the O2 -evolving photolytic cleavage" 
of water and the subsequent fixation of C02 into carbohy- - 
drate. Unlike higher plants, however, during periods of 
nitrogen starvation (when NO; levels in the medium are 
depleted) certain species differentiate specialized cell 
types (heterocysts) which maintain low oxygen tension, a 
prerequisite for their major function: the reduction of 
atmospheric N2 to NH3, prior to its incorporation into 
amino acids and proteins. In addition, cyanobacteria 
possess prokaryotic genetics, which allow the future 
possibility of genetic manipulation. Also, environmental 
factors have been shown in certain strains to modify the 
partitioning of photosynthetic reductant to certain macro- 
molecules ( 3 f 4 )  and storage carbohydrate(5). These factors 
make the cyanobacteria prime candidates for consideration 
in the CELSS program, as either a primary source of bio- 
mass or as a supplement to other algal/higher plant sub- 
systems, to reverse the effect of denitrifying bacteria, 
and recycle atmospheric N2 back into the biological sys- 
tems. 
Research Objectives 
Due to the suitability of cyanobacteria for integra- 
tion as a CELSS subsystem, we propose to investigate 
biomass production in the form of dietary compatible 
macromolecules (protein, lipid, carbohydrate) of nitrogen 
fixing heterocystous cyanobacteria in (commercially 
available) air-lift type fermentors. The research plan is 
to investigate environmental effects of nutrient supply, 
particularly C02 (the major limiting factor for plant 
growth) and the effects of various light regimes on pro- 
ductivity, in particular where it affects N2 fixation. 
Due to the more efficient conversion of W photons to 
photons in the blue region rather than to the full visible 
range by fluorescent discharge tubes, we shall investigate 
growth characteristics, biomass production, and certain 
relevant biochemical functions (photosynthetic O2 evolu- 
tion, respiration, and nitrogen fixation) on cells grown 
under blue light, compared to an equal concentration of 
white light photons. The blue llemission band" overlaps 
absorption bands of the light harvesting antennae pigments 
chlorophyll and phycobiliproteins. 
One of the major factors affecting nitrogenase 
activity is the ambiant oxygen level. Although the 
heterocysts possess a thick cell wall which slows O2 
diffusion to rates that are compatible with the rates of 
O2 removal by the respiratory system, it is quite likely 
that intracellular levels of O2 (particularly in the cells 
directly adjoining the heterocysts) may exceed usual 
environmental levels. To determine the role of O2 in such 
a system, we have been developing an electron spin reson- 
ance (ESR) method which monitors the broadening of a 
paramagnetic spin probe molecule by interaction with 02, 
which is itself a paramagnet in the ground state. Inter- 
action of the spin probe with other paramagnetic species 
causes a measurable broadening of the probe's ESR signal 
line width. Since the probe can be completely broadened 
by adding a cell membrane impermeable paramagntic reagent, 
.- 
the broadening due to O2 inside of the cell can be 
-. 
monitored. Using this technique, we plan to investigate 
the effect of cellular O2 levels, where this may influence 
the rates of nitrogen fixation. 
 ater rials and Methods 
Nostoc muscorum (Anabaena 17119) was grown in BGll 
medium minus nitrate in a 2 litre Bethesda Research 
-1 -2 Laboratories ~irlift Fermentor at 28'~~ i50 u Es m 
light (using Bethesda Research Laboratories 2201 LB day 
light white 300-700 nm, or 2201 LF blue light 400-500 nm) 
with an air flow rate of 2 litres/min, supplemented with 
0.5% C02 . 200 ml aliquots were withdrawn daily, the 
fermentor volume made up by addition of 200 ml of sterile 
medium. Cells were centrifuged at 10,000xg/10 min and 
resuspended to 10 ml in BGll medium suplemented with 10 mM 
Tes buffer pH 7.0. 
O2 evolution was monitored polarographically in BGll 
plus lOmM Tes pH7.0 with a cell density equivalent to 1-2 
ug chlorophyll/ml. Nitrogenase activity was determined in 
whole filaments by monitoring acetylene reduction using a 
Varian Model 3700 gas chromatograph fitted with a Poropak 
T column. Cells (3 ug chlorophyll in 3 ml) were assayed 
in a 5 ml vial under air plus 10% (v/v) C2HZ in a shaking 
T a b l e  I 
CYANOBACTERIA -- (BLUE-GREEN ALGAE) 
Organism Comments Rate of N Reference 
~eduction' 
umoles.mg chlorophyll.hr 
Anabaena sp. filamentous 5-10 Stewart, W.D.P., 1977; 
ATCC 29151 heterocystous Blumwald, E., and 
PCC 7119 frgshwater 
(Nostoc mus- 26 C, 2000-3000 lux 
corun) 
. . 
