- Email: [email protected]
Photoproduction of hydrogen, hydrogen peroxide and ammonia using immobilized cyanobacteria
Int. J. Hydrogen Energy, Vol. 16, No. 5, pp. 313 318, 1991. Printed in Great Britain.
036(~3199/91 $3.00 + 0.00 Pergamon Press plc. International Association for Hydrogen Energy.
PHOTOPRODUCTION OF HYDROGEN, HYDROGEN PEROXIDE A N D AMMONIA USING IMMOBILIZED CYANOBACTERIA I. H. PARK, K. K. Rho* and D. O. HALL Division of Biosphere Sciences, King's College, London W8 7AH, U.K. (Received for publication 13 December 1990) Abstraet--Anabaena azollae cells were immobilized in synthetic polymer foams and cellulosic hollow fibres. The immobilization of cells resulted in stable photosynthetic oxygen evolution activity and MSX-induced ammonia production. In "trickling-medium" photobioreactors using PV-immobilized Anabaena azollae, continuous hydrogen and ammonia production was observed for one week. In a hollow fibre system, cell attachment to hydrophilic cellulosic hollow fibre was better than with hydrophobic polysulfone and polypropylene hollow fibres. Photoproduction of hydrogen peroxide was observed in free and immobilized thermophilic Phormidium laminosum cells. Characteristics of a "trickling-medium" photobioreactor for continuous production of hydrogen peroxide were also studied.
NOMENCLATURE DCMU MSX MV PU PV RF
dichlorophenyldimethylurea L-methionine-D,L-sulfoximine methyl viologen polyurethane polyvinyl riboflavin
INTRODUCTION Over the past few years, much attention has been given to the design of systems capable of conversion, storage and usage of solar energy into chemicals, such as high energy compounds and valuable secondary metabolites [1-2]. One of the possible ways of storing solar energy is to transform it into chemical potential via production of energy rich compounds such as hydrogen, hydrogen peroxide and ammonia [3-4] using photosynthetic processes. The theoretical maximum efficiency for photosynthetic solar energy conversion, defined as the ratio of stored chemical energy to the incident solar radiation on the photosynthesizer is calculated to be about 13%, and the practical maximum conversion efficiency under optimal condition is 9% on an annual basis (Bolton and Hall, unpublished). However, this maximum conversion efficiency is never reached in the field; for most plant species it seldom exceeds 1% on an annual basis. Therefore there is a need and great interest in developing photobiological/photochemical systems which would mimic the energy conversion processes of natural photo*Author to whom correspondence should be addressed,
synthesis, hopefully with better efficiency. Thus, many photobiological systems comprising whole cells of algae or cyanobacteria, isolated thylakoids, photosynthetic bacterial chromatophores and individual photosystems have been used in the design of photobioreactors for the production of H 2, H202, ATP, NADPH2, NH3 in addition to polysaccharides, oils, etc. [3, 5, 6]. For stability and ease of manipulation it may be advantageous to use the photosynthesiser in an immobilized state in the photobioreactor. Recently many approaches have been made to immobilize cyanobacterial cells for the production of energy-rich compounds and some biochemicals using immobilizing matrices such as PV, PU, alginate etc. [1]. Among these materials, alginate is a good matrix for immobilization, however, it acts as a quencher of nutrient and gas diffusion, and thus synthetic polymers are better suitable for immobilization of algal cells. Although PU and PV easily adsorbed algal cells, the cells may leak out of the foam when the reactor is bubbled with nutrient gas. To avoid this possibility, media flushed with gas can be provided, but in this case the gas supply acts as a limiting factor in the growth and production of cells in the reactor. We report here the results of our studies on immobilized cyanobacteria for the production of H 2, H202 and NH3. The design of novel "trickling-medium photobioreactor" is also described. MATERIALS A N D METHODS Culture o f cyanobacteria Anabaena azollae was grown in medium BG-11 [7] without combined nitrogen under cool white fluorescent lamps (100 #E m 2 s i) with shaking (100 rpm) and 313
I . H . PARK et al.
314
supplied with 5% CO2 at 27°C. Cell growth was determined by chlorophyll concentration in the culture suspension. Thermophilic Phormidium laminosum were grown in medium D [8] under the same conditions as Anabaena except that the temperature was 45°C.
