R.H. Wijffels, R.M. Buitelaar, C. Bucke and J. Tramper (Eds) Immobilized Cells: Basics and Applications © 1996 Elsevier Science B.V. All rights reserved.
Nitrate uptake by immobilized growing Chlamydomonas reinhardtii cells I. Garbayo', C. B^aban^ M.V. Lobato* and C. Vilchez^ 'Departamento de Qufmica, Escuela Polit&nica Superior, Universidad de Huelva, 21819 Huelva, Spain department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen AB9 2UE, Scotland Summary Nitrate uptake by growing cells of the green microalgae Chlamydomonas reinhardtii immobilized in calcium alginate has been studied. Beads containing cells and placed into an airiift loop reactor reached a steady-state phase after 5-6 days of exponential growth. Then, a maximum cell loading of 120 ^g Chl.g'gel was found, and nitrate was consumed at a rate of 1000 nmol.mg'Chl.h"^. Cell release at the end of the exponential growth phase was observed and measured, revealing cell growth in the colonies close to bead surface. Thus, although cell loading in the beads remained constant, measurements of oxygen evolution showed that the immobilized cells were viable at the steady-state. These results suggest that grown immobilized C. reinhardtii cells could be used as stable and viable cell system in continuous processes for contaminant removal. Introduction The presence of inorganic compounds in water constitutes an important problem since the human health can be directly affected. It is well known that conventional treatments for inorganic nitrogen removal use to be expensive and less effective than new methods that involve biological systems. Photosynthetic microalgae are able to consume inorganic nitrogen compounds by an assimilatory pathway which includes the following enzymes that reduce nitrite to ammonium: nitrate reductase (NiR), nitrite reductase (Nir), glutamine synthetase (GS) and glutamate synthase (GOGAT). Nitrate is reduced to ammonium by NiR and Nir, being then incorporated into carbon skeletons by the GS-GOGAT cycle. Nitrite and ammonium follow identical sequence, starting their assimilation by the action of their corresponding assimilatory enzymes (1). On the other hand, immobilized microalgae have been used in biotechnological processes because of their high stability and yields for many biological conversions. In addition, immobilization improves the handling of microorganisms and the process control in bioreactors (2). We have previously reported the use of immobilized C. reinhardtii cells for nitrite uptake in airlift reactors (3), and in this report nitrate uptake by growing immobilized cells of C. reinhardtii is studied, discussing that it could be an interesting system to be used for nitrate
411 removal in bioreactors and, consequently, for water purification processes. Materials and methods Organisms and standard culture conditions Chlamydomonas reinhardtii, wild type, strain 21 gr, was grown at 25''C in 15 mM phosphate (pH 7.5) buffered culture medium containing 10 mM KNO3 as nitrogen source. Standard cultures, in 250-ml conical flasks, were bubbled with air containing 5% (vol/vol) CO2 and continuously illuminated with white fluorescence lamps (250 fiE.m'^.s'^ at the surface of the flask). Cells were harvested during exponential growth phase (15 fig Chl.ml"^) by centrifiigation at 5,000 g for 5 min. Cell immobilization by entrapment in alginate Cells were harvested, washed, and resuspended (0.5-1%, wt/vol) in 20 mM Tricine-NaOH (pH 8,0) buffered culture medium, and were thoroughly mixed with an equal volume of an alginate solution (4%, wt/vol) prepared by mixing 4% alginic acid and 2% alginate sodium salt and NaOH to reach pH 6.5-7.5. The final viscosity (7,000 cp) depended on the proportion of alginic acid and alginate mixed. Beads of 2-3 mm diameter were obtained by dropping the alginate cell mixture into a solution of 0.1 M CaCla or BaCl2 at 4°C, and after 5 h they were rinsed with fresh culture medium and were ready for use. For immobilized cells, a rate of 5% (weight of beads/volume of culture medium) was used in the experiments. Measurement ofphotosynthetic and respiratory activities Photosynthetic activity was determined using a Clark-type electrode to measure the lightdependent O2 production from the alginated-entrapped C reinhardtii cells (10 beads) into 1.5 ml of 20 mM Tricine-NaOH (pH 8.0) buffered culture medium. The measurements were made at 25°C under saturating white light illumination (1,500 /xE.m'^.s"^). Respiratory activity was determined by measuring the O2 uptake in dark by the immobilized cells under the conditions above described. Equipment Nitrate uptake experiments were carried out at 25° C, in an airlift reactor of transparent glass with an internal loop and a useful volume of 1.3 dm^ (Fig. 1). The cells were continuously illuminated with white light (50 W.m"^ at the reactor surface), and the bed was fluidized with air only. Analytical determinations Chlorophyll (Chi) was determined by extracting the free cells with acetone. For immobilized cells, the beads were extracted with acetone overnight. After removing the non-extracted material by centrifiigation, the absorbance at 652 nm was determined in the supernatant (e = 34.5 mg ^ml.cm^). More details as described in Vilchez et al. (5). Optical density was used as cell density indicator in the culture medium, and was determined at 680 nm. Nitrate in the medium was determined according to the method of Cawse (6).
