Sustained photoproduction of ammonia from nitrate by Anacystis nidulans

Vol. 44, No. 5 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, No . 1982, P. 1020-1025 0099-2240/82/111020-06$02.00/0 Copy igh © 1982, Ame ican Socie y o Mic obiology Sus ained Pho op oduc ion o Ammonia om Ni a e by Anacys is nidulans JUAN L. RAMOS, MIGUEL G. GUERRERO,* AND MANUEL LOSADA Depa amen o de Bioquimica, Facul ad de Biologia y Consejo Supe io de In es igaciones Cien i icas, Uni e sidad de Se illa, Apa ado 1095, Se illa, Spain Recei ed 9 Ma ch 1982/Accep ed 19 July 1982 Condi ions ha leng hen he ime du ing which L-me hionine-DL-sul oximine (MSX) p omo es exc e ion o ammonia p oduced by pho osyn he ic ni a e educ ion in Anacys is nidulans ha e been sough . I MSX was added e e y 24 h, maximal a es o ammonia p oduc ion we e main ained o 3 days. A e his ime, ammonia p oduc ion ceased due o a speci ic de iciency o glu amine in he cells, which inally led o cell lysis. The e ec i e ammonia p oduc ion pe iod could be u he ex ended ei he by adding a low amoun o glu amine a he end o he 3- day pe iod o by allowing he cells o eco e o 8 h in he absence o MSX a e e e y 48-h pe iod in he p esence o inhibi o . In his way, a s eady p oduc ion o ammonia las ing o a leas 10 days was achie ed. The MSX- ea ed cyanobac e- ial cells hus ep esen a sys em ela i ely s able wi h ime o he con e sion o ligh ene gy in o chemical ene gy h ough he pho o educ ion o ni a e o ammonia. The pho o educ ion o ni a e o ammonia by pho osyn he ic o ganisms ep esen s a ema k- able p ocess o he con e sion o sola ene gy in o chemical ene gy (4, 7, 8). E icien pho o- p oduc ion o ammonia om ni a e by cells o he cyanobac e ium Anacys is nidulans has been achie ed in ou labo a o y a e speci ically in- hibi ing glu amine syn he ase, he i s enzyme in ol ed in he main ammonia assimila ion pa h- way in his o ganism, wi h he glu ama e analog L-me hionine-DL-sul oximine (MSX) (3, 11). To be conside ed aluable o p ac ical pu - poses, a sys em o he con e sion o sunligh ene gy mus ope a e a conside able a e and e iciency and mus be ela i ely s able in ime. The sys em cons i u ed by MSX- ea ed Ana- cys is cells has been shown o p oduce ammonia a high a es (11) wi h ela i ely high e iciency (12), bu un il now he p ocess has been only ca ied ou du ing a limi ed pe iod o ime. This communica ion epo s he achie emen o a s eady pho op oduc ion o ammonia om ni a e by MSX- ea ed A. nidulans cell suspen- sions, las ing o a pe iod o a leas 10 days. MATERIALS AND METHODS G ow h o cells. A. nidulans 1402-1 was g own pho oau o ophically unde con inuous ligh ing (Syl- ania dayligh luo escen ubes, gi ing an i adiance alue o 25 W/m2 a he su ace o he cul u e essels), a 40°C on a syn he ic medium con aining ni a e as he ni ogen sou ce (5), in a s eam o 2% CO2 in ai ( ol/ ol), which was bubbled h ough he cul u e a a low a e o 1 li e /li e o cell suspension pe h. Ammonia p oduc ion expe imen s. Cells om 2-day old cul u es con aining 20 o 25 ,ug o chlo ophyll a (Chl) pe ml we e used. The cells we e ha es ed by cen i uga ion, washed wi h cul u e medium, and inal- ly suspended in he same medium o a densi y o 8 o 10 ,Lg o Chl pe ml. MSX was added o he cell suspension o a inal concen a ion o 10 FM, and he cell suspensions we e hen incuba ed unde s anda d cul u e condi ions wi h he indica ed modi ica ions. Assays o enzyme ac i i ies. Ni a e educ ase and glu amine syn he ase ( ans e ase) ac i i ies we e measu ed in oluenized cells as p e iously desc ibed (11). Ni i e educ ase and NADH-alanine dehyd o- genase we e de e mined in cell- ee ex ac s. Ni i e educ ase ac i i y was es ima ed acco ding o Man- zano e al. (10). NADH-alanine dehyd ogenase was es ima ed as desc ibed by Rowell