Sustained photoproduction of ammonia from dinitrogen and water by the nitrogen-fixing Cyanobacterium anabaena sp. strain ATCC 33047

Vol. 48, No. 1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1984, p. 114-118 0099-2240/84/070114-05$02.00/0 Copy igh ©D 1984, Ame ican Socie y o Mic obiology Sus ained Pho op oduc ion o Ammonia om Dini ogen and Wa e by he Ni ogen-Fixing Cyanobac e ium Anabaena sp. S ain ATCC 33047 JUAN L. RAMOS, MIGUEL G. GUERRERO,* AND MANUEL LOSADA Depa amen o de Bioquimica, Facul ad de Biologia y Conisejo Suipe io de In es iga(Wiones Cien i icas, Uni e si y o Se ille, Se ille, Spaiin Recei ed 30 Decembe 1983/Accep ed 10 Ap il 1984 Condi ions ha e been de eloped ha leng hen he ime du ing which pho osyn he ic dini ogen ixa ion by ilamen s o he cyanobac e ium Anabaena sp. s ain ATCC 33047 p oceeds eely, whe eas he subsequen con e sion o ammonia in o o ganic ni ogen emains blocked, wi h he esul ing ammonia eleased o he ou e medium. When L-me hionine-DL-sul oximine was added e e y 20 h, maximal a es o ammonia p oduc ion (25 o 30 ,umol/mg o chlo ophyll pe h) we e main ained o abou 50 h. A e his ime, ammonia p oduc ion ceased due o a de iciency o glu amine and o he ni ogenous compounds in he ilamen s, condi ions which inally led o cell lysis. The e ec i e ammonia p oduc ion pe iod could be u he ex ended o abou 7 days by adding a small amoun o glu amine a he end o a 40-h p oduc ion pe iod o by allowing he cells o eco e o 8 h in he absence o L-me hionine-DL-sul oximine a e e e y 40-h pe iod in he p esence o he inhibi o . A mo e p olonged s eady p oduc ion o ammonia, las ing o longe han 2 weeks, was achie ed by al e na ing ea men s wi h he glu amine syn he ase inhibi o s L-me hionine-DL-sul oximine and phosphino h icin, p o ided ha 8-h eco e y pe iods in he absence o ei he compound we e also al e na ed h oughou . The biochemically manipula ed cyanobac e ial ilamen s hus ep esen a sys em ha is ela i ely s able wi h ime o he con e sion o ligh ene gy in o chemical ene gy, wi h he ne gene a ion o a aluable uel and e ilize h ough he pho o educ ion o dini ogen o ammonia. The con e sion o sola ene gy in o sui able edox ene gy h ough pho osyn hesis o he wa e -spli ing ype ha is ca ied ou by whole o ganisms is a p ocess o g ea in e es and signi icance. Ligh -d i en syn hesis o ammonia om dini ogen and wa e by pho osyn he ic o ganisms ep e- sen s an in e es ing sys em o he con e sion o ligh ene gy in o s o ed chemical ene gy, wi h he addi ional in e es ha he esul ing compound is a aluable uel and e ilize (4, 7). Filamen ous cyanobac e ia (blue-g een algae) ha a e able o di e en ia e he e ocys s a e unique o ganisms in ha hey can ix N2 unde ae obic condi ions, wi h ligh as he sole sou ce o ene gy and wa e as he ul ima e educ an , acco ding o he ollowing global equa ion: 6ce->P ATP N2 + 3H20 -'---P 2NH3 + 3/202 (AEo' [pH 7] = -1.10 V; lAG' [pH 7] = +318 kJ/mol o ammonia) whe e AE(4 is he s anda d edox po en ial change and AG' is he s anda d ee ene gy change. The achie emen o e ec i e p oduc ion o ammonia om dini ogen by N2- ixing cyanobac e ia equi es (i) p e en ion o he inco po a ion o ammonia o ca bon skele ons, which akes place mainly h ough he glu amine syn he ase-glu a- ma e syn hase pa hway (18), and (ii) o e coming he an ago- nis ic e ec s o ammonia on dini ogen ixa ion (4). Bo h equisi es ha e been simul aneously ul illed by in e e ing * Co esponding au ho . P esen add ess: Uni o Ni ogen Fixa ion, Uni e si y o Sussex, B igh on BN1 9RQ, Uni ed Kingdom. wi h he