Nitrogen Fixation by Thermophilic Blue-Green Algae (Cyanobacteria ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p. 454-458 0099-2240/79/03-0454/05$02.00/0

Vol. 37, No. 3

Nitrogen Fixation by Thermophilic Blue-Green Algae (Cyanobacteria): Temperature Characteristics and Potential Use in Biophotolysis KAZUHISA MIYAMOTO,t PATRICK C. HALLENBECK, AND JOHN R. BENEMANN* Sanitary Engineering Research Laboratory, College of Engineering, University of California, Berkeley, California 94720 Received for publication 7 January 1979

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 x 104 ergs cm-2 s-1. Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of M. laminosus NZ-86m produced hydrogen at comparable rates to Anabaena cylindrica. The conversion efficiency of light to hydrogen energy at maximum rates of hydrogen production was 2.7%.

Relatively little is presently known about the physiology of nitrogen fixation in thermophilic blue-green algae (cyanobacteria). Mastigocladus laminosus, shown to fix nitrogen in early in vivo studies (7) and in vitro studies (12), has mainly been studied in the field, where the measurement of environmental parameters or studies of geographic distribution have been made (4, 13). In its natural habitat, M. laminosus has been found to fix nitrogen at temperatures up to 550C (4) with a reported temperature optimum of 42.5°C (4, 13). A previous laboratory study established that the temperature optimum under non-nitrogen-fixing conditions was around 450C (9). We have now examined, in laboratory studies, nitrogen fixation and growth of M. laminosus at different temperatures and the effects of light intensity and oxygen tension. Hydrogen production mediated by nitrogenase in heterocystous blue-green algae has been demonstrated in laboratory and outdoor studies (2, 8, 10, 15) and has potential for development as a biophotolysis system. Sustained hydrogen production for 7 to 19 days, using nitrogenstarved cultures of the alga, Anabaena cylindrica 629, was demonstrated by Weissman and

Benemann (15). More recently, the sustained (4week) decomposition of water to produce hydrogen by sunlight was demonstrated with an outdoor culture of A. cylindrica 629 maintained under argon-N2-C02 (98.7%-1%-0.3%) (8). However, for the practical development of biophotolysis, selection of more useful algal strains is necessary. The main reasons are: (i) economic considerations require low operational and maintenance costs for biosolar converters, and (ii) large variations of outdoor conditions, especially in temperature and insolation, are inevitable. Thermophilic, nitrogen-fixing blue-green algae appear to be potentially useful algae for a practical application of biophotolysis. Areas near the tropics are probable sites for future biosolar converters. Temperatures within insulated biophotolysis converters can be expected to reach high values. This may necessitate the use of thermophilic algae. Thermophilic algae have the additional advantage of growing under restrictive conditions, thus minimizing contamination problems.

MATERIALS AND METHODS Organisms and culture methods. Three strains of Mastigocladus laminosus from the culture collect Present address: Department of Biochemical Engineer- tion of R. W. Castenholz (6) were grown in modified ing, Faculty of Pharmaceutical Sciences, Osaka University, Allen and Arnon medium (1) (modifications: 2 ,ug of Osaka, Japan. Fe per ml as ethylenediaminetetraacetic acid complex 454

NITROGEN FIXATION BY M. LAMINOSUS

VOL. 37, 1979

and 20 mM NaHCO3) at 35 to 55°C. Cultures were sparged with 0.3 to 0.6% C02 in air to keep the pH at 8.5 i 0.3. The cultivation vessel was a 1.7-liter glass column (with a working volume of 1.2 liters and an inner diameter of 70 mm) and was equipped with a jacket through which constant-temperature water was pumped (Fig. 1). Due to fluctuations in room temperature, temperatures in the culture vessel fluctuated +10C. Light was supplied by four 40-W Natur-Escent fluorescent bulbs (Duro-Lite Lamps, Inc.) positioned 26 cm from the center of the culture vessels. The light intensity was measured with a Lambda LiCor LI-185 light meter at 5 heights on the illuminated side, and the results were averaged. Average light intensity at the nearest inner surface was 1.5 x 104 ergs cm2 S-1. For hydrogen production experiments, the culture vessels were illuminated by eight UHO Vital-Lite fluorescent lamps placed at a distance of 30 cm. The average light intensity at the nearest inner surface was 3.0 x 10' ergs cm2 s'- with this experimental arrangement. Growth was measured routinely with a model 800 Klett-Summerson photoelectric colorimeter with a no. 66 red filter. Dry-weight determinations were made as previously reported (15). The conversion factor between dry weight and Klett readings was dependent on the Klett reading, perhaps due to the tendency of this alga to clump. An empirical correlation for the strain NZ-86-m was found to be: conversion factor (milligrams/liter) per Klett units = 2 + 0.005 x Klett units. Acetylene reduction assays. Acetylene reduction assays were done to determine in vivo nitrogenase activity. Samples (2 ml each) from different cultures were pipetted into Fernbach flasks (6.9 ml), which were then fitted with serum stoppers. Flasks were preincubated in the light at the specified temperatures for 10 min. Acetylene gas (0.65 ml), prepared daily by adding water to calcium carbide, was injected, after Gas

