Indian Journal of Experimental Biology Vol. 37, July 1999, pp. 696-700

Immobilization caused physiological and biochemical changes in NaCI-resistant (NaClr

) mutant strain of diazotrophic cyanobacterium Anabaena variabilis

v S Chauhan', S Singhh, R K Gour· & P S Bisen'·

"Institute of Microbiology & Biotechnology, Barkatullah University, Bhopal 462026, India bSchool of Studies in Botany, Jiwaji University, Gwalior 4740 II, India

Received 9 November 1998; revised 31 March 1999

Growth (in term of ~ and G), heterocyst differentiation, nitrogenase activity, ammonia production, ammonia uptake, glutamine synthetase (GS) activity, 14C02 -fixation and Hill activity have been studied in the wild-type Anabaena variabilis and its NaCI-resistant (NaCl') mutant strain immobilized in calcium alginate gel. NaCl' mutant strain grew well both in the presence as well as absence of NaCI (300 mM) at a rate similar to ·its wild-type strain in NaCI-free medium. 'Further, NaCl' mutant strain had generation time of 33 .3 and 71.4 hr, respectively in its free-living and immobilized conditions. Immobilization of the cells in calcium alginate gel increased heterocyst differentiation and nitrogenase activity in both wild­type and its NaCl' strain . GS activity in NaCl' mutant strain was altered by mutation causing liberation of ammonia in the ex ternal medium. No detectable ammonia was however, liberated by the free-living and immobilized wild-type strain . NaCI (300 mM) significantly inhibited the growth, heterocyst differentiation, nitrogenase activity, ammonia uptake, GS activity, carbon-fixation and Hill activity in the wild-type strain, whereas these activities were stimulated in NaCl' mutant strain. Further immobilization of NaCl' mutant strain whereas caused decrease in ammonium uptake and GS acti vity, it stimulated carbon-fixation and Hill activity in this strain . Thus it is suggested that increased ammonium photoproduction by immobilized aCI' mutant strain was due to decrease in ammonium uptake, GS activity and susceptibility of nitrogenase to repression by ammonia and subsequent increase in heterocyst differentiation, nitrogenase activity, carbon-fixation and Hill activity.

Heterocystous cyanobacteria are diazotrophic, oxygenic, photosynthetic prokaryotes which have long been recognized as having potential in biotechnology. One of the biotechnological aspects rccei.ving considerable attention, especially by using diazotrophic cyanobacteria, is the photobiological production of ammoma. Since, free-living diazotrophic cyanobacteria fix dinitrogen sufficient fo r .their own needs and . do not generally liberate significant amounts of ammonia into their external environments, attempt are being made to induce ammol1la production either by the addition of glutamine synthetase (GS) inhibitor or glutamate ana logues such as L-methionine-DL-sulfoximine' (MSX), 5-hydroxylysine2

, and phosphinothricin3 or by producing ammonia liberating mutant strains . By using MSX, continuous photoproduction of ammonia by diazotrophic cyanobacterium Anabaena have been reportedH

.

In the absence of either a natural or ammonia liberating mutant stTain, treatment of N2-fixing cyanobacterial cells with GS inhibitors seem to be the preferred way for induced ammonia liberation.

' Correspondent author

However, prolonged exposure to MSX has been reported to be toxic for cyanobacteria, causing nitrogen starvation and inhibition in growth and protein synthesis7

. By using chemical mutagens such as N-methyl-N'-nitro-N"-nitrosoguanidine (NTG) , several cyanobacterial mutant strains resistant to MSX or defective in GS have been isolated and shown to liberate ammonia in the external mediums.". However, these mutant strains are slow-growing and cannot withstand the environmental extremes like NaCI stress. Recently yve have isolated a NaCl­resistant (NaCI') mutant strain of the cyanobacteriuJ1! Anabaena variabilis by NTG mutagenesis which possess high NaCI tolerance t2

. NaCl' mutant strain shows impairment in its GS activity and liberates a considerable amount of ammonia in the external medium' 2. By using the same strain, we have now studied the response of immobilization on heterocyst di fferentiation ,. nitrogenase and GS achvlhes, ammonium uptake, carbon-fixation, Hill activity and photobiological production of ammonia and provide evidence that immobilization influences the physiological, biochemical and metabolic activities of the cells, leading to Increase in heterocyst differentiation, nitrogenase ac tivity and photo-

CHAUHAN el al. : IMMOBILIZATION CAUSED PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN NaCl 697

biological production .of ammonia in NaCl r strain.