ope a ion o he main ammonia assimila ion pa h- way by using glu ama e analogs, such as MSX (L-me hio- nine-DL-sul oximine) o PT (phosphino h icin [2-amino-4- (me hylphosphinyl)-bu anoic acid]) (6, 9, 12, 17). To be conside ed p ac ical, a sys em o he con e sion o sunligh ene gy mus ope a e a a conside able a e and e iciency and mus be ela i ely s able wi h ime. The sys em cons i u ed by MSX- o PT- ea ed Anabaena sp. s ain ATCC 33047 ilamen s has been shown o p oduce ammonia a high a es, in he ange o 25 o 30 pLmol/mg o chlo ophyll pe h (6, 9, 12), wi h ela i ely high e iciency, ca. 10% o i s heo e ical maximum, bu un il now he p ocess has been only ca ied ou o sho pe iods o ime. This communica ion epo s he achie emen o s eady pho op oduc ion o ammonia om dini ogen by he cyano- bac e ium Anabaena sp. s ain ATCC 33047, las ing o longe han 2 weeks. MATERIALS AND METHODS G ow h o cells. Anabaena sp. s ain ATCC 33047 was g own pho oau o ophically, unde con inuous illumina ion (Syl ania day ligh luo escen ubes, wi h 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 modi ied om BG11 medium as desc ibed p e iously (16), excep ha 25 mM NaHCO3 was added. A s eam o 2% ( ol/ ol) CO, in ai was bubbled h ough he cul u e a a low a e o 1.2 li e s/li e o cell suspension pe h. Ammonia p oduc ion expe imen s. Anabaena sp. s ain ATCC 33047 ilamen s om 2-day-old cul u es, con aining 20 o 25 1Lg o chlo ophyll a (Chl) pe ml, we e used. The ilamen s we e ha es ed, washed wi h cul u e medium, and inally esuspended in he same medium o each a cell densi y o 8 o 10 pLg o Chl pe ml. The ilamen suspensions we e incuba ed o 60 o 90 min in he ligh unde s anda d 114 SUSTAINED AMMONIA PRODUCTION FROM DINITROGEN cul u e condi ions. Expe imen s we e s a ed by adding enough MSX o he cell suspension o achie e a a io o 3.5 o 4 nmol o MSX pe ,ug o Chl. Assays o enzyme ac i i ies. Ni ogenase ac i i y was mea- su ed in whole cells by he ace ylene educ ion echnique (9). Glu amine syn he ase ( ans e ase) ac i i y was de e - mined in oluenized ilamen s (12). Alanine dehyd ogenase was es ima ed in cell- ee ex ac s as p e iously desc ibed (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 0WC, he samples we e cen i uged a 40,000 x g o 15 min, and he supe na an was neu alized a 0°C wi h KOH. A e emo al o KCl04 by cen i uga ion (10,000 x g o 10 min), he supe na an s we e used o he es ima ion o me aboli es. Analy ical me hods. Ammonia was de e mined by he phenol-hypochlo i e me hod as desc ibed p e iously (15). Ca bohyd a es we e es ima ed by he phenol-sul u ic me h- od (5). Chl was de e mined spec opho ome ically in me h- anolic ex ac s by using he ex inc ion coe icien gi en by MacKinney (8). E hylene was measu ed by using a PYE UNICAM 204 gas ch oma og aph equipped wi h a column illed wi h Po apak Q and a lame ioniza ion de ec o . Glu ama e and glu amine we e de e mined acco ding o Be gmeye (3). y-Glu amil hyd oxama e was es ima ed a e i s eac ion wi h FeCl3 in acid medium (14). Cellula p o ein was de e mined by he Low y p ocedu e as modi ied by Bailey (2), by p e ea men o he ilamen s wi h 10% (w / ol) ichlo oace ic acid. Fo d y-weigh de e mina ion, 30-ml samples o he cell cul u e we e il e ed h ough p eweighed d y il e s (Wha - man GF/C). The il e s we e washed wi h 2 olumes o dis illed wa e and d ied a 90°C un il cons an weigh was eached. Rela i e phycocyanin le els we e es ima ed spec- o luo ime ically by using an Aminco-Bowmnan spec o- pho o luo ime e equipped wi h a HTV pho omul iplie ube, ype R44GS. Fo luo