and S ewa (13). Ex ac ion o cellula me aboli es. Pe chlo ic acid was added o a sample o he cell suspension o gi e a concen a ion o 0.6 N. A e 10 min a 0°C, he samples we e cen i uged a 40,000 x g o 15 min. The supema an was neu alized a 0°C wi h KOH. A e emo al o he KC104 p ecipi a e by cen i uga ion (10,000 x g o 10 min), he supema an s we e lyophi- lized. The lyophilized ma e ial was suspended o a small olume wi h wa e and used o he es ima ion o me aboli es (3). Analy ical me hods. Ni a e, ni i e, ammonia, -y- glu amyl hyd oxama e, glu ama e, and glu amine we e es ima ed as desc ibed p e iously (11). Ca bohyd a es we e es ima ed by he phenol-sul u ic me hod (6), o al ni ogen by he Kjehldal p ocedu e, packed cell ol- ume by cen i uga ion o he cell suspensions in cali- b a ed hema oc i ubes, and cellula p o ein by a modi ica ion o he Low y p ocedu e (2) a e p e ea - ing he cells wi h 10% (w / ol) ichlo oace ic acid. Chl was de e mined spec opho ome ically in me hanolic 1020 on June 23, 2015 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://aem.asm.o g/Downloaded om SUSTAINED AMMONIA PHOTOPRODUCTION 1021 0 au_o_ -° o e o I 0 D. Z 20 40 ( h)1.5w z 0~~~05 ~~~~~~z FIG 1. Tiecus ee nslaig m ea ed AnuncsAe 00 i 10 Fd- E~~~~~~~~A ~~~~~~~~~~~10 0~~~~~~~~~ : cod osDape eew dana h ie indc d an amoi n h eim(),dniyo C -A-A ~A-A-A A------- _-- 0 20 40 TIME (h) FIG. 1. Time cou se o he e en s leading o am- monia p oduc ion and o i s la e cessa ion in MSX- ea ed A. nidulans cells. A cell suspension (150 ml) wi h a densi y o 9 ,ug o Chl pe ml and supplemen ed wi h 10 pe M MSX was incuba ed unde s anda d cul- u e condi ions. Samples we e wi hd awn a he imes indica ed, and ammonia in he medium (6), densi y o he cell suspension (A), and cellula ac i i y le els o ni a e educ ase (0) and glu amine syn he ase (A) we e de e mined. ex ac s wi h he ex inc ion coe icien gi en by MacKinney (9). The Chl and p o ein con en s o MSX- ea ed A. nidulans cells we e ai ly cons an , wi h alues o 5.8 o 6.2 pLg o Chl and 110 o 130 p.g o p o ein pe dJ o packed cell olume (11). Measu e- men s o he abso bance alues a 628 and 680 nm o he cell suspensions o es ima ion o he phycocyaninl Chl a io we e pe o med by placing he sample- con aining cu e e in he u bid-samples compa men o a Pye Unicam SP 1750 spec opho ome e . RESULTS AND DISCUSSION The main e en s aking place a e he addi- ion o MSX o suspensions o A. nidulans cells in ni a e medium ha e p e iously been de- sc ibed (11) and can be summa ized as ollows. Glu amine syn he ase becomes apidly and com- ple ely inac i a ed, and ammonia appea s in he medium concomi an ly wi h his ac i i y loss. Cell g ow h is p e en ed, and he a es o ni a e up ake and educ ion inc ease abou wo old. Ammonia accumula es in he medium a a con- s an a e o abou 25 pLmolUmg o Chl pe h. The p ocess o ammonia p oduc ion con inues o abou 30 o 35 h wi hou u he addi ions, p o ided ha ni a e is no limi ing, and ni a e educ ase ac i i y emains a i s o iginal high le el du ing his pe iod. As Fig. 1 shows, a e his ime, glu amine syn he ase ac i i y eco - e ed, and he accumula ion o ammonia in he medium ceased, ollowed by i s u iliza ion by he cells, which s a ed o g ow. Once glu amine syn he ase became ac i e, he an agonis ic e - ec s o ammonia (3, 5, 11) came in o play; ni a e u iliza ion was inhibi ed (da a no shown), and he ni a e educ ase le el de- c eased apidly (Fig. 1). Since, unde s anda d cul u e condi ions and in he absence o cells, MSX does no lose i s ac i i y o a leas 48 h, he cessa ion o i s e ec on ammonia p oduc- ion can be ela ed o he ans o ma ion o he inhibi o by he algal cells a he han o i s spon aneous deg ada ion. I seems, he e o e, ha he empo a y ceas- ing o ammonia pho op