Materials and Methods

Organisms and culture conditions-Anabaena I'ariabilis (a rice field isolate) and its NaCl r mutant strain were axenically grown in BG-Ilo medium13

without any nitrogen source. The cultures were incubated at 24°± I °C and illuminated with day-light fluorescent tubes (i rradiance 50 JlIDol m-2 S-I at the surface of the vessels) for 14 hr/day . Unless otherwise stated. all the experiments were carried out with expcmentiallygrowing cultures having a cell density of 20 Ilg ChI a ml"l. The medium was buffered to pH 7.5 with 10 mM 4-(2-hydroxy ethyl)-piperazine ethane sulfonic acid (HEPES)/NaOH. Whenever required. NaCI (300 mM) was added in to the medium, since thi s is the concentration at which the growth of the wild-type strain IS completely arrested l".

Immobili-:;atiOl/-lmmobilization of the wild-type-A. variabilis and its NaCl r mutant strain was carried out by alginate entrapment method l4. Exponentially growing cells were harvested by centrifugation (5000 g for 10 min) at 25°C and resuspended in sterile Nrmedium. The cell suspension was mixed with an equal volume of 3% alginic acid sodium salt (Sigma) and added drop-wise from a syringe canula into 0.1 M CaCI} solution. The alginate beads were kept for 30 min at 4°C for hardening. The beads were then harvested, washed thoroughly with sterile basal medium. The growth medium was free of phosphate to prevent the formation of calcium phosphate which cou ld result the disruption of the beads.

Growlh-0rowth of free-living as well as immobilized wild-type A. variabilis and its NaCl r

mutant strain was measured by measuring the increase in chlorophyll a (ChI a) content following the method of Mackinneyl .l and was expressed in terms of specific growth rate (Jl) and generation time (G) . For determination of ChI a from Ca-immobilized cells, they were first dissolved in 0.1 M tri sodium citrate. Specific growth rate (Jl) and generation time (G) were computed following the method of Guillard l5 .

Ammonillm uptake-Ammonium uptake was assayed by measuring its depletion from the external medium. Exponentially growing cells of free-living and immobilized wild-type A. variabilis and its NaCl r

mutant strain were harvested by centrifugation, washed thoroughly with sterile Nrmedium and resuspended in the same medium buffered to pH 7.5

with 10 mM HEPESlNaOH to a final density of 20 Jlg ChI a ml·l. Uptake assay was carried out in open flasks under aerobic condition with continuous shaking at 24°± I °C and at a photon fluence rate of 50 Jlmol m·2 S·I. Uptake was initiated by the addition of NH4CI (final concentration 200 JlM) to the cell suspension at zero time. Samples were collected at different time intervals and cell-free supernatants were analysed for the residual ammonium. The linear portions of the curves were used to calculate the uptake rate.

Ammonia production--For assay of extracellular ammonia production, alginate immobilized (I ()() beads) and/or free-living cells were placed in conical flasks (100 ml) containing Nr medium (20 ml) and were incubated in light with photon fluence rate of 50 I1-mol m-2 S-I. Samples were harvested after 24 hr and ammonia concentration was estimated by colorimetric method l6. The rate of ammonia production was expressed as Tlmole ammonia produced mg-I protein h(l.

GS activity---GS (transferase) activity was assayed following the method of Sampaio et af. 17 and was expressed in terms of Tlmole y-glutamyl hydroxamate formed mg- I protein min-I.

Heterocyst frequency--The heterocyst frequency of free-living and immobilized cells was determined microscopically in ten independent replicates, and expressed as number of heterocysts per hundred vegetative cells . For determining the heterocyst frequency of immmobilized cells, they were first solubilized by treatment with 0.1 M of trisodium citrate.

Nitrogenase activity--Nitrogenase activity was assayed by acetylene reduction techilique l8. Five ml cell suspension and/or 20 beads were placed in 15 mi' serum vials with 10% acetylene (v/v). After incubating vials for I hr at 25°C and a photon fluence rate of 50 I1-mol m-2 S-I, l'm1 gas sample was analyzed for estimating C2H4 produced with a gas chromatograph (Shimadzu, Japan) fitted with a flame ionization detector and porapak-T column. Nitrogenase activity was expressed in terms of Tlmole C2H4 I1-g-1 ChI a h(l.