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455

which the flasks were vented to atmospheric pressure, rendering the gas phase 11% acetylene by volume. Assays were done in air except in the oxygen inhibition study, in which argon-flushed flasks were made up to the appropriate concentrations of oxygen, the samples were injected, and the reaction was initiated by injection of acetylene. Light intensity (illumination was from the bottom) was 3.3 x 10' ergs cm-2 s-l. Under these conditions, the reaction proceeded linearly with time for at least 30 min. After a 20-min incubation on a reciprocal shaker (Gilson differential respirometer, 160 rpm), the flasks were injected with 0.25 ml of 25% trichloroacetic acid solution to stop the reaction. Ethylene produced was measured as previously described (15). Hydrogen production assays. Effluent gas lines of culture vessels were connected via a gas sample valve to an Aerograph (model A-90-P3) gas chromatograph equipped with a stainless-steel column (packed with a molecular sieve 5-A) and a thermal conductivity detector. Hydrogen, oxygen, and nitrogen could be measured simultaneously by using argon as the carrier gas.

RESULTS AND DISCUSSION Growth and acetylene reduction activity. Algae .that would be used in a biophotolysis converter should be easily handled because of the very low operational and maintenance costs that are required in practical biosolar converters (3). M. laminosus is a branched filamentous alga, has a strong tendency to form clumps or pellets in liquid media, and also exhibits wall growth. These problems were overcome to some extent in this study by the use of strong agitation with a magnetic stirring bar at the bottom of the culture vessel. In some old cultures or with poor agitation, some wall growth occurred. A typical growth curve for this alga is shown in Fig. 2, together with nitrogenase specific ac5dt pH 8. 5

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FIG. 2. Growth and in vivo nitrogenase activity. Nitrogenase activity was measured by acetylene reduction at 50°C and under a light intensity of 3.3 x 104 ergs cm-2 s-'. Symbols: 0, cell density; 0, acetylene reduction activity. Other details are given in the

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456

APPL. ENVIRON. MICROBIOL.

MIYAMOTO, HALLENBECK, AND BENEMANN

tivity, measured by acetylene reduction. Patterns of growth and nitrogenase activity were similar to those of the well-studied heterocystous blue-green algae such as Anabaena (14). Nitrogenase specific activity reached a maximum during the exponential growth phase, usually at Klett values of 20 to 100. Strain survey. The maximum specific activities of three strains ofM. laminosus were measured during exponential growth (Table 1). Among the three strains tested, strain NZ-86-m showed somewhat higher specific activity, which was comparable to the values obtained with A. cylindrica (15). All three strains were similar in the temperature dependency of their nitrogenase activity. Some characteristics of the strain NZ-86-m were further investigated. Temperature effects on growth and in vivo nitrogenase activity. One method for obtaining knowledge about the temperature range for growth is through adaptation experiments. Algal suspensions (2 ml each) grown continuously at 450C and at a dilution rate of 0.025 h-1, were incubated in serum-stoppered 6.9-ml Fernbach flasks at temperatures from 25 to 570C and for times ranging from 10 min to 24 h. After a period of adaptation, acetylene reduction activity was measured. From the results shown in Fig. 3, this strain fixes nitrogen at temperatures up to 540C, with optimum nitrogen fixation occurring at about 45°C. A rather rapid adaptation of acetylene reduction was observed. This could be due to temperature effects on nitrogenase activity itself, or to indirect effects on photosynthesis. Meeks and Castenholz (11) found that adaptation of photosynthesis to super-optimum temperatures in the thermophile, Synechococcus lividus, required 1 h. Thus, the initial rapid changes of acetylene reduction to new initial temperatures probably reflects the temperature dependence of the nitrogenase activity. Although adaptation experiments can give an estimation of both the optimal temperature for growth and the maximum permissible tempera-