Immobilization of cyanobacteria Immobilization of cyanobacteria in PV and PU polymer foams was performed according to Brouers et al. [9]. Determination of ammonia For chemical determination of ammonia, Anabaena cells were suspended in fresh culture medium with 100 g M MSX. After incubation for 3 h, the cell suspension was centrifuged at 2500 g for 10 rain and the content of ammonia in the supernatant was determined according to Solorzano [10]. For continuous determination of ammonia, 10#mole MSX was injected into the reactor (pulse treatment, about 20 min). The eluate from the reactor was mixed with nine times volume of 1 N NaOH and then passed through an ammonia probe (EIL8002-8, Kent Industrial Measurement, U.K.) for polarographic assay, Nitrogenase activity Nitrogenase activity was determined by acetylene reduction [11] under aerobic or anaerobic conditions. Hydrogen was measured using a gas chromatograph fitted with a thermal conductivity detector, Hydrogen peroxide production Photoproduction of hydrogen peroxide was performed in 50 mM Tricine-KOH (pH 7.5) buffer containGC sampling
~1- ~
~
r
Chlorophyll determination Chlorophyll content was calculated from the absorbance at 665 nm after extraction with 95% methanol [12]. Bioreactor "Trickling-medium" column bioreactor using PVimmobilized A. azollae cells (Fig. 1) was made with a water jacketed Pyrex glass column (18 mm I.D., 280 mm L). The column was filled with 100 pieces of sterilized PV-foam (5 mm cubes). Immobilization was initiated by flushing with free cell culture suspension. Air (101min - l ) and the medium (6.25mlh ~) trickling through the foams were supplied from the top of the column. Illumination was with fluorescent lamps (100 #E m -2 s -1) and temperature was 27°C (Table 1). Hollow fibre bioreactors were composed of cuprophane cellulosic hollow fibre (from Green Cross Medical Industry Co., Korea) or polysulfone hollow fibre (donated by Professor S.-C. Wang, Tianjin Univ., China). The free cell suspension was flushed into the interfacial space of the hollow fibre, the medium flowed
I-'-I
1
Glass beads
V.n
Humidifier
~ ~
Medium
Gas Trap
oP2
l"'c°'<"rt
i pH meter
/-1 PV-immobilized cell O
Photosynthetic oxygen evolution Photosynthetic oxygen evolution activity was determined with Clark type oxygen electrode (Rank Brothers, U.K.) at 27°C with illumination of incandescent light (2000 ~tE m -2 s ~) through an orange filter.
0 P~
Air or Argon gas water bath
ing 10mM NaC1, 5 m M NH4CI and 2 5 # M MV under a supply of pure oxygen [4] at 27°C. Hydrogen peroxide was determined by H202-dependent N A D H oxidation in 0.1 M phosphate buffer (pH 7.5).
~ k
Ammonia probe
T Waste
P3
r P4
~
[
_--.
Fig. 1. Schematic diagram of "trickling-medium" reactor for production of H2 and NH 3 using PV-immobilized A. azollae cells. For H2, stopcock 2 and pump 2 were operated. For NH3, stopcock 1 and pumps l, 3 and 4 were operated.
PHOTOPRODUCTION OF H2, H 2 0 2 AND N H 3 Table 1. Characteristics of trickling-medium bioreactor for hydrogen photoproduction using PV-immobilized Anabaena azollae cells
ae 140
Column size Gas phase volume PV-foam Total column volume Solid and liquid phase volume Cell density/reactor volume Medium flow rate Air supply Chlorophyll content
"~ 100 o ~" ._~ 80 ~ 60 E E 40 < 0
Temperature Light intensity
ID 18mm × L. 280mm cm 3 2.47 g dry wt 64 ml 33
31 c m 3 169/lg Chl ml R 6.25 ml h ' 101 min i l 0.82 mg 4.4 mg Chl g 1dry wt PV-foam 27°C 100#E m--' s
through the lumen side of the fibre and air was supplied through the outer space of the fibre. The column was incubated under the same conditions as the previous glass column bioreactor,
Scanning electron microscopy Immobilized cyanobacterial cells were examined by the critical point drying method. Cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) conraining 6% sucrose (w/v) for 2 h at r o o m temperature, The samples were washed in the same buffer, transferred to 1.0% osmium tetroxide in buffer under the same conditions. The specimens were washed and dehydrated in graded ethanol (50-100%) and dried in a Samdri-780 Critical Point Drying Apparatus (Tousimis Research Corp., U . S . A . ) u s i n g liquid carbon dioxide. Dried specimens were coated in a Sputter Coater (EM-Scope, U.K.) and examined in a model S-510 Scanning Electron Microscope (Hitachi Scientific Instruments Co., Japan) at an accelerating voltage of 15 or 25 kV. RESULTS AND DISCUSSION Cyanobacteria are able to fix atmospheric nitrogen via ATP-dependent nitrogenase activity. Nitrogenase is located in the heterocysts which provide 02 protection for the enzyme. U n d e r normal physiological conditions the nitrogen fixed in the heterocysts is transported to the
d .2
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b
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120
i 1
, 2
Free batch culture PV-foam in batch Floating foam Settled foam Mixed* PV-foam in reactor Immersedt Non-immersed (trickling-medium) * From [18]. ? From [191.
Alga
Fig. 2. Stability of MSX-induced ammonia production rate in free (O) and PU-immobilized (Q) A. azollae cells. adjacent vegetative cells as glutamine, which then enters into the cellular metabolic pool. The conversion of a m m o n i u m to glutamine is catalysed by glutamine synthetase [GS] also located in the heterocysts. In the presence of MSX, which is an analogue of glutamate and which functions as an irreversible inhibitor of GS, a m m o n i u m is excreted into the medium [13, 14].
