Glycerol photoproduction by free and Ca-alginate entrapped cells of Chlamydomonas reinhardtii

ELSEVIER

Journal of Biotechnology 42 (1995) 61-67

Glycerol photoproduction by free and Ca-alginate entrapped cells of Chlamydomonas reinhardtii R. L&n, F. Galvfin Departamento

de Bioquimica

*

Vegetal y Biologh Molecular, Facultad de Quimica, Apartado Spain

553, Universidad de Sevilla, 41080-Sevilla,

Accepted 2 May 1995

Abstract For the purpose of saving energy and raw materials required in glycerol production, an immobilized cell system was prepared and its performance in a continuous bubble column bioreactor was evaluated. Cells of the freshwater green alga Chlamydomonas reinhardtii, that excretes glycerol into the medium in response to an osmotic shock (200-250 mM NaCl), were immobilized in various supports. Based on physical and chemical properties of the Ca-alginate beads, this immobilization method was improved and the operation of the continuous system was partially optimized. The performance of this system was evaluated by measuring the glycerol photoproduction for a period of 21 d, showing a glycerol concentration of about 7 g I-’ and a productivity of about 2 g I-’ d-’ corresponding to 23 mg per mg chlorophyll per d. Keywords: Alginate-immobilized

cell; Bubble column; Chlamydomonas reinhardtii; Glycerol photoproduction

1. Introduction

One goal of photochemistry is to emulate the light-driven transport of green plant photosynthesis. Thus, solar energy could be used to generate products of high value such as fuels, industrial and pharmaceutical materials. Glycerol is mainly produced by chemical synthesis from the petrochemical industry or it is a by-product of the soap industry. Limited petrol reserves and replacement of soaps by detergents make the biochemical process for glycerol photoproduction an ex-

* Corresponding author.

tremely attractive possibility (Chen and Chi, 1981). Glycerol is an important osmoregulatory solute in some halophilic green algae such as Dunaliellu (Ben-Amotz and Avron, 1981) and several species of Chlamydomonas (Ben-Amotz and Avron, 1983). Dunaliellu is a marine alga well known for its potential to produce glycerol, but so far all the processes proposed for industrial glycerol production have assumed that there is no leakage of the polyol by this microorganism (Borowitzka and Borowitzka, 1988). Different from Dunaliellu and other halotolerant algae, the freshwater Chlumydomonas reinhardtii excretes the majority of glycerol into the medium at a low saline concentration (Lebn and Galvin, 1994). In contrast to

0168-1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1656(95)00069-O

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green algae that have the added advantage of requiring only cheap inorganic sources and light, osmotolerant yeast produce extracellular glycerol from glucose in alkaline solutions or in the presence of sodium bisulfite (Vijalkishore and Karanth, 1987). Immobilized microorganisms in natural or synthetic polymers are a simple and effective system for biotransformation and biosynthesis of secundary metabolites (Trevan and Mak, 1988; Tramper, 1990; Rao and Hall, 1992). In recent years, entrapment of cells within spheres of Caalginate has became the most widely used technique for immobilizing living cells. The advantages of alginate for cell immobilization include its low price and the capacity to produce transparent beads ideal for entrapping whole photosynthetic cells (Smidsrod and Skjak-Braek, 1990). Entrapment of C. reinhardtii cells in alginate beads for ammonium and glycolate photoproduction has been studied in our laboratory (SantosRosa and Gal&n, 1989; Vilchez et al., 1991), and in all cases biological viability of the immobilized cells was maintained for long periods of time (Santos-Rosa et al., 1989a). Immobilization techniques by entrapment in matrix alginate have also been used to improve extracellular glycerol production by Dunaliella tertiolecta (Grizeau and Navarro, 1986) and yeasts (Bispin et al., 1990). In this work, using a bubble column bioreactor, we have studied the glycerol photoproduction by C. reinhardtii cells entrapped in Ca-alginate beads, which may be of biotechnological interest for industrial activities demanding glycerol as a raw material.

