Effect of Carbon Sources on Growth and Nitrogen Fixation of Aulosira fertilisssima Ghose

Biochem. Physiol. Pflanzen 183, 51-58 (1988) VEB Gustav Fischer Verlag .Jena

Effect of Carbon Sources on Growth and Nitrogen Fixation of Aulosira fertilissima Ghose MEENAKSHI BANERJEE and H. D. KUMAR Centre of Advanred study in Botany, Banaras Hindu University, Varanasi, India K ey T erm Ind ex: Hetcrotrophy, nitrogenase, glutamine synthetase; Aulosira fertilissitna

Summary Sucrose, glucose and fruetose stimulated the growth of the blue-grcen alga Aulosira ferin issima and also increased its heterocyst frequenc y, nitrogenase activity and glu tamine synthetase activity in light and dark. Ribose, laetosc, aeet atc and formate were inhibitory. Sucrose reversed the toxic effect of lactose. Low but prolonged rates of nitrogen asp activity were also obtained in cultures without the supplementation of any sugars.

Iutroduction

Assimilation of organic compounds by blue-green algae was originally studied with the aim of understanding the general problem of obligate photoautotrophy. Obligate photoautotrophs, defined as organisms which are unable to grow on organic compounds as a sole or major source of cell carbon include some groups of bacteria and most of the blue-green algae. Some exceptions to this generalization have been studied by KHOJA and WHITTON (1971, 1975) ; WHITE and SHILO (1975); BOTTO)iLEY and VAN BAALEN (1978a, b) ; THOMPSONand CASSAY (1981) and MISHRA et al. (1985). Cyanobacteria can be divided into 2 categories in terms of their response to organic compounds: some strains only grow in light with carbon dioxide as the carbon source while others also grow on organic media. It has been suggested that these should be designated as "specialist" and "versatile" strains, respectively (SMITH and HOARE 1977). Specialist and versatile strains possess the same complements of enzymes for the metabolism of glucose, but they exhibit marked quantitative differences in their ability to convert this substrate to carbondioxide (PELROY et al. 1972). The specialist character has been attributed to failure of potential growth substrates to enter the cell at rates sufficient to support significant growth. On of the most distinctive features of heterotrophic growth in cyanobacteria is its low rate relative to growth under photoautotrophic conditions. Although the rate of protein synthesis is greatly reduced in organic media in the dark, the usual metabolic route operates though at a much lower rate. A rapid flow of carbon from exogenous carbohydrate into monophosphates in the dark, is apparently not balanced by the removal of metabolites at a comparable rate. This is possibly due to partial block, early in glucose metabolism, and this could, ill turn have the effect of greatly reducing the growth rate of the alga on organic media in the dark. 4*

51

Reports of FAY (1965, 1976), BOTTOMLEY and STEW ART (1977) and WOLK and SHAFFER (1976) give us ample proof that some blue-greens are able to fix nitrogen in the dark when supplemented with exogenous carbon sources. There has been a great deal of work on nitrogen fixing heterotrophs in the rice rhizosphere (YOSHIDA and ANCAJAS 1971, 1973; DOJ\HIERGUES et al. 1973) but there is scarcity of information concerning the non-rhizosphere microorganisms especially the role of blue-green algae in this respect. Aulosira fertilissima is a dominant cyanobacterium of many rice fields and is known to be an important contributor of soil nitrogen (SINGH 1961). Virtually nothing is known about the basic characteristics of heterotrophic growth and metabolism of this organism. In the present work we present some observations on heterotrophic growth, heterocyst frequency and activities of nitrogenase and glutamine synthetase in A. fertilissima.

Materials and Methods A. fertilissima Ghose was grown axenitally in Hughes' medium (HL'GIIES et al. 1958) lacking combined nitrogen. Cultures were maintained at 27 ± 2°C and 2,500 ± 200 Ix. Growth was estimated by extracting Chlorophyll a in 80 % acetone and measuring the absorbanee at 663 nm. Chlorophyll (( content was caltlliated from the absorbanee values by means of the equation of lVhcKIN "EY (1941). Generation time was detl'rmined by the growth equation of KRATZ and Mn:l{s (1955). Hetcroeyst freq llen(·y was ml'asured as a percentage of the total tells by counting at least ten filaments. l'Iitrogenase activity was measured by the atetylene reduction assay (STEW.lltT et al. 196H). Ethylene formed was measured in a CIS Gas Liquid Chromatograph fitted with a Porapak R eolumn and a H2 flame ionization detector. The whol!) cell transferase ac:tivity of glutamine synthetase was ml'aslUed by the method of SHAPIRO and STAD'DI\N (1970) as modified by STACEY et al. (1977). All carbon sourecs were dissolved in double distilled water, filtered through sterile millipore filters (OAD/lm) and aseptieally added to the mlturc tubes to obtain the desired final eoncentration. Dark eondition was LTeatpcl by wrapping the l'ultnre tubes with black paper and imubating in the culture roolll. D-glutose, D-frudose, D-sml'ose, D-Iadose and D-ribose were produds of Sigma Chemical Co., St. Louis, USA. Sodium salts of al'etate and formate were of highest purity "Analar grade" of British Drug Honses (Glaxo) Bombay.