Immobilization of cyanobacteria For immobilization several matrices were used alginate, carrageenan, PU, PV etc. A m o n g these matrices, synthetic polymers appear as best candidates for immobilization because the price is lower than natural polymers and diffusion of nutrients and gases is better. However, some polymers are slightly toxic to the cell. Another problem encountered with polymer foams is that cell entrapment is very loose (although mucilage was excreted and some attachment to the surface of polymers occurs), and the cells can easily leak out from the foam when the reactor is bubbled or stirred. Many studies have described bioreactors using the "immersed system", wherein the immobilized foams are soaked in the medium. Table 2 shows the cell density of the several immobilized and free batch culture systems. It is clear that the cell density in both batch and "tricklingmedium" reactors using immobilized cells was higher than that of free cells, and the cell density of the non-immersed "trickling-medium" reactor reached 1.6-4.4 mg chl g ' dry wt. This is an eight times higher cell density than that of PV-immobilization in a batch system.
Cell density
Anabaena
15/tg Chl cm 3 culture flask volume
Anabaena
0.75 mg Chl g 1 dry wt PV foam 0.31 mg Chl g t dry wt PV foam 0.37 mg Chl g i dry wt PV foam
Phormidium Cyanospira Anabaena
4
Dark Treatment, Day
Table 2. Comparison of cell density in several immobilization systems Type of culture or reactor
L 3
4 #g Chl (5 mm cube PV foam 0 . 7 3 m g C h l g ' dry wt PV foam) 1.6-4.4mg Chl g i dry wt PV foam 62.5-169/~g Chl cm 3 reactor volume 129-380.7/~g Chl ml ~ medium
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Stability o f immobilized cells Immobilized Anabaena cells showed continued stabilities on long storage. We tested the short-term stability of some physiological activity. When the ammonia productivity in the presence of MSX (100ktM) was measured after dark incubation, the rate rapidly decreased in free cells but in PU-immobilized cells, the activity was almost same or even increased after dark incubation (Fig. 2). Figure 3 shows photosynthetic oxygen evolution activities in free and PU-immobilized Anabaena cells during a similar dark treatment, It shows higher stability of PU-immobilized cells compared to the free cells, Continuous production o f ammonia Figure 4 shows continuous ammonia photoproduction in a "trickling-medium" bioreactor using PV-immobilized A. azollae cells. When cells were treated with MSX (10/Lmole pulse treatment, 20min) the ammonia concentration in the eluates reached 500/~M and no cell leakage was observed from the "tricklingmedium" column reactor. Nitrogenase activity and ammonia production were higher after MSX treatment, Newton and Cavins [15] reported that nitrogenase activity in Anabaena spp increased in the presence of MSX, and Reich et al. [16] showed that MSX treatment
27.2ul C2H2/moJh
20
V
,
40
50
Fig. 5. Effect of CO on the production ofH 2 by nitrogenase in a "trickling-medium" reactor using PV-immobilized A. azollae cells. Arrow shows CO (4%) addition.
prevented inactivation of nitrogenase by exogenous ammonia in Anabaena spp. Continuous production o f hydrogen by nitrogenase Hydrogen evolution from the "trickling-medium" reactor was tested under anaerobic conditions. Although hydrogen accumulation in the reactor was observed in the absence of an uptake hydrogenase inhibitor (CO), after the first day the hydrogen concentration gradually decreased. When CO was injected into the column reactor, hydrogen evolution increased significantly (Fig. 5). In the presence of CO, hydrogen evolving rate increased for 4 days and thereafter the rate decreased gradually (Fig. 6), Total hydrogen production reached 2 ml H 2 per reactor (total 4 mg Chl) after 6 days (Fig. 7). In studies of hydrogen production by nitrogenase using the cellulosic hollow fibre reactor, hydrogen evolution was observed for 3 days under anaerobic conditions, in the presence of CO. Thus there is a potential for the use of hollow fibre in the production of some chemicals using symbiotic filamentous cyanobacteria, as the separation of products from the cells is easy and there is less chance of contamination in working reactors. Hydrogen peroxide production in free cells o f P. laminosum We have observed light-dependent hydrogen peroxide production in P. laminosum via the Mehler reaction.
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400
,
30
Time, h
Fig. 3. Stability of photosynthetic oxygen evolution activity in free (C)) and PU-immobilized ( 0 ) A, azollae cells.
500
,
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Dark Treatment,
=
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40
on
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~
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200
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~ 100
,A
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250
R e a c t i o n T i m e , hr
Fig. 4. Continuous production of NH 3 in "trickling-medium" reactor using PV-immobilized A. azollae cells. A, MSX (10/~mole) addition; /X, light,
lO 0
' 0
1
2
3
4
5
'
6
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Fig. 6. H 2 production rate of the "trickling-medium" reactor using PV-immobilized A. azollae cells.
PHOTOPRODUCTION OF H2, H202 AND NH 3 2000
317
Table 3. Hydrogen peroxide production and oxygen evolution civyn
r
and
Vimmobi
d
a /o
Phormidium Free cell PV-immob. 1000
Oxygen evolution (pmolmg t Chlh i)
0~---,--~" . . . . 0
1
2
I , 3
, ' 6
4
5
Reaction Time, Day
7. Continuous H 2 production in "trickling-medium" reactor (4 mg Chl) using PV-immobilized A. azollae cells. Arrow shows CO (4%) point, Fig.