14COrfixation--Exponentially growing free-living and immobilized cells were suspended in 25 mM of HEPESlNaOH (PH 7.5) and incubated in dark for 24 hr to render them deficient in cellular A TP reserve. NaH I4C03 (final concentration 0.05 I1-ci mrl) was added to the cell suspensions and the cell suspensions

698 INDIAN 1. EXP. BIOL., JULY 1999

were incubated in light. Samples were withdrawn at regular time interva ls and the reaction was terminated by adding 0 . 1 ml 2 N HCI. 2.5 m] scintillation cocktail contammg four parts of 0 .8% PPO (2,5-diphenylox azole) plus 0.01 % POPOP (l,4-bis(4-methyl-5-phenyl-2-oxazole)-benzene) in toluene and three parts of ethanol was added to each sampie vial. Reaction mixtures were surface blown for 5 min to remove 14C02 in a liquid scintillation counter (Wall~c 1409, Finland) and the activity was expressed in terms of rate of 14COr fixation (CPM I1g,l Chi a min,I) .

Hill activity (H20-DCPIP~Hill activity was measured in terms of 2,6-dichlorophenol-l­indophenol (DCPIP) reduct ion following the method of Holt and French 19. Cells were made permeable by 0 .5% lysozyme .(w/v) prior to the measurement of DCPIP reducti on as descri bed by Wards and Mayers20

and the activity was expressed in terms of llmole OCP'lP reduced mg' l Chi a min' l.

Chemicals-NTG , HEPES, PPO, POPOP and OCPIP were purchased from Sigma Chemical Co. St. Loui s, Mi ssouri (USA). NaH I4CO, was obtained from BARC, Bombay, India and other chemicals were of hi gh purity grade available from BDH, Poole, England .

Results and Discussion

The data in Table I shows the growth (in terms of spec ific growth rate and generation time) of the free­living and Ca-alginate immobilized wild-type A. variahilis and its NaCI' mutant strain in the presence and absence of NaCI. It is evident that both the strain s grew reasonably well in Nrmedium by havi ng almost s imilar growth rate and generation time. The wild-lype and its NaCl' mutant strain in their immob ili sed condition had a generat ion time of · 68.0 and 7 1.4 hI', respectively in N2-medium, indicating th at immobilization of the cells reduced the growth of both wild-type and its NaCl' mutant strain by abou t 47 %. Add iti on or' NaCI (300 mM) in to the wild-type cultures, caused cell lysis and ultimately death of the cel ls, whereas it stimulated the growth of NaCl' strain signi f icantly (Table I).

Since a mmonta production by diazotrophic cyanobacteria is not a routine physiological function except in so me sy mbi otic associations21 and GS defec tive stra in s. Immobilization is shown to increase the ha lf-life of cyanobacteria in ammonia production

. 2??1 h d h reactions -.- . In t e present stu y eterocyst differentiation, nitrogenase activity and ammonia production have been studied in both wild-type

Table I-Specific growth rate (Ilk l) and generation time (G) of wild-type and NaCl r mutant strain of A. variabilis grown under different growth conditions in presence and absence of NaCI*. These parameters were monitored after incubation with NaCI for 24 hr

Growth conditions 11 (h(l ) G (hr)

Free-living culture

Wild-type 0.03 1 32.9 Wild-type + NaCI 0.0 0.0 NaCl r 0.030 33.3 NaClr + NaCI 0.034 29.4

Immobilized culture

Wild-type 0.0 147 68.0 Wild-type + NaCI OJXll4 7 14.0 NaCl r 0.0 14 71.4 NaCl r + NaCI 0.0 144 69.4

• NaCI was added in the growlh medium at the final concentration of300 mM. The data in each column are means of two independent experiments with four replicates each. The -maximum variati on from the mean value was less than 5%.