escence measu emen s, ilamen suspensions (9 pLg o Chl pe ml) we e illumina ed wi h g een ligh (590 nm, 5 W/m2), which is mainly abso bed by phycocyanin, wi h he ene gy hen ans e ed o chlo o- phyll. Chlo ophyll luo escence is p opo ional o he le el o phycocyanin in he ilamen suspensions. Ligh in ensi y measu emen s we e done wi h a YSI-Ke e ing model 65A adiome e . Chemicals. MSX was pu chased om Sigma Chemical Co., S . Louis, Mo. PT (6) was kindly supplied by P. J. Lea (Ro hams ed, Uni ed Kingdom). O he chemicals we e p od- uc s o E. Me ck AG, Da ms ad , Fede al Republic o Ge many. RESULTS AND DISCUSSION The addi ion o MSX o suspensions o Anabaena sp. s ain ATCC 33047 ilamen s ha we e g owing on ai (78% N2) as he ni ogen sou ce caused a apid inac i a ion o cellula glu amine syn he ase, wi h cell g ow h consequen ly p e en ed. Ni ogenase ac i i y inc eased g adually in e- sponse o MSX, eaching a s able le el, which was a ound wo old highe han he ini ial le el, abou 4 h a e i s addi ion. Mos o he ammonia (ca. 90%) esul ing om N2 ixa ion by he cells was eleased and accumula ed in he ou e medium (9; see also Fig. 1). The a e o ammonia p oduc ion inc eased concomi an ly wi h he inc ease in ni ogenase ac i i y men ioned abo e, un il i eached a maximum in co espondence wi h he es ablishmen o a s able high ni ogenase le el. The maximal a es o ammonia p oduc ion unde hese condi ions we e 25 o 30 p.mol/mg o Chl pe h, and he p ocess con inued o be ope a i e o abou 20 h (Fig. 1). Figu e 1 also shows ha abou 20 h a e MSX addi ion glu amine syn he ase ac i i y s a ed o eco - e and accumula ion o ammonia in he medium ceased. These changes we e ollowed by he u iliza ion o he ammonia ha was eleased by he ilamen s, wi h g ow h s a ing again. In pa allel wi h he occu ence o ammonia up ake, he ni ogenase ac i i y le el dec eased sligh ly. Since unde s anda d cul u e condi ions and in he ab- sence o ilamen s MSX keeps ac i e o a leas 48 h, he cessa ion o he e ec o he inhibi o on ammonia p oduc- ion could be ela ed o i s ans o ma ion by he cyanobac- e ial cells a he han o i s spon aneous deg ada ion. The empo a y in e up ion o ammonia p oduc ion is appa en ly 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. Acco dingly, o leng hen he pe iod o ammonia p oduc ion, esh MSX a i s op imal a io wi h espec o he amoun o cells (3.5 o 4 nmol/Lg o Chl) was added o suspensions o Anabaena sp. s ain ATCC 33047 in which he MSX e ec had been los . This addi ion esul ed again in inac i a ion o p eexis ing glu amine syn he ase and allowed anew maximal a es o ammonia p oduc ion (da a no E I n w I z I D 0 I I 4 C-) .- z cm 0 I-J -J w C-) 100 w .s ZA 0 4 w E 50 8 x _ x b- E 0 30 w In 04- w I O i Z _ ) = 15 w c z E 4 D PI- %-'o 0- 0 0 0 12 24 TIME (h) FIG. 1. Time cou se o he e en s leading o ammonia p oduc- ion and i s la e cessa ion in MSX- ea ed Anabaena sp. s ain ATCC 33047 ilamen s. A ilamen suspension (150 ml) wi h a densi y o 9 ,ug o Chl pe ml ha was supplemen ed wi h 35 ,uM 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 (A), densi y o he ilamen suspension (0), and cellula ac i i ies o ni ogenase (0) and glu amine syn he ase (A) we e de e mined. VOL. 48, 1984 115 116 RAMOS, GUERRERO, AND LOSADA shown). Following his app oach, successi e addi ions o MSX o he ilamen suspension e e y 20 h allowed maximal a es o ammonia p oduc ion o be main ained o abou 48 h (Fig. 2). Du ing his pe iod, glu amihe syn he ase emained ully inac i e, and he le el o ni ogenase ac i i y was a leas wo old highe han ha in un ea ed ilamen s. Ne e - hele,ss, a e 48 h in he p-esence o MSX, he ammonia p oduc ion a e dec eased p og essi ely, in spi e o he ac ha glu amine syn he ase emained inac i e. This cessa ion in ammonia p oduc ion seems o be a consequence o a loss o ni ogenase ac i i y (da a no shown). 