Electron microscopic studies on immobilized growing Chlamydomonas reinhardtii cells Maria J. Wchez, Javier Vigara, In& Garbayo, and Carlos Vilchez Departamento de Quimica y CCMM., Escuela Polittknica Superior, Universidad de Huelva, Huelva, Spain Electronmicroscopicobservationsof Chlamydomonas reinhardtii cells immobilizedin agar beads revealed that many colonies are assembled over the gel sulfate and are enlarged during growth. When the microalga reached the stationary phase of growth, the chlorophyll content in the gel beads remained constant, but the gel sutiace showed microinterstices that allow cell release to the culture medium. The viability was maintained by the cells; 0 1997 by Elsevier Science thus, this system could be interesting in applied processes operating continuously. Inc. Keywords:
Microalgae; immobilization; electron microscopy;
Introduction Immobilization of microbial cells is increasingly applied in biotechnological processes. Since the presence of residual nitrate and/or nitrite, heavy metals, and other chemicals in drinking water is a potential health hazard, the use of immobilized cells for drinking water and wastewater treatment is one of the most interesting areas for investigators.’ When adsorbed or entrapped, microorganisms were used for wastewater treatment. The matrix was first inoculated with the cells which were then allowed to multiply and fill the surface and/or available space.* We previously reported the possibility of using immobilized Chlamydomonas reinhardtii cell systems for nitrite removal from wastewater.3*4 In order to get information concerning the biological viability of these immobilized systems, we have studied using scanning electron microscopy the cell distribution in the growing gel beads and also the evolution of associated photosynthetic and/or respiratory activities.
Materials and methods Organisms and standard culture conditions Wild-type C. reinhardrii strain 21 gr. was grown at 25°C in 1.5 mu phosphate-buffered culture medium pH 7.5 containing 10 mM
Address reprint requests to Prof. Carlos Vflchez, Departamento de Qufmica y CC.MM., Escuela Politkcnica Superior, Universidad de Huelva, Palos de la Frontera. 21819 Huelva, Spain Received 30 November 1995; accepted 10 October 1996
Enzyme and Microbial Technology 21:45-47, 1997 0 1997 by Elsevier Science inc. 655 Avenue of the Americas, New York, NY 10010
KNO, as the nitrogen source.s The standard cultures (about 200 ml) in 250-ml conical flasks were bubbled with air containing 5% (v/v) CO, and continuously illuminated witi white fluorescence lamps (250 kE.rnm2 s-* at the surface of the tube).
Immobilization in agar
of C. reinhardtii cells by entrapment
The cells were harvested at the exponential phase of growth (15 p.g Chl ml-‘), washed and resuspended (0.5-l% w/v) in 20 mu Tritine-NaOH-buffered culture medium pH 7.5 and thoroughly mixed with an equal volume of an agar (Bacteriological agar, ADSA-Micro, Barcelona, Spain) suspension (3% w/v), at 35°C. When the preparation became solid, the agar was cut into small cubes (8 mm3) which were rinsed with fresh culture medium before use. The volume of liquid medium us&d for incubating immobilized cells was 100 ml containing 10% (w/v) of agar blocks.
activity was determined using a Clark-type electrode to measure light-dependent 0, production from the agarentrapped C. reinhardtii cells (ten beads) into 1.5 ml of 20 mu Tricine-NaOH-buffered culture medium p&l 7.5. The measurements were made at 25°C under saturating white light illumination (1,500 p_E mm2 s-l). The respiratory activity was determined by measuring the 0, uptake in the dark by the immobilized cells under the conditions described above.
Chlorophyll was determined by extracting the free cells with acetone. For immobilized cells, the beads wdre extracted with ac-
0141-0229/97/$17.00 PII SOl41-0229(96)00223-2
Papers etone overnight. After removing the nonextracted material, the
absorbance at 652 nm was determined in the supematant (E = 34.5 mg ml-’ cm-‘). Further details are in Vilchez et ~1.~
Cell counting The sample was prepared treating 1 ml cell suspension with ethyl alcohol (50 ~1). The counting plate (Neubauer chamber) was filled with the cell preparation and the number of cells was determined using a Nikon 102 optical microscope. Sample preparation
The beads were prepared as described by Van Neerven et aL6 The beads were washed for 10 min in 0.1 M Na-cacodylate buffer pH 7.3. The first fixation was performed in the same buffer containing 2% (w/v) glutaraldehyde for 3 h. The beads were then washed in the buffer for more than 1 day prior to dehydration in a series of gradually increasing ethanol concentrations: 10, 20,30,40,50,60, 70, 80, 90, and two times 100%. Each step was allowed to equili-
brate for 10 min. The beads were dried using the CO, critical point drying technique. Thin sections of gel were prepared with a knife blade and coated with gold. The sections were then observed with a scanning electron microscope (Hitachi HHS-2R).