oduc ion is due o he eco e y o glu amine syn he ase ac i i y a he han o he induc ion o any o he ammonia assimila ion pa hway. Thus, o leng hen he pe- iod o ammonia p oduc ion, esh MSX a i s op imal concen a ion wi h espec o he densi- y o he cell suspension (1 nmol/,lg o Chl) (12) was added o he Anacys is cell suspension in which he e ec o he o iginal MSX had been los . This addi ion esul ed again in inac i a ion o he p eexis ing glu amine syn he ase, allow- ing new maximal a es o ammonia p oduc ion (da a no shown). Following his app oach, successi e addi ions o 10 ,uM MSX o he cell suspension e e y 24 h allowed maximal a es o ammonia p oduc ion o be sus ained o 3 days. Du ing his pe iod, cellula glu amine syn he ase emained ully inac i e, he ni a e educ ase ac i i y le el being high, and abou 90%o o he ni a e aken up appea ing in he medium as ammonia (Fig. 2). A his s age, he addi ion o MSX no longe esul ed in main enance o he p ocess o ammonia p oduc ion, and cell lysis o he suspension was obse ed sho ly a e wa ds. To de e mine he cellula si ua ion leading o he cessa ion o ammonia p oduc ion by Anacys- is, he le els o a ious cell componen s, enzy- ma ic ac i i ies ela ed o ni a e me abolism, and amino acids in ol ed in he glu amine syn- he ase-glu ama e syn hase pa hway we e s ud- ied in Anacys is cells which had been ea ed wi h MSX o 68 h and we e compa ed wi h hose in no mal cells. The analysis o Anacys is cells subjec ed o MSX ea men o 68 h yield- ed da a simila o hose p e iously ound in expe imen s o sho e du a ion (11). The cellu- la le els o p o ein, Chl, phycocyanin, and o al ni ogen emained cons an and equal o hose o un ea ed cells, whe eas he ca bohyd a e le el inc eased linea ly. The inc ease in he ca bohy- d a e le el, om 6.2 ,ug/,ul o cells a ime ze o o 39 pug/Ll o cells a 68 h, was in e p e ed as due o con inued pho osyn he ic CO2 ixa ion while VOL. 44, 1982 on June 23, 2015 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://aem.asm.o g/Downloaded om 1022 RAMOS, GUERRERO, AND LOSADA 0 w w a. a- z _ CZ E o W w1 E C.) =. 0 0 a- z 0 4 C1 2- N w Ln w 4 z 20 C- 8 E "I w 0 z 10 w z Ln zS I. D O (D 24 48 72 TIME (h) FIG. 2. Con inuous ammonia pho op oduc ion by MSX- ea ed A. nidulans cells o 3 days. Condi ions we e hose desc ibed in he legend o Fig. 1, excep ha MSX (1.5 ,umol) was added e e y 24 h. A 48 h, KNO3 (1.5 mmol) was added. Samples we e wi hd awn a he imes indica ed, and ammonia (0) and ni a e (0) in he medium and cellula ac i i y le els o ni a e educ ase (A) and glu amine syn he ase (A) we e de e mined. MSX p e en ed he inco po a ion o ammonia in o ca bon skele ons and subsequen cell g ow h (11). I is in e es ing ha he phycocyanin le els ypical o no mal cells we e main ained in MSX- ea ed Anacys is cells when he medium con- ained ni a e bu no when i was ee o ni o- 1 6E 7 16 U w A gen. As a consequence, MSX- ea ed Anacys is cells ac i ely p oducing ammonia om ni a e did no su e enhanced phycocyanin deg ada- ion, as is he case o cells subjec ed o ni ogen s a a ion (1). This con as s wi h he si ua ion obse ed o MSX- ea ed ilamen ous cyano- bac e ia unde dini ogen- ixing condi ions, as 4 Sn E ^ ~~~~~2 A ~~~w z A I- D 0 0o 30 60 TIME ( h ) FIG. 3. Cellula le els o glu amine and glu ama e in MSX- ea ed and un ea ed A. nidulans cells. Cells g own on ni a e medium we e ans e ed o media con aining KNO3 (20 mM) (open symbols) o KNO3 (20 mM) and MSX (10 FM) (closed symbols). Samples we e wi hd awn a he imes indica ed, and he cellula con en in glu amine (ci cles) and glu ama e ( iangles) was es ima ed. O he condi ions we e hose desc ibed in he legend o Fig. 1. APPL. ENVIRON. MICROBIOL. on June 23, 2015 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://aem.asm.o