2. Materials

and methods

2. I. Chemicals

Alginic acid and alginate sodium salt (medium viscosity) from Macrocystis pyriyeru and Tricine were obtained from Sigma, St. Louis, MO, USA. Agar from Difco, Detroit, MI, USA. Glycerokinase and glycerol-3-phosphate dehydrogenase from Boehringer, Mannheim, Germany. All other

of Biotechnology 42 (1995) 61-67

reagents were supplied Germany.

by Merck, Darmstadt,

2.2. Microorganism and culture conditions Chlamydomonas reinhardtii, wild strain 21 gr from Dr. R. Sager (Sidney Farber Cancer Center, Boston, MA) and Monoraphidium braunii and Chlorella vulgaris, wild strains from Sammlung von Algenkulturen (Pflanzenphysiologisches Insitut der Universitat Gottingen, Deustschland), were grown at 25°C in 15 mM K-phosphate (pH 7.0) buffered medium (Sueoka et al., 1967) containing 10 mM KNO, as nitrogen source. The standard cultures in 250-ml conical and l-l Roux flasks, were bubbled with air containing 5% (v/v) CO,, and continuously illuminated with cool white and daylight fluorescent lamps (150 PE rnp2 s-l, at the surface of the flasks). All cultures were grown in axenic conditions.

2.3. Cell immobilization The cells were harvested at the beginning of the exponential phase (15-20 pg ml-’ chlorophyll (Chl)), washed and resuspended in a 20 mM Tricine-NaOH (pH 8.0) buffered culture medium (6%, w/v). They were thoroughly mixed with an equal volume of a sterile alginate solution (6%, w/v), prepared by autoclaving (20 min a 120°C) a mix of 2% alginic acid (pH 6.5-7.5 adjusted with NaOH) and 4% alginate sodium salt (medium viscosity). The final viscosity, about 7000 centipoise, depended on the proportion of the mixed alginic acid and alginate sodium salt, and on the autoclaving process. Beads of about 3 mm diameter were obtained by dropping the alginate cell mixture into a solution of 0.1 M CaCl, or 0.1 M BaCl, at 4°C. After 5 h they were rinsed with fresh culture medium and were ready for use (Smidsrod and Skjak-Braek, 1990). The whole process was carried out in a laminar flow cabin. Immobilization in agar was carried out as described above for alginate, except that 4% (w/v) agar cooled to 50°C after the sterilizing process was used, then the beads were obtained by dropping the agar cell mixture (1:l) into vegetable oil

R. Lebn, F. Gal&

/Journal

3 I 12)

I 4 5

6 DL-

7

a

12

-

I

Fig. 1. Bubble column (250 ml) for glycerol photoproduction with immobilized cells. Numerical notation represents: 1, air reservoir; 2, CO* reservoir; 3, flow meters; 4, gas inlet; 5, feed reservoir; 6, pump; 7, influent medium; 8, effluent medium; 9, sampling; 10, effluent reservoir; 11, gas sparger; 12, illumination system.

at 4°C. After 5 h they were rinsed with fresh culture medium and were ready for use. 2.4. Standard conditions for glycerol photoproduction

The cells were harvested at the beginning of the exponential phase (15-20 pg ml-’ Chl), washed and resuspended in a 15 mM K-phosphate (pH 7.5) buffered culture medium (about 20 pg ml-’ Chl) containing 10 mM KNO, and 200 mM NaCl. For the immobilized system, the beads (about 150 pg Chl g-‘) were resuspended in a 20 mM Tricine-NaOH (pH 8.0) buffered culture medium (15%, w/v> containing 10 mM KNO, and 250 mM NaCl. For long-term experiments, 10 mM CaCl, was added to the medium to prevent bead degradation. The corresponding suspensions, in a 250-ml conical flask (Fig. l), were bubbled at 25°C with air containing 5% (v/v> CO, and were continuously illuminated with cool white and daylight fluorescent lamps. The light intensity was 500 I.LE mP2 s-i, at the surface of the culture. Sterile conditions were maintained for the whole process. 2.5. Analytical determinations Free cell chlorophyll was determined by heating and extracting with acetone &non, 19491,

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63

using an absorbance coefficient at 652 nm of 34.5 mgg’ ml cm- ‘. For immobilized cells, chlorophyll was determined by heating and extracting with methanol (Marker, 1972), using an absorbance coefficient at 665 nm of 76 mg- ’ ml cm-‘. Glycerol was determined enzymatically by the UV-method of Wieland (19841, using glycerokinase and glycerol-3-phosphate dehydrogenase. Excreted glycerol (extracellular) was determined in 2-ml aliquots of cell free medium. Accumulated glycerol was determined in similar aliquots from cells exhaustively washed with isotonic medium, followed by boiling for 10 min to extract the intracellular glycerol.