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 Anabaena sp. s ain ATCC 33047, he le els o a ious cell componen s, enzyma ic ac i i ies ela ed o ni ogen me abolism, and amino acids in ol ed in he glu amine syn he ase-glu ama e syn hase pa hway we e measu ed ih ilamen s which had been ea ed wi h MSX o 36 h and we e compa ed wi h hose in un ea ed ilamen s. Table 1 shows he esul s ob ained. The alues o he chlo ophyll and p o ein con en we e only sligh ly lowe in MSX- ea ed ilamen s han i n he un ea ed ones. Howe e , ema kable di e ences we e ound ega d- ing he le els o ca bohyd a es and phycocyanin. The ca bo- hyd a e and phycocyanin le els in MSX- ea ed ilamen s we e h ee old highe and i e old lowe , espec i ely, han hose in un ea ed ilamen s. The educ ion in he phyco- cyanin le el in MSX- ea ed cells was also e iden om he change in he abso bance a 628 nm/abso bance a 680 nm a io o he cell suspensions, which dec eased om 0.91 o 0.72 as a consequence o he p olonged ea men wi h MSX. The di e ences in ca bohyd a e and phycocyanin con en we e in e p e ed as being due o con inued pho osyn he ic CO2 ixa ion in he p esence o MSX, which o he wise o-' E %-. 0 Lii 0 Q 0- 0 6 4 2 0 20 40 60 TIME(h ) FIG. 2. Con inuous ammonia pho op oduc ion by MSX- ea ed Anabaena sp. s ain ATCC 33047 ilamen s. Condi ions we e he same as hose desc ibed in he legend o Fig. 1, excep ha MSX (35 ,uM) was eadded e e y 20 h. The ammonia in he medium was de e mined a he imes indica ed. TABLE 1. E ec o MSX on he le els o di e en cell componen s in Anabaena sp. s ain ATCC 33047" Cell componen Time (h) and ea men Chlb Phyco- P o einh Ca bo- cyaniin" hyd a esb 0 31 100 700 79 36 (wi hou MSX) 30 100 650 94 36 (wi h MSX) 26 20 550 284 ' A suspension o Anabaena sp. s ain ATCC 33047 ilamen s con aining 7.9 ,ug o Chl (250 pLg [d y weigh ]) pe ml was di ided in o wo hal es, o one o which MSX (35 FM) was added a ze o ime and 20 h la e . A e 36 h o incuba ion unde s anda d g ow h condi ions, he cell densi y alues eached we e 37.0 jig o Chl (1,214 ,ug [d y weigh ]) pe ml and 7.8 ,g o Chl (304 ,ug [d y weigh ]) pe ml o he MSX- ee and MSX-con aining suspensions, espec i ely. Values a e in mic og ams pe millig am (d y weigh ). Values a e pe cen ages o he con ol a ze o ime. p e en ed he inco po a ion o ammonia de i ed om N2 ixa ion in o ca bon skele ons. Ac ually, he C/N a io inc eased in MSX- ea ed Anabaena sp. s ain ATCC 33047 ilamen s om 4.2 a ze o ime o 6.8 a 36 h. The MSX- ea ed cells hus appea o be ni ogen s a ed (1). Such an induc ion o ni ogen s a a ion by MSX has been also epo ed o N2- ixing Anabaena cylind ica (19), bu i is in con as wi h he si ua ion no iced in MSX- ea ed Anacys is cells p oducing ammonia om ni a e, which did no exhibi enhanced phycocyanin deg ada ion (10, 11). MSX- ea ed Anabaena sp. s ain ATCC 33047 ilamen s lacked glu amine syn he ase ac i i y and exhibi ed low, al hough ep oducible, le els o NADH-alanine dehyd oge- nase (ca. 14 mU/mg o p o ein), abou wo old highe han hose ound in un ea ed ilamen s. This ac i i y migh be esponsible o he assimila ion o low amoun s o ammonia, since he ni ogen de iciency was mo e se e e in MSX- ea ed Anabaena sp. s ain ATCC 33047 kep unde a gon a mosphe e han in ha main ained