g/Downloaded om SUSTAINED AMMONIA PHOTOPRODUCTION 30 1- 23 1- 16 31 O c 0~~~~~ 0~~~~~~~ 0 0 _ o -- o-o W8e" I,o o/~~~~ IA ,.o I/0 72 96 10 48 TIME (h) FIG. 4. E ec o glu amine addi ion on he p olonga ion o he ammonia p oduc ion pe iod by MSX- ea ed A. nidulans cells. Condi ions we e hose desc ibed in he legend o Fig. 2, excep ha glu amine (0.5 mM) was added a 70 h (a ow) o one-hal o he suspension (0); he o he hal was used as a con ol wi hou any addi ion (0). ound o Anabaena cylind ica (14) and Ana- baena sp. s ain ATCC 33047 (unpublished da a). Al hough MSX- ea ed Anacys is cells lacked glu amine syn he ase, hey exhibi ed low bu ep oducible le els o NADH-alanine dehyd o- genase (a ound 7 mU/mg o p o ein), le els o he same o de o magni ude as hose ound in no mal cells. This inding sugges s ha ammonia assimila ion pa hway(s) ha may be seconda y unde no mal condi ions, can play an impo an ole in he main enance o he ni ogen balance in MSX- ea ed cells. The enzymes esponsible o ni a e educ ion, namely, ni a e educ ase and ni i e educ ase, in MSX- ea ed Anacys is cells we e always p esen a abou he same le els as in he un ea ed cells. As a consequence o he inac i a ion by MSX o glu amine syn he ase, he glu amine le el in ammonia-p oducing cells dec eased apidly wi h ime and became p ac ically negligible 30 h a e he addi ion o he inhibi o . This beha io was in con as wi h ha o un ea ed cells, which, unde he same condi ions, main ained hei no mal glu amine le els. The le el o glu ama e in MSX- ea ed Anacys is cells also dec eased wi h ime, bu much slowe ; i s alue a e 68 h was 35% ha o no mal cells (Fig. 3). This amino acid analysis indica es ha Anacys is cells sub- jec ed o ea men wi h MSX become speci i- cally de icien in glu amine and p obably also in glu amine de i a i es. Such a me abolic si ua- ion migh e en ually lead o he cessa ion o ammonia p oduc ion. To o e come he MSX-p omo ed glu amine de iciency and hus p olong he ammonia p o- duc ion pe iod, wo di e en a emp s we e made. One o hem consis ed o he addi ion o he cell suspension o glu amine a low concen- a ions (0.2 o 0.5 mM) simul aneously wi h he no mal eaddi ion o MSX a he end o he 3- day pe iod. The da a in Fig. 4 show ha his glu amine ea men esul ed in sus ained ammo- nia p oduc ion a cons an a e o a leas 24 h, a phenomenon ha did no occu in a con ol wi hou glu amine addi ion, in which he a e o ammonia p oduc ion slowed wi h ime un il i became negligible. In e es ingly enough, he glu- amine ea men did no esul in cell g ow h (measu ed as Chl) o in any inc ease in glu a- mine syn he ase ac i i y, bu he le els o ni a e and ni i e educ ases doubled. No inc emen in he ammonia p oduc ion a e was obse ed, howe e , sugges ing ha he no mal ni a e and ni i e educ ase le els a e high enough o main- ain he p ocess a i s maximal a e. Glu ama e could no eplace glu amine in sus aining ammo- nia p oduc ion, a esul which con i ms he assump ion ha he cessa ion o he p ocess is speci ically ela ed o glu amine de iciency. I E I :3 w I z z 0 I I 1023 VOL. 44, 1982 on June 23, 2015 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://aem.asm.o g/Downloaded om 1024 RAMOS, GUERRERO, 0 E E 0 w 0 0 a- 4 z 0 -J I- 0 6 3 0 100 200 TIME ( h ) FIG. 5. E ec o he ecu en addi ion and emo al o MSX on he pho op oduc ion o ammonia by A. nidulans cells. A cell suspension wi h a densi y o 9 p.g o Chl pe ml was main ained o 48 h in ni a e cul u e medium supplemen ed wi h 10 puM MSX. A e his ime, cells we e ha es ed and washed h ee imes wi h esh ni a e medium lacking MSX. A e 8 h in he absence o he inhibi o , and once he cell loading had been co ec ed o 9.0 + 0.2 p.g o Chl pe ml, MSX (10 puM inal concen a ion) was eadded. Successi e pe iods o ammonia p oduc ion (a