412 Results and discussion The study of nitrate uptake by immobilized C. reinhardtii cells in a continuous flow reactor is the final goal of our investigation. We have previously reported that C. reinhardtii cells could be an interesting system suitable to be used for nitrite removal processes (4). The characterization of C reinhardtii cells immobilized in calcium alginate for nitrate uptake showed that they could be used in a wide range of different environmental conditions, maintaining high capacity of nitrogen consumption (data not shown). Alginate provides the cells with special protection against pH or temperature changes in the culture medium, in comparison with the answer of freely suspended cells under identical conditions (4). In this report, cells were grown in an airlift loop reactor (Fig. 1), operating in discontinuous mode, and nitrate uptake, biological activities and cell growth were followed. Main reactor parameters are described in Table 1. The reactor was filled up with standard culture medium suplemented in 1 mM KNO3. An initial cell loading of 40 /ig Chl.g' was immobilized, because higher values did not allow the cells to reach the levels of photosynthetic and respiratory activities observed for the freely suspended ones growing under standard conditions (4). Thus, initial nitrate uptake rate decreased as function of the chlorophyll content in the beads (data not shown). Exponential growth phase started at the third day of growing, reaching a steady-state phase sustained between the days 8 and 10, as can be concluded from the cell loading data (Fig. 2). Medium
Figure 1. Reactor configuration for nitrate uptake experiments
T i m e (days) Figure 2. Nitrate uptake by growing immobilized cells of C. reinhardtii in an airlift loop reactor. When indicated, cell loading ( • ) and nitrate in the culture medium ( • ) were determined. Cell loading is expressed as Mg Chl.g'gel and nitrate in the medium as mM.
Table 1 Airlift loop reactor Parameter
Two arms, internal loop
Nitrate consumption at the end of the exponential growth phase was observed. It was sustained for three days, and decreased becoming zero when cell loading also decreased. Since cell release ocurred into the medium and free cell growth in the culture medium took then place (see optical density, Table 2), cell growth was then not observed in the beads because both substrate and light limitations avoided it. Chlorophyll inside the beads started to decrease and nitrate uptake rate became almost zero. It is important to note two interesting facts when exponential growth phase was finishing: 1. Cell loading in the beads remained constant for three days 2. In parallel, cell release from the beads increased Although the chlorophyll data indicates that immobilized biomass does not change, since the number of freely suspended cells increased into the culture medium cell growth in the beads could be taking place. In that way, new cells would occupy the place leaved by the old ones, which would be released into the medium. We have previously reported for C reinhardtii cells immobilized in agar that the growth biomass is concentrated close to the surface of the beads (7). However, in the centre of the gel beads the cell population did not increase, probably because the availability of nutrients and light for the cells was very low due to diffussional limitations and to shading effect caused by the external colonies over the internal ones (8). Because of the cell growth on the gel bead surface, many micro-insterticies appeared when the culture reached the stationary growth phase (7), probably playing an important role in the steady-state of living immobilized cells, allowing the oldest cells be released into the medium. We conclude that immobilized C reinhardtii grown cells could be an interesting system for nitrate uptake continuous processes. Table 2 Photosynthetic activity (/xmol Oj.mg'Chl.h*) of immobilized growing cells of C. reinhardtii and optical density (680 nm) in an airlift loop reactor Time (h)
Acknowledgements We thank to University of Huelva and Basical and Chemical Industries Association (AIQB) the finantial supporting which allowed our participation in the meeting.
415 References 1 Vega JM, Menacho A, Ledn J. Trends Photochem Photobiol 1991; 2: 69-111 2 Rao KK, Hall DO. In: CSIC, ed. Trends in Photosynthesis Research. Advanced Course. Palma de Mallorca, 1990; 153-158. 3 Vilchez C, Vega JM. Enzyme Microb Technol 1995; 17: 386-390 4 Vflchez C, Vega JM. Appl Microbiol Biotechnol 1994; 41: 137-141 5 Vilchez C, Galv^ F, Vega JM. Appl Microbiol Biotechnol 1991; 35: 716-719 6 Cawse PA. Analyst 1967; 92: 311-315. 7 Vilchez MJ, Vigara J, Garbayo I, Vflchez C. Enzyme Microb Technol. Submitted 8 Wada M, Kato J, Chibata I. J Ferment Technol 1980; 58: 327-331