•=
45.8
Hydrogen peroxide production %umolmg i Chlh i) +MV 19.3
mce
Anacystis * Free cell
26.3
112
17.5
36
-MV
3.6
0
+MV + DCMU
0
0
--
HzO2/O2 ratio 0.4 0.7 0.3 Free cells are 10 days old. PV-immobilized cells are 1 month old and the activity was measured 10 days after media change. MV is 25jtM and DCMU is 5pM. *From [5].
30
.C
o o, E .~ o
20
~:
to o
~
/.__ L /-
L/MV L/RF
D/-
D/MV D/RF
Fig. 8. Photoproduction rate of H202 under various conditions in P. laminosum cells. L, light; MV, methylviologen 25 pM; RF, riboflavin, 25/~M; D, dark.
Hydrogen peroxide production rates showed a linear relationship to the chlorophyll concentration and reached 2 1 . 6 p m o l m g --~ C h l h ~ at 27c'C. This value is lower than the rate in Anacystis nidulans ( 3 6 / ~ m o l m g I Chl h l , Ref. [5]). Initially the rate of hydrogen peroxide production was high but after 20 min it decreased. This may result from the decomposition of hydrogen peroxide produced in the reaction medium as
10 um
well as from a decreased production rate. The increased hydrogen peroxide content can also inhibit cell viability. When the hydrogen peroxide production rate was measured under varying conditions of the reaction system, MV, as the artificial electron accepter, was most effective for the photoproduction of hydrogen peroxide (Fig. 8). Hydrogen peroxide production laminosum cells
in PV-immobilized P.
Table 3 shows photosynthetic oxygen evolution and hydrogen peroxide production activities in free and PV-immobilized P. laminosum cells. PV-immobilized cells showed lower hydrogen peroxide production and photosynthetic oxygen evolution than free cells, but the ratio of hydrogen peroxide production to oxygen evolution in immobilized cells was higher. This implies that although that immobilization process decreased the rate of hydrogen peroxide formation it increased the efficiency of utilization of photosynthetic electron transport. When the ratio, hydrogen peroxide production to oxygen evolution, was calculated from
100 um
Fig. 9. Scanning electron microscopy of A. azollae cells immobilized (A) in PV foam and (B) on cellulosic hollow fibre surfaces.
I . H . PARK et al.
318
Anacystis nidulans experiments [5], it was 0.3 a n d 0.5 at 27°C a n d 45°C, respectively, Electron microscopy o f P V and hollow fibre-immobilized Anabaena cells Figure 9 shows the electron microscopy o f Anabaena cells in PV foam a n d a n cellulosic hollow fibre surfaces. Naturally A. azollae lives symbiotically in the leaf cavity o f Azolla [17]. The surface of cellulosic hollow fibres may resemble s o m e w h a t the n a t u r a l host tissues. The a t t a c h m e n t of cells to fibre surface was better with the cellulosic fibre t h a n with the h y d r o p h o b i c hollow fibres (polysulfone or polypropylene). As s h o w n in Fig. 9, Anabaena cells covered the cellulosic hollow fibre surface almost entirely.
Acknowledgements--l. H. Park thanks the Korea Science and Engineering Foundation for financial support. His permanent address is Department of Biology, Dong-A University, Pusan, Korea. D. O. Hall acknowledges financial support from the EEC. REFERENCES 1. D. O. Hall and K. K. Rao, Chimica Oggi 7, 41~47 (1989). 2. M. D. Trevans and A. L. Mak, Trends Biotechnol. 6, 68-72 (1988). 3. M. Brouers and D. O. Hall, J. Biotechnol. 3, 307 311 (1986).
4. M. A. de la Rosa, K. K. Rao and D. O. Hall, Photobiochem. Photobiophys. 11, 173 187 (1986). 5. I. Morales and F. F. de la Rosa, Solar Energy 43 (6), 373-337 (1989). 6. C. O. P. Patterson and J. Myers, Plant Physiol. 51, 104-109 (1973). 7. R. Y. Stanier, R. Kunisawa, M. Mandel and G. CohenBazire, Bacteriol. Rev. 35, 171 205 (1981). 8. R. W. Castenholtz, Schweiz. Z. Hydrol. 32, 538 551 (1970). 9. M. Brouers, D. J. Shi and D. O. Hall, Methods in Enzymol. 167, 629~i36 (1988). 10. L. Solorzano, Limnol. Oceanogr. 14, 799 801 (1969). 11. W. D. P. Stewart, G. P. Fitzgerald and R. H. Burris, Arch. Microbiol. 62, 336-348 (1968). 12. J. F. Tailing and D. Driver, Proc. lOth Pacific Science Cong. Div. of Technical Information, U.S. Atomic Energy Commission, pp. 142 146 (1961). 13. R. Rozino, W. Rowe and A. Meister, Biochemistry 8, 1066-1075 (1969). 14. W. D. P. Stewart and P. Rowell, Arch. Microbiol. 62, 336-348 (1975). 15. J. W. Newton and J. F. Cavin, Biochim. Biophys. Acta 809, 44 50 (1985). 16. S. Reich, H. Almon and P. Boger, FEMS Microbiol. Lett. 34, 53 56 (1986). 17. D. J. Shi, M. Brouers, D. O. Hall and R. J. Robinson, Planta 172, 298-308 (1989). 18. C. Garbisu, H. Wen, D. O. Hall and J. L. Serra, in M. Baltscheffsky (ed.), Current Research in Photosynthesis, Vol. II, pp. 699 702. Kluwer, Utrecht (1990). 19. Vincenzini, M. Brouers, D. O. Hall and R. Materassi, Photobiochem. Photobiophys. 13, 85-94 (1986).