Table 2-Heterocyst frequency (%), nitrogenase activity (T] mole C2H4 produced ""g-I Chi a 11(1 ) and photoproduction of ammoni a

(T]mole NH/ mg' l protein h( l) in the free-living and immobilized wild,type and NaClr mutant strain of A. variabilis in presence and absence of NaCI*- These parameters were monitored after incuba-

tion with NaCl for 24 hr

Culture Heterocyst Ni trogenase Ammoni a conditi ons freq uency activity production

Free-living culture

Wild-type 6.0 10.5 NO Wild-type + NaCI 3.0 NO NO

NaCl r 8.0 13.5 252.0 NaClr + NaCl 11.0 16.7 282.0

Immobilized culture

Wild-type 9.8 16.5 NO Wild-type + NaCl 4.5 NO NO NaClr 12.5 194 367.0 NaClr + NaCI 17.8 22.7 386.0 NO = Not detectable • NaC I was added in the growth medi um at the final concentration of300 mM. The data in each column are means of two independent . experiments with four replicates each. The maximum variation from the mean value was less than 5%.

A. variabilis and its NaCl' mutant strain in their free­living and immobilized conditions (Table 2). It - is evident from the data in Table 2 that NaCi r mutant strain in its free-living state produced more heterocysts and nitrogenase to that of its wild-type counterparts in the absence of NaCI. These activities

CHAUHAN el al.: IMMOBILIZATION CAUSED PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN NaCI 699

were further increased upon immobilization. Thus, by increasing the frequency of functional heterocyst, the rate of N2-fixation can be enhanced. Similar increase in heterocyst frequency and nitrogenase activity have also been observed in other cyanobacteria22

.24

.

Whereas, the supplementation of NaCI (300 mM) to the free- living and immobilized cells of the wild-type strain resulted in the significant inhibition of heterocyst differentiation and nitrogenase activity, however, these activities were stimulated in the NaCI' mutant strain (Table 2). The; inhibitory effect of NaCI on heterocys t differentiation and nitrogenase activity have been found to be variable. Whereas NaCI inhibited the heterocyst differentiation by 50%, it caused a complete inhibition in nitrogenase activity, sugges ting that nitrogenase system was more sensitive to NaCI than the syste m involved in heterocyst formation.

Furthermore, NaCI' mutant strain in its immobilized state ex hibited heterocyst differentiation and nitrogenase activity even in the presence of ammonium-nitrogen (data not shown). This may be due to reduced uptake and/or metabolism of ammonium by immobilized cells of NaCl' strain .

Tahle 3-Ammonium uptake (TJmole NH: mg·1 protein h(I) , GS (transferase) activity (llmole y-glutamyl hydroxamate formed mg·1 protein min ' '), '~COr fixation (CPM Ilg.1 Chi a min,i) and Hill ac ti vity (qmole DCPIP reduced mg' l Ch i a min" ) in free­living and immobilized wild-type and NaCl' mutant strain of A. m /'iabilis in the presence and absence of NaCI*' These parameters were monitored after incubation with NaCI for 24 hr

Cult!]re cond itions

Wild-type Wild-type + NaCI NaCl' NaCl ' + NaCi

Wild-type Wild-type + NaCI NaCl' NaCl' + NaCi

Ammo- GS nium activity

uptake

Free-living culture

108.5 243.5 1

5R.5 32. 15 50.0 116.38 49.5 115.98

Immobilized culture

60.0 203.5 30.0 28.5

20.4 103.2 10.0 101.9

14COr Hill fixa ti on ac tivit y

12.6 34.0

3.9 9.0 10.5 35.0 15 .0 40.0

14.0 36.0 9.2 11 .0

14.8 43.2 !7.3 46.7

NaCI was added in the growth medium in the final concentration 01'300 mM. The data in each co lumn are means of two independent experiments with four replicates each. The m:lximum variation from the mean value was less than 5%.

Similar results on nitrogenase activity even in the presence of ammonium has also been reported in Anabaena sp.CA 25

. Since, in the presence of external nitrogen source the process of Nr fixation may be redundant , while NaCI' mutant strain exhibited a unique behaviour and would be a better biofertilizer for reclamation of salt-affected soils . Further to see the impact of immobilization and NaCI on photoproduction of ammoma by wild-type A. variabilis and its NaCI' mutant strain, the free­living and immobilized cells of both the strains were incubated for 24 hr in combined N-free medium with and without NaCI under continuous illumination and shaking and ammonia liberated into the ex ternal medium was assayed. Interestingly, the ammonia production was only confined to free-Ii ving and immobilized cells of NaCI' mutant strain both in the presence as well as absence of NaCI. While no detectable ammonia was liberated by identical cells of the wild-type strain (Table 2). NaCI' mutant strain in its immobilized state was capable of fixing N2 even in the presence of ammonical-nitrogen and has been reported earlier that immobilization also c hanges the metabolic activity and efficiency of the ce ll s 24.26. It is expected that the increased ammonia production by immobilized NaCI' mutant strain may be a consequence of the c hanges in the levels of ammoni a uptake, activity of the ammonium ass imilating enzyme (GS), carbon-fixation and Hill activity .