ound 48 h) and cell eco e y (a ound 8 h) (a ows) we e al e na ed as indica ed. The las eaddi ion o MSX was o each a inal concen a ion o 100 puM. The second app oach o ex end he ammonia p oduc ion pe iod consis ed o allowing he cells o eco e om glu amine de iciency by hem- sel es. This goal was achie ed by al e na ing exposu e o MSX wi h pe iods in which he glu amine syn he ase inac i a o was emo ed. Figu e 5 shows ha his sequen ial ea men allowed con inuous p oduc ion o ammonia a high a es o a leas 10 days. The long pe iod in he p esence o MSX (a ound 48 h) esul ed in ne ammonia p oduc ion, whe eas he sho e pe iod in i s absence (a ound 8 h) did no . Du ing he eco e y pe iods, glu amine syn he ase ac- i i y inc eased om 0 o 150 o 300 mU/mg o p o ein (10 o 2b% o he le el in no mal cells), which allowed glu amine syn hesis and a sligh g ow h ( om 9.0 o abou 10.6 ,ug o Chl pe ml in 8 h). Su p isingly, a e day 8 o ammonia p oduc ion, esis ance o MSX appea ed. This phenomenon was p obably due o an adap a ion o he cells o MSX, making i necessa y o inc ease he MSX concen a ion up o 100 ,uM o again achie e comple e inac i a ion o cellula glu amine syn he ase. LITERATURE CITED 1. Alien, M. M., and A. I. Smi h. 1%9. Ni ogen chlo osis in blue-g een algae. A ch. Mik obiol. 69:114-120. 2. Bailey, J. L. 1%7. Techniques in p o ein chemis y, 2nd ed., p. 340. Else ie , Ams e dam. 3. Flo es, E., M. G. Gue e o, and M. Losada. 1980. Sho - e m ammonium inhibi ion o ni a e u iliza ion by Ana- cys is nidulans and o he cyanobac e ia. A ch. Mic obiol. 128:137-144. 4. Gue e o, M. G., J. M. Vega, and M. Losada. 1981. The assimila o y ni a e- educing sys em and i s egula ion. Annu. Re . Plan Physiol. 32:169-204. 5. He e o, A., E. Flo es, and M. G. Gue e o. 1981. Regula- ion o ni a e educ ase le els in he cyanobac e ia Ana- cys is nidulans, Anabaena sp. s ain 7119, and Nos oc sp. s ain 6719. J. Bac e iol. 145:175-180. 6. Koche , G. 1978. Ca bohyd a e de e mina ion by he phenol-sul u ic me hod, p. 95-97. In J. A. Hellebus and J. S. C aigie (ed.), Handbook o phycological me hods. Physiological and biochemical me hods. Camb idge Uni- e si y P ess, Camb idge, England. 7. Losada, M. 1979. Pho op oduc ion o ammonia and hyd o- gen pe oxide. Bioelec ochem. Bioene g. 6:205-225. 8. Losada, M., and M. G. Gue e o. 1979. The pho osyn he - ic educ ion o ni a e and i s egula ion, p. 366-403. In J. Ba be (ed.), Pho osyn hesis in ela ion o model sys ems. Else ie , Ams e dam. 9. MacKinney, G. 1941. Abso p ion o ligh by chlo ophyll solu ions. J. Biol. Chem. 140:315-322. 0 0 / 0-0 0 0/ 0-0 / A0 0 I --I 1 -I AND LOSADA APPL. ENVIRON. MICROBIOL. on June 23, 2015 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://aem.asm.o g/Downloaded om SUSTAINED AMMONIA PHOTOPRODUCTION 1025 10. Manzano, C., P. Candau, C. G6mez-Mo eno, A. M. Re- l inpio, and M. Losada. 1976. Fe edoxin-dependen pho- osyn he ic educ ion o ni a e and ni i e by pa icles o Anacys is nidulans. Mol. Cell. Biochem. 10:161-169. 11. Ramos, J. L., M. G. Gue e o, and M. Losada. 1982. Pho op oduc ion o ammonia om ni a e by Anacys is nidulans cells. Biochim. Biophys. Ac a 679:323-330. 12. Ramos, J. L., M. G. Gue e o, and M. Losada. 1982. Op imiza ion o condi ions o pho op oduc ion o ammo- nia om ni a e by Anacys is nidulans. AppI. En i on. Mic obiol. 44:1013-1019. 13. RowelU, P., and W. D. P. S ewa . 1976. Alanine dehyd o- genase o he N2- ixing blue-g een alga, Anabaena cylin- d ica. A ch. Mic obiol. 107:115-124. 14. Wood, N. B., and R. Haselko n. 1980. Con ol o phyco- bilip o ein p o eolysis and he e ocys di e en ia ion in Anabaena. J. Bac e iol. 141:1375-1385. VOL. 44, 1982 on June 23, 2015 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://aem.asm.o g/Downloaded om