036(~3199/91 $3.00 + 0.00 Pergamon Press plc. International Association for Hydrogen Energy.
PHOTOPRODUCTION OF HYDROGEN, HYDROGEN PEROXIDE A N D AMMONIA USING IMMOBILIZED CYANOBACTERIA I. H. PARK, K. K. Rho* and D. O. HALL Division of Biosphere Sciences, King's College, London W8 7AH, U.K. (Received for publication 13 December 1990) Abstraet--Anabaena azollae cells were immobilized in synthetic polymer foams and cellulosic hollow fibres. The immobilization of cells resulted in stable photosynthetic oxygen evolution activity and MSX-induced ammonia production. In "trickling-medium" photobioreactors using PV-immobilized Anabaena azollae, continuous hydrogen and ammonia production was observed for one week. In a hollow fibre system, cell attachment to hydrophilic cellulosic hollow fibre was better than with hydrophobic polysulfone and polypropylene hollow fibres. Photoproduction of hydrogen peroxide was observed in free and immobilized thermophilic Phormidium laminosum cells. Characteristics of a "trickling-medium" photobioreactor for continuous production of hydrogen peroxide were also studied.
NOMENCLATURE DCMU MSX MV PU PV RF
dichlorophenyldimethylurea L-methionine-D,L-sulfoximine methyl viologen polyurethane polyvinyl riboflavin
INTRODUCTION Over the past few years, much attention has been given to the design of systems capable of conversion, storage and usage of solar energy into chemicals, such as high energy compounds and valuable secondary metabolites [1-2]. One of the possible ways of storing solar energy is to transform it into chemical potential via production of energy rich compounds such as hydrogen, hydrogen peroxide and ammonia [3-4] using photosynthetic processes. The theoretical maximum efficiency for photosynthetic solar energy conversion, defined as the ratio of stored chemical energy to the incident solar radiation on the photosynthesizer is calculated to be about 13%, and the practical maximum conversion efficiency under optimal condition is 9% on an annual basis (Bolton and Hall, unpublished). However, this maximum conversion efficiency is never reached in the field; for most plant species it seldom exceeds 1% on an annual basis. Therefore there is a need and great interest in developing photobiological/photochemical systems which would mimic the energy conversion processes of natural photo*Author to whom correspondence should be addressed,
synthesis, hopefully with better efficiency. Thus, many photobiological systems comprising whole cells of algae or cyanobacteria, isolated thylakoids, photosynthetic bacterial chromatophores and individual photosystems have been used in the design of photobioreactors for the production of H 2, H202, ATP, NADPH2, NH3 in addition to polysaccharides, oils, etc. [3, 5, 6]. For stability and ease of manipulation it may be advantageous to use the photosynthesiser in an immobilized state in the photobioreactor. Recently many approaches have been made to immobilize cyanobacterial cells for the production of energy-rich compounds and some biochemicals using immobilizing matrices such as PV, PU, alginate etc. [1]. Among these materials, alginate is a good matrix for immobilization, however, it acts as a quencher of nutrient and gas diffusion, and thus synthetic polymers are better suitable for immobilization of algal cells. Although PU and PV easily adsorbed algal cells, the cells may leak out of the foam when the reactor is bubbled with nutrient gas. To avoid this possibility, media flushed with gas can be provided, but in this case the gas supply acts as a limiting factor in the growth and production of cells in the reactor. We report here the results of our studies on immobilized cyanobacteria for the production of H 2, H202 and NH3. The design of novel "trickling-medium photobioreactor" is also described. MATERIALS A N D METHODS Culture o f cyanobacteria Anabaena azollae was grown in medium BG-11 [7] without combined nitrogen under cool white fluorescent lamps (100 #E m 2 s i) with shaking (100 rpm) and 313
I . H . PARK et al.
314
supplied with 5% CO2 at 27°C. Cell growth was determined by chlorophyll concentration in the culture suspension. Thermophilic Phormidium laminosum were grown in medium D [8] under the same conditions as Anabaena except that the temperature was 45°C.