Tel-Or, E., 1982 
Anabaena sp. filamentous 30-60 Stacey, G. & g . ,  1977 
ATCC 33047 heterocystous 
(Sea anabaena) magine 
37 C, 1600 lux 
Gloeothece sp. unicellular 2-4 Kallas, T.,et g . ,  1982; 
ATCC 27152 non-heterocystous Gallon, .J;R, 1980 
PCC 6909 frgshwater 
(Gloeocapsa 26 C ,  2000-3000 lux 
SP.) 
Synechococcus 
sp. 
ATCC 27145 
PCC 6311 
un'icellular" Blumwald, E. g . ,  
concentric thylakoids 1983 
frgshwater 
26 C, 2000-3000 lux -. ,- i : r 
Synechococcus 
SP. 
ATCC 27264 
PCC 7002 
(&qmenellum 
guadrupli- 
catum PR-6) 
-- 
unicellular Van Baalen, C., 1962 
concentric thylakoids 
ma ine I; 39 C, 2000-3000 lux 
6 ,  
PCC 7421 
unicellular Rippka, R. & d., 
lagks thylakoids 1974 
22 C, < 500 lux . * 
T a b l e  I1 
C E U  COMPOSITION OF NOSTOC MUSCORUM GROWN 
UNDER WHITE (300-700nm) AND BLUE (400-5oonm) LIGHT 
Percent Dry Weight 
Light Protein Glycogen Soluble Lipid chlorophyll Total 
Quality Sugars 
White 3 6 28 10 13 1 .,05 8 8 
Blue 25 22 10 9 0.46 6 7 
Samples taken after seven days of growth 
345 
waterbath at 2 8 O ~  under 50uEs -Im-2 blue or white light. 
r 
Light intensities were measured using a Li-Cor inc. 
Integrating quantum/radiometer/photometer Li-188B, with a 
Li-190SB quantum sensor. 
Oxygen-broadening experiments: 
Dense suspensions (1-2 mg chl/ml) of the cyanobac- 
terium Agmenellum quadruplicatum, in ASP-I1 medium, were 
introduced into 75 ul capillary tubes, together with 0.5 
mM of the nitroxide spin-probe 3-hydroxy-2,2,5,5-tetra- 
methyl 3-pyrrolin-l-oxyl (PCA-01). The ESR signal was 
monitored by using a varian El09 EPR spectrometer. The 
sample was illuminated inside the cavity with an Oriel 
Universal Xenon Air Lamp. 
Ex~erimental Results 
Representative strains of cyanobacteria are shown in 
Table I, along with their nitrogen fixing capabilities and 
growth conditions. The heterocystous filamentous strain 
Nostoc muscorum (Anabaena 7119) was chosen for the present 
study. , . 
Growth of Nostoc muscorum under blue (400-500nm) and 
2 
white (300-700nm) light at 150uE/s/m , is shown in Figure 
1. Clearly the cells grow readily under the blue light, 
Fig. 1) Growth of Nostoc 1 00 
muscorum under white 
(300-500 nm) and blue 
(400-500 nm) light, 
measured by absorbance 
changes at 550 nm. E 
G=cell doubling time. C % 0.10 
V) 
a 
001 
0 1 2 3 4 5 6 7  
Days of Growth 
J 
- white cells 
- G=22hrs 
G=56hrs 
28OC 150 ~ ~ / s / r n ~ -  
I I I 
even though the cell doubling time (generation time, G) is I 
increased by a factor of 2.5. Analysis of photosynthetic 
O2 evolution and nitrogenase activity during growth under 
blue light reveal very little difference from those grown 
5~ 
under white light when rates were calculated on a total 
1 1 
protein basis (Figure 2 and 3, upper sections). Calcula- 
tion of rates (particularly photosynthesis) on a total 
chlorophyll basis reveal an apparent increase in photo- 
synthetic rate (Figure 2, lower section). However, this 
reflects a decrease in total cell chlorophyll, a chromatic 
adaption to blue light (see Table 11). Analysis of cellu- 
lar products after a seven day growth period under blue 
and white light reveals that little change occurs in 
soluble sugars, glycogen, or lipids, while total protein 
decreases only by 10% (Table 11). An nunknownff component 
which does increase (see "Totalff column in Table 11), is 
probably cell wall material, and is under further charac- 
terization. 
14 
blue cells,whrte l~ght 
z Iu 
loo - 2a°C 150 ~ ~ / s / r n 2  
0 1 2 3 4 5 6 7  
0 1 2 3 4 5 6 7  
Days of Growth 
Days of Growth 
Fig. 2) Photosynthetic O2 Fig. 3)-Nitrogenase 
evolution of cells grown activity, as measured by 
under white or blue light, acetylene reduction based 
based on total protein (upper on total protein (upper 
secion) and chlorophyll section) and chlorophyll 
(lower section) conditions as (lower section) conditions 
in Materials and Methods. as in Materials and 
Methods. 