Results and discussion Biological
viability of immobilized
The chlorophyll content in the beads shows a lag during the first day of growth (about 15 kg Chl g-’ gel), but increased rapidly up to a maximum value of 90 pg Chl g-’ gel during the exponential growth phase and remained stable in the stationary phase of growth. Free cells in the culture medium were perceptible after the second day of growth. They increased linearly with time (Figure IA). The cell release rate to the medium was constant in the stationary growth phase. This indicated the generation of new cells in the beads and the subsequent release of the oldest ones. Free cells appearing in the culture medium were grown in new medium with nutrients. They showed a very low growth rate; this indicated that they could hardly divide themselves. It is evident that most of the free cells appearing in the medium come from the growth of immobilized cells. All incubations were performed under sterilization conditions, thereby avoiding bacterial contamination. On the other hand, the photosynthetic activity of the immobilized cells decreased with time until a constant value (80%) at the stationary phase of growth (Figure 1B) was obtained. This may be due to the shading effect and diffusion limitations for the release of 0, when cells are growing in the beads.4 The respiratory activity was constant during the experiment, thereby indicating cell viability. These data suggest that after the sixth day of growth, a dynamic equilibrium was reached in the beads which is characterized by a good biological viability of immobilized cells, a constant number of cells in the beads, and a continuous release at constant rate of free cells to the medium. Distribution growth
cells in gel beads with
Apparently the cell distribution in the gel bead stays uniform just after immobilization was performed. The cells are sparsely located at the surface of the gel beads (Figure 2A). After three days of growth, the biomass was concentrated close to the surface of the beads which showed a bumpy 46
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Figure 1 Growth of C. reinhardtii cells immobilized in agar. Immobilized cells were incubated under continuous illumination in culture medium supplemented with 10 mM nitrate. At the indicated times, chlorophyll in the beads (A, pg g-’ gel), free cells in the culture medium (A), light-dependent 0, production (0, pmol mg-’ Chl h-l) and dark-dependent 0, uptake (0, pmol mg-’ Chl h-l) were determined. More details in MATERIALS AND METHODS
aspect (Figure 2B); however, in the center 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 diffusional limitations and a shading effect caused by the external cell colonies over the internal ones.7 This growth pattern shown by immobilized microalgae is in good agreement with the results reported by Wijffels and Tramper8 for the nitrifying bacteria Nitrosomonas europea. immobilized in carrageenan. The micrographs show the cell colonies covered by a thin skin of polymer. Cell division was clearly observed (Figure 3A). Because of the growth of cell colonies on the gel bead surface, many microinterstices appeared when the culture reached the stationary growth phase (Figure 3B). A size for the microinterstices between 5-10 p,m could be calculated from the micrographs which is very similar to the C. reinhardtii wild-type cell size. They probably played an important role in the steady-state of living immobilized cells, thereby allowing the oldest cells to be released from the surface to the medium.
studies on immobilized
M. Vilchez et al.
2b Figure 2 Cell population in the gel beads with growing. Aspect of the gel surface just after immobilization (A) or 72 h of growth (6)
The biomass content in the gel beads, thus, would be constantly maintained at the stationary phase of growth while the immobilized C. reinhardtii cells closing the block surface are continuously regenerated. This is a determinant factor for applied processes using immobilized microalgae cells operating continuously.
Acknowledgments We are thankful for the financial support from the University of Huelva and DGICYT (Research Grant PB93-0735 to Dr. Jose M. Vega). We are also grateful to J.M.V. for critically reading the manuscript.
Figure 3 Cell growth on the surface of the gel beads. Cellular division occurred under a thin skin of polymer (A) and microinterstice in the gel surface (B)
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