Immobilization of cyanobacteria Immobilization of cyanobacteria in PV and PU polymer foams was performed according to Brouers et al. [9]. Determination of ammonia For chemical determination of ammonia, Anabaena cells were suspended in fresh culture medium with 100 g M MSX. After incubation for 3 h, the cell suspension was centrifuged at 2500 g for 10 rain and the content of ammonia in the supernatant was determined according to Solorzano [10]. For continuous determination of ammonia, 10#mole MSX was injected into the reactor (pulse treatment, about 20 min). The eluate from the reactor was mixed with nine times volume of 1 N NaOH and then passed through an ammonia probe (EIL8002-8, Kent Industrial Measurement, U.K.) for polarographic assay, Nitrogenase activity Nitrogenase activity was determined by acetylene reduction [11] under aerobic or anaerobic conditions. Hydrogen was measured using a gas chromatograph fitted with a thermal conductivity detector, Hydrogen peroxide production Photoproduction of hydrogen peroxide was performed in 50 mM Tricine-KOH (pH 7.5) buffer containGC sampling
~1- ~
~
r
Chlorophyll determination Chlorophyll content was calculated from the absorbance at 665 nm after extraction with 95% methanol [12]. Bioreactor "Trickling-medium" column bioreactor using PVimmobilized A. azollae cells (Fig. 1) was made with a water jacketed Pyrex glass column (18 mm I.D., 280 mm L). The column was filled with 100 pieces of sterilized PV-foam (5 mm cubes). Immobilization was initiated by flushing with free cell culture suspension. Air (101min - l ) and the medium (6.25mlh ~) trickling through the foams were supplied from the top of the column. Illumination was with fluorescent lamps (100 #E m -2 s -1) and temperature was 27°C (Table 1). Hollow fibre bioreactors were composed of cuprophane cellulosic hollow fibre (from Green Cross Medical Industry Co., Korea) or polysulfone hollow fibre (donated by Professor S.-C. Wang, Tianjin Univ., China). The free cell suspension was flushed into the interfacial space of the hollow fibre, the medium flowed
I-'-I
1
Glass beads
V.n
Humidifier
~ ~
Medium
Gas Trap
oP2
l"'c°'<"rt
i pH meter
/-1 PV-immobilized cell O
Photosynthetic oxygen evolution Photosynthetic oxygen evolution activity was determined with Clark type oxygen electrode (Rank Brothers, U.K.) at 27°C with illumination of incandescent light (2000 ~tE m -2 s ~) through an orange filter.
0 P~
Air or Argon gas water bath
ing 10mM NaC1, 5 m M NH4CI and 2 5 # M MV under a supply of pure oxygen [4] at 27°C. Hydrogen peroxide was determined by H202-dependent N A D H oxidation in 0.1 M phosphate buffer (pH 7.5).
~ k
Ammonia probe
T Waste
P3
r P4
~
[
_--.
Fig. 1. Schematic diagram of "trickling-medium" reactor for production of H2 and NH 3 using PV-immobilized A. azollae cells. For H2, stopcock 2 and pump 2 were operated. For NH3, stopcock 1 and pumps l, 3 and 4 were operated.
PHOTOPRODUCTION OF H2, H 2 0 2 AND N H 3 Table 1. Characteristics of trickling-medium bioreactor for hydrogen photoproduction using PV-immobilized Anabaena azollae cells
ae 140
Column size Gas phase volume PV-foam Total column volume Solid and liquid phase volume Cell density/reactor volume Medium flow rate Air supply Chlorophyll content
"~ 100 o ~" ._~ 80 ~ 60 E E 40 < 0
Temperature Light intensity
ID 18mm × L. 280mm cm 3 2.47 g dry wt 64 ml 33
31 c m 3 169/lg Chl ml R 6.25 ml h ' 101 min i l 0.82 mg 4.4 mg Chl g 1dry wt PV-foam 27°C 100#E m--' s
through the lumen side of the fibre and air was supplied through the outer space of the fibre. The column was incubated under the same conditions as the previous glass column bioreactor,
Scanning electron microscopy Immobilized cyanobacterial cells were examined by the critical point drying method. Cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) conraining 6% sucrose (w/v) for 2 h at r o o m temperature, The samples were washed in the same buffer, transferred to 1.0% osmium tetroxide in buffer under the same conditions. The specimens were washed and dehydrated in graded ethanol (50-100%) and dried in a Samdri-780 Critical Point Drying Apparatus (Tousimis Research Corp., U . S . A . ) u s i n g liquid carbon dioxide. Dried specimens were coated in a Sputter Coater (EM-Scope, U.K.) and examined in a model S-510 Scanning Electron Microscope (Hitachi Scientific Instruments Co., Japan) at an accelerating voltage of 15 or 25 kV. RESULTS AND DISCUSSION Cyanobacteria are able to fix atmospheric nitrogen via ATP-dependent nitrogenase activity. Nitrogenase is located in the heterocysts which provide 02 protection for the enzyme. U n d e r normal physiological conditions the nitrogen fixed in the heterocysts is transported to the
d .2
/
b
315
~
~
J
120
i 1
, 2
Free batch culture PV-foam in batch Floating foam Settled foam Mixed* PV-foam in reactor Immersedt Non-immersed (trickling-medium) * From [18]. ? From [191.