Results

Sucrose, glucose and fructose stimulated the growth of Aulosira both in light and dark. Among the concentrations tested (0.5 -4 %), sucrose and fructose caused maximum stimulation at 0.5 % and glucose at 2 %. Lower concentrations of lactose, ribose, acetate and formate did not cause any significant change in growth while higher concentrations were inhibitory. The generation time of air grown cultures in light was about 65 h, which fell to 48 h with sucrose, 50 h in glucose and 54 h in fructose at the above stated concentrations. Other carbon sources had no significant effect, on generation time. In the dark the generation time was 133 h in control. It was 98 h, 104 h, and 109 h in the presence of same concentrations of sucrose, glucose and fructose (Table 1). Other carbon sources did not support dark growth. 52

BPP 183 (19H8) 1

Table 1. Groll:lh of .lilllosiTa under nitrogen fixing conditions suppleme nted lrith carbon compounds. Concentrations taken here are those which support maximum growth. Treatments

Generation time in hours (Light)

Generation time in hours (Dark)

Control Aulosim Sucrose (0.5 Glucose (2 %)) Frue t os(' (0.5 %) Acetate (0.05 %) Formate (0.05 %) Lactose (0.05 %) Ribose (0.05 %)

65 48 50 54 62 61

133 98 104 109 NG NG NG NG

G2 62

XG = Xo. growth

Table 2 A. In Light. Nitrogenase activity, heterocyst fr equency alld glutamine synthetase activity of

Aulosira supplemented with carbon sources on the SI:xth day. E xperimental sets

Pen-ent heterocyst fn'qnency

11 Control Sncrose Glucose Fruetose Ribose Lactose Formate Acetate

± SE

3.5 [>. 8

± 0.09 ± 0.12 ± 0.06 ± 0.01 ± 0.07 ± 0.08

5.4 4. 8 1.2 1.6 1.4 ± 0.14 1.8 ± 0.21

Nitrogenase activity nnwl C2 H 4 /,ug ChI. a)

11

± SE

21.06 ± 0.2 46.48 ± 0.12 30.06 ± 0.1 28.09 ± 0.16 4.76 ± 0.2 5.44 ± 0.38 5.32 ± 0.22 5.83 ± 0.18

Glutamine synthet ase m11 y-glutamyl hydroxamate),ug ChI. a) 30 min 11 ± SE 2.3 ± 0.05 3.2 ± 0.01 2.8 ± 0.07 2.75 ± 0.02 0.50 ± 0.005 0.55 ± 0.007 0.58 ± 0.001 0.62 ± 0.001

Concentrations of surrose, glucose and fructose were 1, ,3 and 1 %, resp ectively. Concentration of other carbon sources was 0.05 %.

Table 2 B. In dark. Nitrogenase activity, heterocyst frequ ency and glutamin e synthetase activity of Aulosira wUh carbon sources on the sixth ' day. Expf'rimental sets

Control Sucrose G\uC'ose Fructose

11 ± SE

Nitrogenase activity Glu tamine synthetase mM nnwl C2 H4 /,ug ChI. a/h y-glntamyl hydroxamatel ,ug ChI. a 130 min }f ± SE 11 ± SE

3.5 ± 0.09 H.4 ± 0.08 cUI ± 0.09 3.4 ± 0.08

2.4 ± 0.08 15.3 ± 0.04 12.8 ± 0.06 EO ± 0.03

Percent heterocyst frequ ency

0.25 ± 1.60 ± 1.38 ± 1.30 ±

0.001 0.005 0.002 0.001

Coneentrations of sucrose, glucose and fructo se were 1, 3 and 1 %, respectively.

BPP 183 (1988) 1

53

•

100

01

•

U 01 ::1

50

. ~

:z::

u

Glucose Sucrose

"0 E c

E

10

Fructos.

5

01 :J.

.,1

----0-"::::::==* Sucros. Glucose

~

u

Fructos.

