Appl Microbiol Biotechnol (1989) 31:55-58
Applied Microbiology Biotechnology © Springer-Verlag 1989
Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii Francisco Santos-Rosa and Francisco Galvfin Departamento de Bioqulmica Vegetal y Biologia Molecular, Facultad de Quimica, Apartado 553, Universidad de Sevilla, 41080 Sevilla, Spain
Summary. Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of Lmethionine-D,L-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 ~tmol NH~-.mg chlorophyll (Chl) -1, with an average production rate of 14 ~tmol NH4+.mg Ch1-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 gmol NH + -mg Ch1-1 and 6.7 ~tmol NH~-.mg Chl -~ per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.
Introduction
The photosynthetic reduction of nitrite to ammonium and its incorporation into carbon skeletons in green algae occurs in two steps: (a) nitrite is reduced to ammonium in a six electron reaction catalysed by the metalloenzyme ferredoxin-nitrite reductase (Losada et al. 1981); (b) incorporation of ammonium into carbon skeletons, which occurs via the glutamine synthetase-glutamate synOffprint requests to: F. Galvfin
thase cycle (Lea and Miflin 1975; Vega et al. 1987). The addition of L-methionine-D,I.-sulphoximine (MSX), which inhibits glutamine synthetase activity, induces, after a lag period of 2 h, effective ammonium excretion into the medium (Cullimore and Sims 1981; Hipkin et al. 1982; Florencio and Vega 1983). The effect of several relevant environmental factors influencing the ammonium photoproduction rate might be investigated to improve the yield of this process. In this respect, strain selection of a mutant with maximal expression of nitrite reductase activity (Sosa et al. 1978), optimal medium pH for nitrite uptake and photoreduction (C6rdoba et al. 1986), and overcoming the antagonistic effects of ammonium concentration on nitrite assimilation are of relevant interest. Since 1978 immobilization processes have emerged as an important tool for increasing the longevity of photosynthetic cells as biocatalysts. There is some evidence that immobilization of cells may lead to increased yields in the biotransformation of natural products (Brodelius 1985; Brouers and Hall 1986). Entrapment in alginate beads seems to be adequate for immobilization of photosynthetic cells because it is a transparent material which preserves cellular viability and stabilizes its biocatalysts properties (Tamponet et al. 1985; Bailliez et al. 1986; Dainty et al. 1986). This technique has been commonly used for immobilization of microalgae and applied to continuous photoproduction processes (Robinson et al. 1986). In this paper, results are reported on the effect of MSX on ammonium assimilation and the effect of ammonium on nitrite uptake. The photoproduction of ammonium from nitrite by MSXtreated free-living and immobilized cells of C. reinhardtii is shown.
F. Santos-Rosa and F. Galvfin: Ammonium photoproduction by C. reinhardtii cells
56
Materials and methods
extracted with acetone at 60 ° C in a ground glass homogeniser by hand disruption of the beads for 5 min. Nitrite was estimated by the diazo-colorimetric assay of Snell and Snell (1949) and ammonium was measured by the method of Solorzano (1969).
Organism and growth conditions. Chlamydomonas reinhardtii, mutant straint 104 (Fernandez and Matagne 1984), was grown at 25°C in a liquid medium (Sueoka et al. 1967) containing 6 mM KNO2 as the nitrogen source. Cultures were bubbled with air supplemented with 5% (v/v) CO2 and continuously illuminated with white fluorescent lamps (50 W.m-2). The ceils were used for experiments in their exponential phase of growth.
Results and discussion
Ammonium photoproduction from nitrite using MSX to block glutamine synthetase has been reported in free-living C. reinhardtii cells (Florencio and Vega 1983). In our experiments, excretion of ammonium into the medium began 2 h after the addition of 1 m M MSX, once the blockage of glutamine synthetase occurred, and this inactivation was prolonged for at least 24 h. It is interesting to emphasize that a significant increase of nitrite reductase activity was observed during ammonium excretion, and if MSX and nitrite were restored in the medium every 24 h this process could be prolonged. Similar behaviour was observed using C. reinhardtii cells immobilized by entrapment in Ca alginate with a lag for ammonium excretion around 3 h, probably because of the diffusional restriction of MSX throughout the gel (data not shown). Ammonium has an antagonistic effect on the assimilation of nitrite by C. reinhardtii cells by promoting rapid inhibition of nitrite uptake and repression of nitrite reductase synthesis (Guerrero et al. 1981). The presence of MSX in the medium makes C. reinhardtii cells less sensitive to this effect (Cullimore and Sims 1981; Florencio and Vega 1983). However, when the concentration of
Cell immobilization. A 6% cell suspension (w/v) in 20 m M Tricine-NaOH (pH 8)-buffered medium was mixed (1 : 1 (v/v) ratio) with 6% (w/v) sodium alginate (from Macrocystis pyrifera, Sigma, St. Louis, USA) solution (pH 6.0-7.5; viscosity ca. 2500 centipoises) and the mixture added dropwise into a 0.1 M CaClz solution at 4 ° C to form beads of 3 mm diameter. The beads were ready for use after at least 5 h. Ammonium photoproduction. The cells or beads were incubated in 250-ml open conical flasks at 25°C with continuous illumination from white fluorescent lamps (50 W.m -2) and stirring with air bubbled at the bottom of the flasks (12 1-h-I); 15 m M K-phosphate (pH 7.5)-buffered medium for free cells or 20 mM Tricine-NaOH (pH 8.0)-buffered medium for immobilized ones were used. The free cells or beads were incubated for 2 or 3 h in medium containing 1 m M MSX, prior to addition of 6 m M KNO2.