Alga
Fig. 2. Stability of MSX-induced ammonia production rate in free (O) and PU-immobilized (Q) A. azollae cells. adjacent vegetative cells as glutamine, which then enters into the cellular metabolic pool. The conversion of a m m o n i u m to glutamine is catalysed by glutamine synthetase [GS] also located in the heterocysts. In the presence of MSX, which is an analogue of glutamate and which functions as an irreversible inhibitor of GS, a m m o n i u m is excreted into the medium [13, 14].
Immobilization of cyanobacteria For immobilization several matrices were used alginate, carrageenan, PU, PV etc. A m o n g these matrices, synthetic polymers appear as best candidates for immobilization because the price is lower than natural polymers and diffusion of nutrients and gases is better. However, some polymers are slightly toxic to the cell. Another problem encountered with polymer foams is that cell entrapment is very loose (although mucilage was excreted and some attachment to the surface of polymers occurs), and the cells can easily leak out from the foam when the reactor is bubbled or stirred. Many studies have described bioreactors using the "immersed system", wherein the immobilized foams are soaked in the medium. Table 2 shows the cell density of the several immobilized and free batch culture systems. It is clear that the cell density in both batch and "tricklingmedium" reactors using immobilized cells was higher than that of free cells, and the cell density of the non-immersed "trickling-medium" reactor reached 1.6-4.4 mg chl g ' dry wt. This is an eight times higher cell density than that of PV-immobilization in a batch system.
Cell density
Anabaena
15/tg Chl cm 3 culture flask volume
Anabaena
0.75 mg Chl g 1 dry wt PV foam 0.31 mg Chl g t dry wt PV foam 0.37 mg Chl g i dry wt PV foam
Phormidium Cyanospira Anabaena
4
Dark Treatment, Day
Table 2. Comparison of cell density in several immobilization systems Type of culture or reactor
L 3
4 #g Chl (5 mm cube PV foam 0 . 7 3 m g C h l g ' dry wt PV foam) 1.6-4.4mg Chl g i dry wt PV foam 62.5-169/~g Chl cm 3 reactor volume 129-380.7/~g Chl ml ~ medium
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Stability o f immobilized cells Immobilized Anabaena cells showed continued stabilities on long storage. We tested the short-term stability of some physiological activity. When the ammonia productivity in the presence of MSX (100ktM) was measured after dark incubation, the rate rapidly decreased in free cells but in PU-immobilized cells, the activity was almost same or even increased after dark incubation (Fig. 2). Figure 3 shows photosynthetic oxygen evolution activities in free and PU-immobilized Anabaena cells during a similar dark treatment, It shows higher stability of PU-immobilized cells compared to the free cells, Continuous production o f ammonia Figure 4 shows continuous ammonia photoproduction in a "trickling-medium" bioreactor using PV-immobilized A. azollae cells. When cells were treated with MSX (10/Lmole pulse treatment, 20min) the ammonia concentration in the eluates reached 500/~M and no cell leakage was observed from the "tricklingmedium" column reactor. Nitrogenase activity and ammonia production were higher after MSX treatment, Newton and Cavins [15] reported that nitrogenase activity in Anabaena spp increased in the presence of MSX, and Reich et al. [16] showed that MSX treatment
27.2ul C2H2/moJh
20
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,
40
50
Fig. 5. Effect of CO on the production ofH 2 by nitrogenase in a "trickling-medium" reactor using PV-immobilized A. azollae cells. Arrow shows CO (4%) addition.
prevented inactivation of nitrogenase by exogenous ammonia in Anabaena spp. Continuous production o f hydrogen by nitrogenase Hydrogen evolution from the "trickling-medium" reactor was tested under anaerobic conditions. Although hydrogen accumulation in the reactor was observed in the absence of an uptake hydrogenase inhibitor (CO), after the first day the hydrogen concentration gradually decreased. When CO was injected into the column reactor, hydrogen evolution increased significantly (Fig. 5). In the presence of CO, hydrogen evolving rate increased for 4 days and thereafter the rate decreased gradually (Fig. 6), Total hydrogen production reached 2 ml H 2 per reactor (total 4 mg Chl) after 6 days (Fig. 7). In studies of hydrogen production by nitrogenase using the cellulosic hollow fibre reactor, hydrogen evolution was observed for 3 days under anaerobic conditions, in the presence of CO. Thus there is a potential for the use of hollow fibre in the production of some chemicals using symbiotic filamentous cyanobacteria, as the separation of products from the cells is easy and there is less chance of contamination in working reactors. Hydrogen peroxide production in free cells o f P. laminosum We have observed light-dependent hydrogen peroxide production in P. laminosum via the Mehler reaction.
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lO 0
' 0
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2
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4
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Fig. 6. H 2 production rate of the "trickling-medium" reactor using PV-immobilized A. azollae cells.
PHOTOPRODUCTION OF H2, H202 AND NH 3 2000
317
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7. Continuous H 2 production in "trickling-medium" reactor (4 mg Chl) using PV-immobilized A. azollae cells. Arrow shows CO (4%) point, Fig.