2

3

4

5

Concentratio ns (.0/.,) Fig. 1. N ega.live corre!a.lion bel/cecil nitrogenase activity and chlorophyll a content tf!wn grown l:n different concentl'lllions of the 3 sugars.

There was significant increase in nitrogenase activity in light with the supplementation of the 3 sugars. With sucrose (1 %) there was nearly 2.5-fold stimulation followed by 1.5-fold in glucose (3 %) and i-fold in fructose (1 %). In the dark also all these 3 carbon sources (same concentration) supported dark nitrogen fixatio n significantly (Table 2A and B). Even in the absence of these sugars there was some activity in the dark. It was observed that with decrease in chlorophyll a content the nitrogenas e activity increased (Fig. ]). In the case of sucrose and fructose maximum stimulation of nitrogenase activity occurred at the concentration of 1 % while chlorophyll a content was highest at 0.5 %. Similarly in the case of glucose 3 % elicited maximum activity whereas the chlorophyll a was highest at 2 %. With increasing nitrogenase activity there was increase in glutamine synthetase activity both in light and dark. Other carbon sources inhibited both nitrogenase and glutamine synthetase activity. Heterocyst frequency increased by the addition of the 3 carbon compounds (same concentrations which enhanced nitrogenas e activity) from 3.5 % in control to 5.8 % with sucrose, 5.4 % in glucose and 4.8 % in fructo se. Other carbon sources inhibited the heterocyst frequ ency. In the dark t here was no significant change in heterocyst frequency with the addition of the 3 carbon compounds. Heterocyst frequency, nitrogenase activity and glutamine synthetase activity was observed on the sixth day. The nitrogenase activity which was totally inhibited by lactose (1 %) was restored partially with the addition of 1 % sucrose. Maximum stimulation was observed with 54

EPP 183 (1988) 1

Table 3. Reversal oflactose inhibited nitrogenase activity by sucrose

(1111101

C2 H4(f.1g ChI. a (h).

Nitrogenase activity after 6 d ± SE

Experiment sets

l\I Control Sucrose 1 % Lactose 1 % 1 % Laetose + 1 % SueTose 1 % Laetose + 1 % Su('!'ose (grown in laetosc for 6 d and thm 1 % SlH'TOSe added before assay). 1 % Sucrose + 1 % Ladose (grown in Sllerosc, with Jaetose being added before assay).

22.06 43.08

± ±

16.33 6.67

± 0.17

15.60

± 0.18

0.14 0.12

± 0.22

sucrose alone. Cultures grown in lactose for 6 days, to which sucrose was added 1 h before assay, showed less reversal of enzyme activity than the set which was grown in sucrose for 6 d and to which lactose had been added 1 h before assay (Table 3). Discussion

From our observations it appears that Aulosira has the capacity to grow and fix nitrogen in light and dark when supplied with sucrose, glucose and fructose, and therefore can be denoted as a versatile strain. Sucrose was the most suitable carbon source supporting maximum growth and nitrogen fixation. The failure to observe stimulation of growth and nitrogenase activity in light with ribose, lactose, acetate and formate may be due to the lack of requisite enzymes to metabolise these compounds. These organic compounds did not support heterotrophic growth and nitrogen-fixation. The concentration dependent stimulation of growth by glucose may possibly be due to the slow rate of entry into the cells whereas in the case of sucrose and fructose the entry rate seems to be faster. The impermeability to potential growth substrates is unlikely to be the only barrier to heterotrophic growth (DOOLITTLE 1979). The metabolic priority of blue-green algae in the dark is to restrict carbohydrate metabolism in order to ensure survival of cells throughout the period of darkness. From this it follows that the cells must be able to distinguish substrate molecules on the basis of their origin, if they are to metabolise exogenous carbon sources at a higher rate than the endogenous reserves. This is unlikely and therefore versatile blue-green algae grow in the dark on organic media at rates much lower than under photoautotrophic conditions. They therefore can possibly distinguish between conditions when an exogenous growth substance is available and those which are wholly dependent on their endogenous reserves. A very limited respiratory rate would account for the inability of certain cyanobacteria to use organic compounds although they can exploit them as a source of carbon during photo heterotrophic growth. A direct role of these compounds in stimulation of nitrogenase activity cannot be ruled out since non-availability of photosynthates also limits nitrogen-fixation. Supply of photosynthetic products by exogenous addition is bound to induce some increase EPP 183 (1988) 1