unde ai . In ac , a e 36 h o ea men , he o me had los mos o i s phycocya- nin, whe eas he la e s ill e ained ca. 20% o he ini ial le el. As a consequence o he inac i a ion o MSX o glu amine syn he ase, he glu amine le el in ammonia-p oducing ila- men s dec eased apidly wi h ime and became p ac ically negligible 9 h a e he addi ion o he inhibi o . This beha - io con as ed wi h ha o un ea ed ilamen s, which, unde he same condi ions, main ained hei ini ial glu amine le el. The le el o glu ama e in MSX- ea ed Anabaena sp. s ain ATCC 33047 ilamen s also dec eased wi h ime bu did so much mo e slowly han ha o glu amine, i s alue a e 36 h being 75% o ha in un ea ed ilamen s (Fig. 3). This amino acid analysis indica es ha he Anabaeha sp. s ain ATCC 33047 ilamen s ha we e subjec ed o ea men wi h MSX, in addi ion o su e ing om gene al ni ogen s a a ion, became speci ically 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 de iciencies and hus p olong he ammonia p oduc ion pe iod, wo di e en a - emp s we e made. One o hem was he addi ion o he cell suspension o glu amine (0.2 mM) simul aneously wi h he no mal eaddi ion o MSX 40 h a e he expe imen had been ini ia ed. The da a in Fig. 4 show ha his glu amine ea men esul ed in sus ained ammonia p oduc ion a a cons an a e o an ex a pe iod o a leas 32 h, a phenome- non ha did no occu in a con ol ha ecei ed he no mal / I 0 */ 0 a / APPL. ENVIRON. MICROBIOL. SUSTAINED AMMONIA PRODUCTION FROM DINITROGEN 117 'a E C w z 4 0 12 24 36 0-% 2 c u 1 S O" 0 E I. CD TIME (h) FIG. 3. Cellula le els o glu amine and glu ama e in MSX- ea ed and un ea ed Anabaena sp. s ain ATCC 33047 ilamen s. An ai -g own suspension o ilamen s con aining 9 ,ug o Chl pe ml was ans e ed ei he o a medium ee o combined ni ogen sou ce (open symbols) o o he same medium supplemen ed wi h 35 ,uM MSX (closed symbols). Samples we e wi hd awn a he imes indica ed, and he cellula con en o glu amine (ci cles) and glu a- ma e ( iangles) was es ima ed. O he condi ions we e he same as hose desc ibed in he legend o Fig. 2. addi ion o MSX (35 ,uM) bu did no ecei e glu amine. The glu amine ea men did no esul in cell g ow h (measu ed as an inc ease in Chl) o in any inc ease in glu amine syn he ase ac i i y bu allowed he main enance o he high ni ogenase ac i i y le el. Glu ama e (0.2 mM) could no eplace glu amine in sus aining ammonia p oduc ion, a esul which sugges s ha cessa ion o he p ocess is ela ed o glu amine de iciency. The second app oach o ex end he ammonia p oduc ion pe iod was o allow he ilamen s o eco e om hei E 0 w - cD 0 0. c 0- z 0 S 18 9 0 24 48 72 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 Anabcue naJ sp. s ain ATCC 33047 ilamen s. Condi ions we e he same as hose de- sc ibed in he legend o Fig. 2, excep ha glu amine (0.2 mM) was added a e 40 h (a ow) o one-hal o he suspension (@). wi h he o he hal used as a con ol wi hou glu amine addi ion (0). = 2 E E ( 0 2 ~~~~~~00 0 4 40 80 120 160 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 Anihbaeni sp. s ain ATCC 33047 ilamen s. A ilamen suspension (150 ml) wi h a densi y o 9.8 p.g o Chl pe ml was main ained o 40 h in he s anda d cul u e medium supplemen ed wi h 35 ,uM MSX. A e his ime cells we e ha es ed and washed wi h esh medium lacking MlSX. A e 8 h in he absence o he inhibi o , and once he cell load had been co ec ed o 10 + 0.2 p.g o Chi pe ml, MSX (35 F.M inal concen a ion) was eadded. Successi e pe iods o ammonia p o- duc ion (ca. 40 h) and cell eco e y (ca. 8 h) (a ows) we e al e na ed as indica ed. de