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30
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Fig. 8. Photoproduction rate of H202 under various conditions in P. laminosum cells. L, light; MV, methylviologen 25 pM; RF, riboflavin, 25/~M; D, dark.
Hydrogen peroxide production rates showed a linear relationship to the chlorophyll concentration and reached 2 1 . 6 p m o l m g --~ C h l h ~ at 27c'C. This value is lower than the rate in Anacystis nidulans ( 3 6 / ~ m o l m g I Chl h l , Ref. [5]). Initially the rate of hydrogen peroxide production was high but after 20 min it decreased. This may result from the decomposition of hydrogen peroxide produced in the reaction medium as
10 um
well as from a decreased production rate. The increased hydrogen peroxide content can also inhibit cell viability. When the hydrogen peroxide production rate was measured under varying conditions of the reaction system, MV, as the artificial electron accepter, was most effective for the photoproduction of hydrogen peroxide (Fig. 8). Hydrogen peroxide production laminosum cells
in PV-immobilized P.
Table 3 shows photosynthetic oxygen evolution and hydrogen peroxide production activities in free and PV-immobilized P. laminosum cells. PV-immobilized cells showed lower hydrogen peroxide production and photosynthetic oxygen evolution than free cells, but the ratio of hydrogen peroxide production to oxygen evolution in immobilized cells was higher. This implies that although that immobilization process decreased the rate of hydrogen peroxide formation it increased the efficiency of utilization of photosynthetic electron transport. When the ratio, hydrogen peroxide production to oxygen evolution, was calculated from
100 um
Fig. 9. Scanning electron microscopy of A. azollae cells immobilized (A) in PV foam and (B) on cellulosic hollow fibre surfaces.
I . H . PARK et al.
318
Anacystis nidulans experiments [5], it was 0.3 a n d 0.5 at 27°C a n d 45°C, respectively, Electron microscopy o f P V and hollow fibre-immobilized Anabaena cells Figure 9 shows the electron microscopy o f Anabaena cells in PV foam a n d a n cellulosic hollow fibre surfaces. Naturally A. azollae lives symbiotically in the leaf cavity o f Azolla [17]. The surface of cellulosic hollow fibres may resemble s o m e w h a t the n a t u r a l host tissues. The a t t a c h m e n t of cells to fibre surface was better with the cellulosic fibre t h a n with the h y d r o p h o b i c hollow fibres (polysulfone or polypropylene). As s h o w n in Fig. 9, Anabaena cells covered the cellulosic hollow fibre surface almost entirely.
Acknowledgements--l. H. Park thanks the Korea Science and Engineering Foundation for financial support. His permanent address is Department of Biology, Dong-A University, Pusan, Korea. D. O. Hall acknowledges financial support from the EEC. REFERENCES 1. D. O. Hall and K. K. Rao, Chimica Oggi 7, 41~47 (1989). 2. M. D. Trevans and A. L. Mak, Trends Biotechnol. 6, 68-72 (1988). 3. M. Brouers and D. O. Hall, J. Biotechnol. 3, 307 311 (1986).
4. M. A. de la Rosa, K. K. Rao and D. O. Hall, Photobiochem. Photobiophys. 11, 173 187 (1986). 5. I. Morales and F. F. de la Rosa, Solar Energy 43 (6), 373-337 (1989). 6. C. O. P. Patterson and J. Myers, Plant Physiol. 51, 104-109 (1973). 7. R. Y. Stanier, R. Kunisawa, M. Mandel and G. CohenBazire, Bacteriol. Rev. 35, 171 205 (1981). 8. R. W. Castenholtz, Schweiz. Z. Hydrol. 32, 538 551 (1970). 9. M. Brouers, D. J. Shi and D. O. Hall, Methods in Enzymol. 167, 629~i36 (1988). 10. L. Solorzano, Limnol. Oceanogr. 14, 799 801 (1969). 11. W. D. P. Stewart, G. P. Fitzgerald and R. H. Burris, Arch. Microbiol. 62, 336-348 (1968). 12. J. F. Tailing and D. Driver, Proc. lOth Pacific Science Cong. Div. of Technical Information, U.S. Atomic Energy Commission, pp. 142 146 (1961). 13. R. Rozino, W. Rowe and A. Meister, Biochemistry 8, 1066-1075 (1969). 14. W. D. P. Stewart and P. Rowell, Arch. Microbiol. 62, 336-348 (1975). 15. J. W. Newton and J. F. Cavin, Biochim. Biophys. Acta 809, 44 50 (1985). 16. S. Reich, H. Almon and P. Boger, FEMS Microbiol. Lett. 34, 53 56 (1986). 17. D. J. Shi, M. Brouers, D. O. Hall and R. J. Robinson, Planta 172, 298-308 (1989). 18. C. Garbisu, H. Wen, D. O. Hall and J. L. Serra, in M. Baltscheffsky (ed.), Current Research in Photosynthesis, Vol. II, pp. 699 702. Kluwer, Utrecht (1990). 19. Vincenzini, M. Brouers, D. O. Hall and R. Materassi, Photobiochem. Photobiophys. 13, 85-94 (1986).