55

in nitrogen-fixation. Another possible explanation for the enhanced nitrogenase activity may be the decreasing chlorophyll a content. This sort of a negative correlation has been observed in Anabaena variabilis (HAURY and SPILLER 1981). The decrease in chlorophyll a content may cause a decline in oxygen evolution thus providing more favourable conditions for nitrogen-fixing machinery by creating microaerobic states. The nitrogenase activity in the dark is dependent upon a low potential reductant (NADPH) and a supply of energy. In Aulosira the enhanced nitrogenase activity in the dark with sucrose, glucose and fructose suggests that these 3 carbon sources can supply the alga with the necessary requirements for nitrogen-fixation in the dark. The abrupt decline in nitrogenase activity after transfer of Aulosira from light to dark may be associated with the speedy depletion of energy and reductant supplies for nitrogenase action. Continued nitrogen fixation in the dark would therefore depend on mobilization of reserve produce and on the rate at which carbon moves from its reservoir to the site of nitrogenase activity. It is noteworthy that the decrease as compared to light control is less dramatic when the alga is supplemented with carbon sources. Addition of carbon sources would be expected to support and stimulate nitrogenase activity for prolonged periods in the dark. This is observed in our case. In the dark ferredoxin NADP+ reductase may function to reduce not NADP but ferredoxin. The energy supply in the form of ATP for nitrogen fixation in the dark is probably supplied by oxidative phosphorylation and the observation that in the dark or in low light intensities oxygen stimulates the nitrogenase activity lends credence to this inference (WOLK 1970; BOTHE et al. 1977; PETERSON and WOLK 1978). Another evidence is the observation that ATP production is limited in the dark in the absence of oxygen (BOTTOMLEY and STEW ART 1976) and under these conditions nitrogenase activity is low and undetectable (STEWART 1977). The prolonged activity obtained in the dark in the absence of the three sugar supplements could be due to a large store of reductants generated during the preceding light period. Such dark nitrogen fixation has been reported by LEX and STEWART (1973) and FAY (1976). Another possibility is that the alga may have a large store of endogenous reserve which is metabolised in the dark for nitrogen fixation. The observed increase in glutamine synthetase activity can be correlated with the high nitrogenase activity. Ammonia assimilation is an energy dependent process and added carbon sources may enhance ATP synthesis, giving the glutamine synthetase additional energy, and hence increasing its activity. This is further supported from the 14C kinetic studies by LAWRIE et al. (1976). The exact basis of lactose toxicity in this alga is unknown. The inhibition of nitrogenase activity by lactose could be due to the fact that the alga is unable to assimilate lactose and this in turn limits the availability of photosynthate for the process. Another possible reason could be that the bypro ducts of lactose breakdown may be toxic to the cells. Sucrose, which is readily taken up by the alga helps it to overcome the inhibition by lactose. The stimulatory effect of carbon sources on heterocyst production can be attributed to a temporary preponderance of intermediates of carbon metabolism to which ammonium may be linked. The observed increase in heterocyst frequency with exogenous addition of carbon compounds may be because of the enhanced consumption of inhibitor of heterocyst differentiation by the addition of sucrose, glucose and fructose thereby reducing its exogenous level.

56

TIPP 183 (1988) 1

The observations on Aulosim prompt us to infer that this alga may be at an advantage in low light intensities. Although other aspects of cyanobacterial metabolism have been studied, their metabolism in t he dark has been relatively neglected. Adaptation 10 low light intensities can be perceived as a strategy of storing much of the carbon fixed in the light for subsequent metabolism in the dark. This faculty may be extended to enable cells to exist for long periods in the dark, or in low light environments. Acknowledgements We thank Dr. ASHoK l\nuR for Ya.lnable suggestions. 'Financial assistance in the form of Senior Research FplJowship of the Co 1Illl' i I of Sticntific and Industrial Research, New Delhi is gratefully atlmowledgcd by l\LH.