Enzyme activities. All enzymic assays were performed in vivo using cells made permeable by treatment with toluene. Immobilized cells were released by shaking the alginate beads in 0.5 M K-phosphate solution until complete degradation of the gel occurred. The assay of reduced methyl viologen-nitrite reductase was performed as described by Vega et al. (1980). The glutamine synthetase was measured by its transferase activity, following colorimetrically the 7/-glutamyl-hydroxamate formed during the reaction according to Shapiro and Stadtman (1970).
Analytical determinations. Chlorophyll (Chl) was determined by the method of Arnon (1949). For immobilized cells Chl was
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Fig. 1. Effect of ammonium in the medium on nitrite consumption by L-methionineD,L-sulphoximine (MSX)-treated free-living Chlamydomonas reinhardtii ceils. A cell suspension of 30 ixg chlorophyll (Chl).m1-1 was used. After 24 h ammonium excretion, 6 m M KNO2 and 1 m M MSX were added, and after 48 h the cells were harvested, washed and resuspended in fresh standard medium containing 6 m M KNO2 and 1 m M MSX. Nitrite and ammmonium were determined in the medium at the times indicated. Other experimental conditions are given in Materials and methods
F. Santos-Rosa and F. Galvfin: Ammonium photoproduction by C. reinhardtii cells
ammonium in the medium reached 8 m M or higher, nitrite consumption by MSX-treated cells was completely inhibited (Fig. 1). When the cells were removed and resuspended in fresh medium containing 6 m M KNO2 and 1 m M MSX, nitrite was again consumed and ammonium excretion restored (Fig. 1), so a discontinuous process can operate under these conditions. A similar performance was attained when C. reinhardtii cells entrapped in Ca alginate were involved in such experiments (data not shown). Ammonium photoproduction by MSX-treated C. reinhardtii cells has been conducted using a discontinuous batch operation which involved ammonium removal from the medium, when required, to avoid nitrite uptake inhibition (Fig. 2). A stoichiometric bioconversion from nitrite to ammonium was exhibited over 96 h and 18 m M of ammonium was accumulated (Fig. 2A). The rate of nitrite assimilation increased during the first 48 h, which is consistent with the increase observed in nitrite reductase activity. Then a signifi-
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Fig. 2. Discontinuous ammonium photoproduction from nitrite by MSX-treated free-living (A) and immobilized (B) C. reinhardtii cells. A cell suspension of 28 ~tg Chl-ml-~ (A) or entrapped cells in Ca alginate (210 txg Chl.g beads-1) suspended at 10% (w/v) in the appropiate standard medium (B) were used. At each point the cells or beads were harvested, washed and resuspended in fresh standard medium containing 6 mM KNO2 and 1 m M MSX. Nitrite and ammonium were determined in the medium at the times indicated. Other experimental conditions are given in Materials and methods
57
cant decrase of nitrate uptake took place, since cell degradation occurs under prolonged restriction of ammonium assimilation. Immobilized cells were used under similar conditions to those described for the free-living system with 28 m M ammonium accumulated at the end of the process (Fig. 2B). The rate of nitrite assimilation increased significantly from 48 h to 84 h which promoted a medium renovation every 12 h instead of every 24 h for the free system. The biocatalytic performance of free cells showed an average production rate of 6.7 l.tmol NH + . mg C h l - ~ per hour and a productivity of 650 ~tmol N H + . m g C h i - ] , calculated on the basis of the initial Chl concentration, while immobilized cells raised 14 ~tmol NH~-.mg Ch1-1 per hour and 1300 ~tmol NH + .mg Ch1-1. It can be concluced that immobilization favours the nitrite uptake rate of entrapped cells which have about a 2-fold higher ammonium photoproduction rate than free-living ones. This immobilization yield is consistent with that of Bailliez et al. (1985) who found a marked improvement in hydrocarbon production when Botryococcus braunii cells were immobilized in alginate. High yields of ammonium production were obtained from cyanobacteria immobilized in polyvinyl foam, and a similar yield increase was observed when the membrane permeability of cells was altered by acetone pretreatment, whereas that of untreated cells remained low. This suggests that the increase in ammonium production by immobilization in polyvinyl foam is associated with changes in membrane permeability induced by cell-matrix interactions (Brouers and Hall 1986). In this way, we have observed that C. reinhardtii cells after 24 h immobilization, treated and untreated with toluene, show the same permeability to an in vivo nitrite reductase assay. The results reported in this paper demonstrate the biocatalytic advantages of immobilized cells over free-living ones when MSX-treated C. reinhardtii cells are used for ammonium photoproduction from nitrite. At an operational level, this information might be useful, but if immobilization technology is to advance it seems likely that a greater understanding of the actual effects of immobilization is required. Acknowledgements. This work has been supported by Research Grant no. BT87-0028-C02 from the Comisi6n Interministerial de Ciencia y Tecnologia. The authors thank Mrs. Rosa Maria Reina for helpful secretarial assistance.