Further to ascertain whether the increased ammonium production by immobilized cells could be due to change in ammonium uptake, GS act ivity. carbon-fixation and Hill ac tivity , these parameters were measured in both wild~type and its NaCl' mutant strain in presence and absence of NaCI (Table 3). It is evident from the data of Table 3 that NaCI ' mutant strain in its free- living state had only about 50% GS activity both in presence as well as absence of NaCI as compared to its free-living wild-type counterpart (w ithout NaCI) . NaCI' mutant strain in its free- li vi ng state ass imilated ammonia at a slower rate as compared to it s wild-type strain in the absence of NaCI. Whereas the addition of NaCI s ign ifican tl y inhibited the GS as well as ammonium uptake actIvItIes in the wild-type strain, it, however, could not do so in NaCI' mutant strain (Table 3) . Ammonium uptake and GS activities, thus showed parallel behaviour in both wild-type and its NaCI' mutant strain. Immobilization of the cells further decreased the ammonium uptake and GS activity in both the strains, indicating that the reducti on in

700 INDIAN 1. EXP. BIOL. , lUL Y 1999

ammonium uptake and GS activity ·may be the main cause for increased ammonium production in NaCl ' mutant strai n.

Since, di azotrophic organisms require carbon skeleton and energy (in the form of ATP) for efficient N2-fixation and as it has also been shown above that immobilizati on of the cells resulted in the increase in heterocyst differentiation and nitrogenase acti vity, it is plausible to presume that carbon-fixation and Hill activ ity should also increase following immobilization to support more efficient Nr fixation and ammonia production. Further to find out more conclusive evidence regard ing the above proposition, the carbon­fixation and Hill activity were measured in both free­li ving and immobi lized cells of wild-type A. variabilis and its NaCl' mutant strain (Table 3). The levels of carbon-fixati on and Hill activity in the free-living

aC I' strain were found to be slightly lower as compared to its free-living wild-type couTlterpart (Tab le 3). Whereas NaCI caused significant inhibit ion in carbon-fixation and Hill activity in the free-living and immobili zed wild-type strain, it, however, stimulated these activities in NaCl' mutant strain. This

aC I-induced inhibition in the wild-type strain may be ex plained by a decrease in the supply of ATP and reductants due to NaCI-induced inhibit ion in c:arbon­fixation and Hill activity. In contrast, the presence of enhanced levels 6f carbon-fixation and Hill act ivity in NaCl -treated a ' we ll as untreated free-living and immobili zed ce ll s of NaCl' mutant strain , support more heterocyst differentiation, nitrogenase activity and as a consequence more ammonia production in NaC I' mutant strain . Thus, increased ammonia production by immobili zed ce ll s of NaCl' mutant strain can be exp lained on the basis of increased heterocyst different iation, nitrogenase actl Vlty, carbon-fixation. Hill activity and subsequent decrease in ammon ia uptake and GS activity.

It is co nc luded that ( I) impairment in GS ac ti vi ty and dec reased susceptibility of the enzyme nitroge nase by ammonium are the main reasons for the hi gh nitrogenase activity and ammonia production In NaC I' mutan t strain of A. variabilis, (2) imlllobilizat ion caused red ucti on in ammonium uptake and GS ac ti vi ty resulted in furth er increase in photoproducti on of allllllon ia by immobilized NaCl' l1luta"nt ~ train. (3) NaCI-cau'sed in c rea~e in heterocyst dilTcrc nti ati oll. nitroge nase activity, carbon-fix at ion and Hill ac ti vit y arc the main reasons for the optimum growth of th e free-living and immobilized NaCI'

mutant strain even in the presence of NaCI , and (4) NaCl' mutant strain has all the characteristics to be a novel strain for the photoproduction of ammoni.a under complete photoautotrophic condition and would be considered a better biofertilizer for reclamation of salt-affected soil s.

Acknowledgement The authors thank lCAR, New Delhi , India for

financial assistance.

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