ture, these experiments are complicated by the fact that conditions such as pH and oxygen tension in the flasks might change considerably. Because of these potential problems, another method was also used. A study of the growth of batch cultures of NZ-86-m in the vessel shown in Fig. 1 was made. Both growth and in vivo nitrogenase activity were the highest at around 450C, and the maximum tolerable temperature was 550C (Fig. 4). Effect of oxygen on acetylene reduction activity. Inhibition of nitrogenase by oxygen is variable and can be significant, especially with old cultures of Anabaena (14). The effects of oxygen on the acetylene reduction activity of NZ-86-m were studied at three different growth rates. No significant difference in oxygen inhibition was observed at the different growth rates, as shown in Fig. 5. Atmospheric oxygen tension s ,I

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VOL. 37, 1979

NITROGEN FIXATION BY M. LAMINOSUS

was found to be slightly inhibitory (about 20%) to this alga regardless of growth rate. Effect of light intensity. Acetylene reduction activity in the Fembach flasks was measured as a function of light intensity. A light intensity of 1 x 104 ergs cm-2 s-' was found to be saturating for samples up to 300 Klett units (Fig. 6). However, under the present cultivation conditions, growth was light limited at around 100 Klett units. At this cell density, in situ nitrogenase activity was probably also light limited. Open continuous cultivation of NZ-86-m. To test the feasibility of culture maintenance in connection with the problem of contamination, open continuous cultures were conducted (Fig. 7). The continuous culture was operated for 2 months without any significant contamination problems. The fluctuation in cell density was mainly due to a plugging phenomenon in the overflow tube; thus a rapid intermittent withdrawal method was employed after day 40. Acetylene reduction activity also fluctuated inde45°C pH 8. 5

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pendently ofcell density. Occasionally, acetylene reduction activity fluctuated from day to day, but over the length of the experiment, activity was relatively constant. Hydrogen production. Hydrogen-producing cultures were maintained in the culture vessel shown in Fig. 1 under the same conditions used previously, except that in this experiment an increased light intensity (3.0 x 10' ergs cm-2 s 1) was employed. At this higher intensity, strain NZ-86-m grows exponentially to about 150 Klett units before reaching light-limited, linear growth. The sparged gas was switched, at various growth phases, from air-CO2 (99.5%-0.5%) to various mixtures of argon-N2-CO2. Two to three days afterwards, peak rates of hydrogen evolution were observed. After reaching peak productivity, rates of hydrogen evolution usually declined. The peak rates obtained at various growth phases are shown in Fig. 8 as a function of algal density. The rate of hydrogen evolution was roughly proportional to the algal density when exponentially growing cells were used for hydrogen production, and the volume specific rate reached its highest value when late-exponential-growth phase (ca. 150 Klett units) cultures were used. The conversion efficiency of light energy to hydrogen gas was calculated as 2.7% at the highest production rate. During exponential growth, nitrogenase specific activity is maximal and rather constant (Fig. 2). Decreased rates of hydrogen production at algal densities greater than Klett 150 reflect a lower in vivo specific activity of nitrogenase due, in part, to light limitation on energy supply to nitrogenase. Thus, this strain has potential for use in biophotolysis. However, whether high rates of hydrogen evolution by M. laminosus can be sus-

APPL. ENVIRON. MICROBIOL.

MIYAMOTO, HALLENBECK, AND BENEMANN

458

This work was supported in part by the Solar Energy Research Institute.