References Arnon DI (1949) Copper enzymes in isolated chloroplasts" polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1-15
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F. Santos-Rosa and F. Galv~n: Ammonium photoproduction by C. reinhardtii cells
Bailliez C, Largeau C, Casadevall E (1985) Growth and hydrocarbon production of Botryococcus braunii immobilized in calcium alginate gel. Appl Microbiol Biotechnol 23:99105 Bailliez C, Largeau C, Casadevall E (1986) Immobilization of Botryococcus braunii in alginate: influence on chlorophyll content, photosynthetic activity and degeneration during batch cultures. Appl Microbiol Biotechnol 23:361-366 Brodelius P (1985) The potential role of immobilization in plant cell biotechnology. Trends Biotechnol 3:280-285 Brouers M, Hall DO (1986) Ammonia and hydrogen production by immobilized cyanobacteria. J Biotechnol 3:307321 Crrdoba F, Cgrdenas J, Fernandez E (1986) Kinetic characterization of nitrite uptake and reduction by Chlamydomonas reinhardtii. Plant Physiol 82:904-908 Cullimore JV, Sims AP (1981) Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation. Planta 153:18-24 Dainty JG, Goulding KH, Robinson RP, Simpkins I, Trevan MD (1986) Stability of alginate immobilized algal cells. Biotechnol Bioeng 28:210-216 Fernandez E, Matagne R (1984) Genetic analysis of nitrate reductase deficient mutants in Chlamydomonas reinhardtii. Curt Genet 8:635-640 Florencio FJ, Vega JM (1983) Utilization of nitrate, nitrite and ammonium by Chlamydomonas reinhardtii. Photoproduction of ammonium. Planta 158:288-293 Guerrero MG, Vega JM, Losada M (1981) The assimilatory nitrate-reducing system and its regulation. Ann Rev Plant Physiol 32:169-204 Hipkin CR, Everest SA, Ress TAU, Syrett PJ (1982) Ammonium generation by nitrogen-starved cultures of Chlamydomonas reinhardtii. Planta 154:587-592 Lea PJ, Miflin BJ (1975) The occurrence of glutamate synthase in algae. Biochem Biophys Res Commun 64:856-862
Losada A, Guerrero MG, Vega JM (1981) The assimilatory reduction of nitrate. In: Bothe H, Trebst A (eds) Biology of inorganic nitrogen and sulfur. Springer, Berlin, Heidelberg, New York, pp 30-63 Robinson PK, Mak AL, Trevan MD (1986) Immobilized algae: a review. Process Biochem August: 122-127 Shapiro B, Stadtman ER (1970) Glutamine synthetase (E. coli). Methods Enzymol 17A:910-936 Snell FD, Snell CT (1949) Corolimetric methods of analysis, vol. 2. Van Nostrand, New York, pp 804-805 Solorzano L (1969) Determination of ammonia in natural waters by the phenol-hypochlorite method. Limnol Oceoanogr 14:799-801 Sosa FM, Ortega T, Barea JL (1978) Mutants from Chlamydomonas reinhardtii affected in their nitrate assimilation capability. Plant Sci Lett 11:51-58 Sueoka N, Chian KS, Kates JR (1967) Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardtii. I. Isotopic transfer experiments with a strain producing eight zoospores. J Mol Biol 25:47-66 Tamponet C, Constantino F, Barbotin JN, Calvayrac R (1985) Cytological and physiological behaviour of Euglena gracilis cells entrapped in a calcium alginate gel. Physiol Plant 63:277-283 Vega JM, C/trdenas J, Losada M (1980) Ferredoxin-nitrite reductase. Methods Enzymol 69:255-270 Vega JM, Gotor C, Menacho A (1987) Enzymology of the assimilation of ammonium by the green alga Chlamydomonas reinhardtii. In: Ullrich WR, Aparicio PJ, Castillo F (eds) Inorganic nitrogen metabolism. Springer, Berlin, Heidelberg, New York, pp 132-136
Received July 11 1988/Accepted January 18 1989