In conclusion, the nitrogen fixing strain Nostoc 
muscorum is capable of growth under light at the blue end 
of the visible spectrum. One must evaluate whether the 
energy saving gained by employing blue photons-outweigh 
the slower growth rate (and hence biomass production). 
However, if a system employing a continuous culture (kept 
at a fixed point in the exponential growth phase, linear 
section of Figure 1) for air revitalization, etc., then a 
slower growth rate may be advantageou~~~requiring less - 
- 
addition of growth medium to maintain the constant cell 
348 
- - - - - -- - 
density. 
Nitrogenase, the enzyme responsible for the biolog- 
ical fixation of atmospheric N2, is extremely oxygen- 
.-- 
sensitive(2). One of the.'modes in which cyanobacteria 
llsolvedll the problem of N2-fixation under aerobic condi- 
A. 
- 
tions was the development of the heterocysts mentioned 
above. However, as with other nitrogenase containing 
microorganisms, the mechanisms by which these cells retain 
an oxygen-free internal environment is not completely 
-- 
understood. . . 
We have recently developed an ESR technique which 
enables us to measure introcellular O2 concentrations. We 
hope to utilize-this method to gain a better*understanding 
of the protection mechanisms against the toxic effects of 
o2 to nitrogenase, and thereby of the ways the activity of 
this enzyme may be manipulated to the projectls advantage. 
Fig. 4 )  The center ESR signal of PCA-01 (A) and the 2nd 
derivative (B) , before (solid line) and after (dashed 
line) 1 min illumination ,of A. quadruplicatum suspension 
(lmg chl/ml). PCA-01 concenGation was 0.5 mM. 
~ - -  ~- 
The above technique is based upon the broadening 
effect dissolved O2 has on the nitroxide spin probes ESR 
signal. An example of such an effect is presented in 
Figure 4. ~hotosynthetically-generated oxygen, in a sus- 
pension of the cyanobacterim Agmenellum guadruplicatum, 
causes a broadening of the signal by 0.5 G (Figure 4a). 
The effect is even more pronounced when a higher deriva- 
tive of the spectrum is examined (Figure 4b). According 
to our calculations, this broadening is equivalent to O2 
concentration in the suspension of 2.5 mM. Furthermore, 
by quenching the extracellular signal, it is possible to 
examine only the probe inside the cells (Figure 5 ) ,  and 
using the measured degree of broadening calculate intra- 
Fig. 5) A schematic 
presentation of the mode A. Probe Only 
by which an internal spin- 
probe signal is 
differentiated from the 
external one. @ +id- 
B Probe + Na2Mn-EDTA 
63 
cellular O2 concentrations. Studies are presently under- 
way to compare O2 levels in non-N2-fixing and some N2- 
fixing cyanobacterial cells, as well as to establish the 
02-sensitivity of various photosynthetic activities. 
350 
Future Research 
We propose to continue the investigation of the 
effects of selected wavelengths (and combinations of 
selected wavelengths) on biomass production and the bio- 
chemical functions of O2 evolution and N2 fixation. 
In non-nitrogen fixing strains of cyanobacteria we 
have shown that changes in the environmental conditions, 
particularly increases in salinity, result in a marked 
increase in soluble sugars ( 6  and carbohydrate storage 
products ( 5 )  . We plan to investigate the partitioning of 
photosynthetically derived reductant between the various 
macromolecules (protein, lipid, carbohydrate) under such 
conditions to determine the ability to manipulate the 
relative 1evels.of the various macromolucules by environ- 
mental changes. 
References 
1. MacElroy, R. D., and Bredt, J. (19.84) NASA CP-2378, 
XXV COSPAR Conference, Graz, Austria (MacElroy, Smernoff 
and Klein, ed. s) pp 1-9 ' 
2. Payne, W.J., Rowe, J.E. and Sherr, B.F. (1980) lh; 
Nitrogen Fixation vol 1 (W.E. Newton and W.H. O m e -  
Johnson, ed.s) pp 29-42 Vain Park Press, Baltimore 
3. Reed, R.H., Richardson, D.L., Warr, S.R.C. and Stewart 
(1984) Journal of Genetic Microbiology 130, 1-4 
4. Mackay, M.A., Norton, R.S. and Borowitzka, L.J. (1984) 
Journal of Genetic Microbiology 130, 2177-2191 
5. Fry, I.V., Huflejt, M., Erber, W.W.A., Peschek, G.A. 
and Packer, L. (1985) Submitted-for publication 
6. Blumwald, E., Mehlhorn, R.J. and Packer, L. (1983) 
Plant Physiology 73, 377-380 
7. Blumwald, E., Mehlhorn, R.J., and Packer, L. (1983) 
Proc. National Academy of Science 80, 2599-2602 


Application of photosynthetic N2-fixing cyanobacteria to the CELSS program

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server