iciencies by hemsel es. This 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 ammonia p o- duc ion o a leas 7 days. The long pe iod in he p esence o MSX (a ound 40 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 8 0 E E 0 w u i 0 4 6 4 2 0 3 6 9 12 15 18 TIME (days) FIG. 6. E ec o he al e nance o MSX and PT on he pho op o- duc ion o ammonia by Anabaena sp. s ain ATCC 33047 ilamen s. Condi ions we e he same as hose desc ibed in he legend o Fig. 5. excep ha he successi e pe iods o ammonia p oduc ion p oceed- ed al e na ely in he p esence o MSX (ca. 40 h) and o PT (ca. 20 h). wi h eco e y pe iods (ca. 8 h) in he absence o any inhibi o be ween hem (a ows). / "I si ~~~~/ 0~~~~~~~ ~ ~ ~ . VOL. 48, 1984 118 RAMOS, GUERRERO, AND LOSADA ac i i y inc eased om 0 mU o 140 o 280 mU/mg o p o ein (7 o 14% o he le el in no mal cells), allowing glu amine syn hesis, inc ease o he phycocyanin le el ( om 20 o 25% o 50 o 80% o he ini ial one), and sligh g ow h ( om 9.8 o abou 10.5 p.g o Chl pe ml in 8 h). Su p isingly, a e 7 days o ammonia p oduc ion, esis ance o MSX appea ed. This phenomenon was p obably due o an adap a ion o he ilamen s o MSX and made i necessa y o inc ease he MSX concen a ion up o 100 o 150 FM o again achie e comple e inac i a ion o cellula glu amine syn he ase. The e ec on he du a ion o he ammonia p oduc ion p ocess o al e na ing ea men s wi h wo di e en glu a- mine syn he ase inhibi o s, namely, MSX and PT, has also been es ed. PT is ano he glu ama e analog which e ec i e- ly inhibi s glu amine syn he ase and allows ammonia p oduc- ion a high a es by Anabaenca sp. s ain ATCC 33047 (6). The ea men including he use o bo h MSX and PT consis ed o a i s pe iod in he p esence o MSX (ca. 40 h), ollowed a e a sho in e al o eco e y (ca. 8 h) by a second pe iod (a ound 20 h) in he p esence o PT. Figu e 6 shows ha his sequen ial ea men wi h MSX and PT pe mi ed con inuous ammonia p oduc ion o longe han 2 weeks, p o ided ha eco e y pe iods in he absence o ei he inhibi o we e also al e na ed h oughou . The p ocess o biological ammonia pho op oduc ion om N2 by adequa ely ea ed li ing cyanobac e ial cells ep e- sen s he ne gain o a aluable compound om abundan and inexpensi e subs a es, namely, ai and wa e , a he expense o only sunligh as he sou ce o ene gy. Since he sys em ope a es, once i has been s abilized as desc ibed he ein, a a conside able a e (see e e ence 4 o a compa i- son o ammonium p oduc ion a es by di e en biological sys ems) and wi h a easonable ene gy con e sion e iciency (a ound 2%), i could p o e e en ually aluable o p ac ical pu poses. ACKNOWLEDGMENTS This wo k was suppo ed by g an s om Fundacidn Ram6n A eces and Comisi6n Aseso a In es igacion (Spa in). We hank An onia F iend and Pepa Pe ez de Leon o help ulI assis ance. We a e g a e ul o Pe e J. Lea (Ro hams ed, Uni ed Kingdom) o a gene ous gi o phosphino h icin. LITERATURE CITED 1. Allen, M. M., and A. I. Smi h. 1969. Ni ogen chlo osis in blue- g een algae. A ch. Mic obiol. 69:114-120. 2. Bailey, J. L. 1967. Techniques in p o ein chemis y. 2nd ed., p. 340. Else ie /No h-Holland Biomedical P ess. Ams e dam. 3. Be gmeye , H. U. 1974. Me hoden de Enzyma ischen Analyse. 2nd ed. Ve lag Chemie. Weinheim. Fede al Republic o Ge ma- ny. 4. Gue e o, M. G., J. L. Ramos, and M. Losada. 1982. Pho osyn- he ic p oduc ion o ammonia. Expe ien ia 38:53-58. 5. 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 physiological me hods. Physiological and biochemical me hods. 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