References BOTIn:, n., TEXNIGKEIT, J., and EISIlREXNEIl, G.: Utilization of molecular hydrogen by blue-green alga Anabaena cylindrica. Arch. :Nficrobiol. 114, 43-49 (1977). BOTTOMLEY, P ..T., and STEWART, W. D. P.: ATP pools and transients in the blue-green alga Anabaena cylindrica. Arch. Microbiol. 108, 249- :258 (1976). BOTTO~iLEY, P. J., and STEWAHT, W. D. P.: ATP and nitrogenase activity in nitrogen fixing heterocystous bluc-grepn algae. New Phytol. 79 , 625-638 (1977). BOTTOMLEY, P . .T., and YAN B.HLE N, C.: Charaeieristil's of heterotrophic growth in blne-green alga Nosloc sp. strain ,Mal'. J. Gen. lI'lierobiol. 107, 309- 318 (1978a). BOTTOMLEY, P. J., a.nd VAX IhALEN, C.: Dark hexose metabolism by photoautotrophically and photoh eterotrophiea lly grown eells of blUl'-green alga (eyanobaeterium) Nosloc sp. strain Mac. J. Bacteriol. 135, 885-894 (1978b). DOMMERGUES, Y., BALANDREAU, J ., RINAUDO , G., and WEIXRA1W, P.: :'l"on-symbiotic nitrogen fixation in the rhizosphere of rice, maiz!~ and different tropical grasses. Soil BioI. Biochem. I), 83- 89 (1973). DOOLITTLE, W. F.: The cyanobacterial genome, its expression and the control of that expression. Adv. Mierob iol. Physiol. 20, 1- 102 (1979) . FAY, P.: Heterotrophy and nitrogen fixation in Chlorogloea fritschil:. J. Gen. lIicrobiol. 39, 11-20 (19G5). FAY, 1'.: Factors inflnencing dark nitrogen fixat ion in a blue-green alga. Appl. Enyiron. lIicrobiol. 31, 376-379 (1976). H .wRY, J. P., and SPILL ER, H.: Fructose uptake an d influence 011 growth and nitrogen fixation by Anabaena variabilis. J. Baeteriol. 147, 227-235 (1981). HUGHES, E. 0., CORILUf, P. R, and ZEHNDEH, II.: Toxicity of unialg
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PELROY, R. A., RIPPKA, R. and STANIER, R. Y.: The metabolhm of glucose by unicellular blue-green algae. Arch. Microbiol. 87, 303-322 (1972). PETERSON, R. B., and WOLK, C. P.: High recovery of nitrogenase activity and of 55Fe-Iabelled nitrogenase in heteroeysts isolated from Anabaena variabilis. Prot. Natn. Acad. Sci. U.S.A. 75, 6271 to 6275 (1978). SHAPIRO, P. M., and STADTMAN, E. R.: Glutamine synthetase (Escherichia coli). Methods in Enzymology Vol. 17 A, 910-922 (1970). SINGH, R. N.: Role of blue-green algae in nitrogen economy of Indian agriculture. Indian Couneil of Agricultural Research. New Delhi (1961). SMITH, A. J., and HOA]{E, D. S.: Speeialist phototrophs, lithotrophs and methylotrophs. Microbiol. Rev. 41, 419-448 STACEY, G., TABITA, F. R., and VAN RULEN, C.: Nitrogen and ammonia assimilation in the cyanobacteria: Purifieation of glutamine synthetase from Anabaena sp. strain C. A. J. Bacteriol. 132,

596-603 (1977). STEWART, W. D. P.: Blue-green algae. In: 'A Treatise on Dinitrogen Fixation' (cds.), HARDY, R. W. F. and SILVE]{, W. S. John Wiley & Sons, New York 1977. STEWART, W. D. P., FITZGERALD, G. P., and BURRIS, R. H.: Acetylene reduction by nitrogen fixing blue-green algae. Arch. Microbiol. 62, 336-348 (1968). THOMPSON, J., and CASSAY, B. M.: Uptake and metabolism of sucrose by Streptococcus lactis. J. Bacteriol. 148, 398-402 (1981). WHITE, A. W., and SHILO, M.: Growth of the filamentous blue-green alga Plectonema boryanum. Arch ~licrobiol. 102, 123-127 (1975). WOLK, C. P.: Aspects of the development of a blue-green alga. Ann. N. Y. Acad. Sci. 169, 641-647

(1970). W OLI(, C. P., and SHAFFEH, P. W.: Heterotrophic micro- and macroeultures of a nitrogen fixing cyanobacterium. Arch. Microbiol. 110, 145-147 (1976). YOSHIDA, T., and ANCAJAS, R. R.: Nitrogen fixation by bacteria in the root zone of rice soil. Soil. Sci. Soc. Amer. Proc. 35, 156-158 (1971). YOSHIDA, T., and ANACAJAS, R. R.: Nitrogen fixation activity in upland and flooded rice fields. Soil Sei. Soc. Amer. Proc. 37, 42-46 (1973).

Received December 22, 1986; accepted Mareh 13, 1987 Authors' address: Prof. H. D. KUMAH, Centre of Advanced Study in Botany, Banaras Hindu University, Post Box No. 14, Varanasi - 221 005, India.

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HPP 183 (1988) 1