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uLITERATURE CITED

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1. Allen, M. B., and D. L. Arnon. 1955. Studies on nitrogenfixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol. 30:366~ ~~~~~~~~~~ ~~372. 2. Benemann, J. R., and N. M. Weare. 1974. Hydrogen evolution by nitrogen-fixing Anabaena cylindrica cultures. Science 184:174-175. 3. Benemann, J. R., and J. C. Weissman. 1976. Biopho-

tolysis: problems and prospects, p. 413-426. In H. G. FIG. 8. Hydrogen production by M. laminosus NZSchlegel and J. Barnea (ed.), Microbial energy conver86-m. Algaxl cells grown at an increased light intensity sion. Erich Goltze KG, Gottingen. (3.0 x 104 ergs cm-2 s-) were sparged with various 4. Billaud, V. A. 1967. Aspects of the nitrogen nutrition of mixturesc f argon-N2-C02 at various growth phases. some naturally occurring populations of blue-green al0.1 to 1.0%, and Co2 Percentag,es of N2 varied from p. 35-54. In Environmental requirements of bluewas at 0.5>%. Peak rates of hydrogen evolutioneweregae, green algae. U.S. Department of the Interior, Washington, D.C. observed .2 to 3 days after the initiation of nitrogen 5. Castenholz, R. W. 1969. Thermophilic blue-green algae Imnitation and are shown as a function of algal and the thermal environment. Bacteriol. Rev. 33:476density at that time.

taimed fo r several weeks, as has been demonstrated writh A. cylindrica (8, 15), is not known

Also, even at production at the.highest the hiNghlest production AlSO, at presenlt.it. rate, hyd rogen evolution was less than acetylene even

reductior i which, after nitrogen limitation, was three- to fourfold higher than the values shown in Table 1. 1. This observation suggests the participation of an uptake hydrogenase in this system. Further experiments to extend the length of hydroj gen evolution and to study the effect of uptake hLydrogenase are Il progress. The tI iermophilic, nitrogen-fixing blue-green alga, M. ilaminosus NZ-86-m, was found to grow well in a ltemperature range from 35 to 500C and to exhib:oit high acetylene reduction activity, comparalble to that of Anabaena species. An open, co]ntinuous culture of the alga at 450C could be maintained for 2 months without any contamination problem. A high rate significar it ,t contamination of conver sion of light to hydrogen energy by this alga was obtained under artificial illumination. These re.sults suggest that M. laminosus NZ-86m is a Iproniising alg for use in a practical biophoto converter. ACKNOWLEDGMENTS We

thank William J. Oswald for his support and encour-

agement.

504. 6. Castenholz, R. W. 1970. Laboratory culture of thermophilic cyanophytes. Schweiz. Z. Hydrol. 32:538-551. 7. Fogg, G. E. 1952. Studies on nitrogen fixation by bluegreen algae II. Nitrogen-fixation by Mastigocladus laminosus Cohn. J. Exp. Bot. 2:117-120. 8. Hallenbeck, P. C., L, V. Kochian, J. C. Weissman, and J. R. Benemann. 1979. Solar energy conversion with hydrogen producing cultures of the blue-green Anabaena cylindrica. Biotechnol. Bioeng. Symp. ~~~~~~~~~~~~alga,

8:103-118.

9. Holton, R. W. 1962. Isolation, growth, and respiration of a thermophilic blue-green alga. Am. J. Bot. 49:1-6. 10. Jeffries, T. W., H. Timourian, and R. L Ward. 1978. Hydrogen production by Anabaena cylindrica: effects of varying ammonium and feric ions, pH, and light. Appl. Environ. Microbiol. 35:704-710. 11. Meeks, J. C., and R. W. Castenholk. 1971. Growth and in an extreme thennophile, Synechococphotosynthesis cus lividus (cyanophyta). Arch. Mikrobiol. 78:2541.

12. Schneider, K. C., C. Bradbeer, R. N. Singh, L C. Wang, P. W. Wilson, and R. H. Bursis. 1960. Nitrogen fixation by cell-free preparations from microorganisms. Proc. Natl. Acad. Sci. U.S.A. 46:726-733.

13.

W. D. P. 1960. Nitrogen fixation by blue-green Stewart, algae in Yellowstone thermal areas. Phycologia

9:261-

268. 14. Weare, N. M., and J. R. Benemann. 1973. Nitrogen II. Nitrogenase activfixation by Anabaenaand cylindrica ity during induction aging of batch cultures. Arch. Mikrobiol. 93:101-112. 15. Weissman, J. C., and J. R. Benemann. 1977. Hydrogen production by nitrogen-starved cultures of Anabaena cylindrica. Appl. Environ. Microbiol. 33:123-131.