3. Results and discussion 3.1. Extracellular and intracellular glycerol photoproduction by free and immobilized green algae The freshwater green alga C. reinhardtii tolerates up to 200 mM of saline concentration (Le6n and Galvin, 1994). In response to this osmotic shock, the cells accumulated during the first 24 h the 15% of the total glycerol synthetized as osmoregulatory metabolite, equivalent to 1 mg mg - ’ Chl, to provide the corresponding osmotic balance (Table 1). After this period of time, all the synthetized glycerol was excreted into the medium, reaching a concentration of 4 g 1-l at 120 h. Other freshwater green alga tested, such as Monoraphidium braunii and Chlorella vulgaris, did not accumulate and excrete glycerol (Table 1). In contrast, halotolerant Chlamydomonas species, such as C. pulsatilla, accumulated intracellular glycerol in response to osmotic stress in the range of 350-2000 mM NaCl (Ben-Amotz and Avron, 1983). The halotolerant green alga Dunaliella is able to live in the presence of 5 M NaCl, accumulating glycerol up to 45 mg mg-’ Chl after 10 d, but did not excrete detectable glycerol (BenAmotz and Avron, 1981). Glycerol excretion by Ca-alginate entrapped C. reinhardtii cells in the presence of 250 mM NaCl was about 2-fold higher than that of the free ones (Table 1). This immobilization yield is

R. Le&, F. Gal&

64

Table 1 Extracellular and intracellular glycerol free and immobilized green algae Green

algae

Free cells C. reinhardtii M. braunii C. vulgaris D. salina a Immobilized cells C. reinhardtii D. tertiolecta ’

/Journal

photoproduction

Extracellular (gGlyl-‘)

Intracellular (mg Gly mg

4 0 0 0

1 0 0 45 (10 d, 5 M NaCI)

7 5 (4 d, 4 M NaCl)

nd nd

of Biotechnology 42 (1995) 61-67

by

’ Chl)

NaCl Concentration

Gly, glycerol; d, day; nd, not determined. a Ben-Amotz and Avron, 1981; h Grizeau and Navarro, 1986. Free and immobilized cells in Ca-alginate were resuspended in the corresponding liquid medium for glycerol photoproduction in the standard conditions described in Materials and methods. Except when otherwise indicated, the glycerol produced was determined after 5 days.

consistent whith the data of Bailliez et al. (1985) for hydrocarbon photoproduction by Botryococcus braunii, of Santos-Rosa and Galvan (1989) for ammonium photoproduction by C. reinhardtii, and of Vilchez et al. (1991) for glycolate photoproduction by C. reinhardtii. In general, the production increase by the immobilized system is associated to changes in membrane permeability induced by cell-matrix interactions (Brouers and Hall, 1986; Santos-Rosa et al., 1989a). Immobilization also improves extracellular glycerol production in yeasts (Bispin et al., 1990) and D. tertiolecta (Grizeau and Navarro, 19861, which in normal conditions does not excrete.

Table 2 Glycerol photoproduction

by immobilized

(mM)

Fig. 2. Glycerol excreted over 24 h by Ca-alginate entrapped cells of C. reinhardtii at different NaCl concentrations. Immobilized cells were resuspended in the corresponding liquid medium for glycerol photoproduction in the standard conditions described in Materials and methods, except that different NaCl concentrations were used.

One important advantage of immobilization was an improvement in salt tolerance, probably due to a protective effect of the alginate matrix, similar to that reported by Santos-Rosa et al. (1989a) for nitrite assimilation. The immobilized cells tolerated up to 250 mM NaCl (Fig. 21 vs. 200 mM for free ones (Leon and Galvan, 1994). Microorganisms have previously been immobilized by various methods to provide different environments. Large variations have been reported in activity, yield and storage and functional stability after immobilization (Brodelius and Mosbach, 1987; Rao and Hall, 1992). In our case, we have immobilized C. reinhardfii cells in several supports to study glycerol photoproduction and mechanical stability during the process (Table 2). The results obtained indicate that the Ca-alginate is the most adequate. The Ba-algin-

C. reinhardtii cells in various

supports:

Mechanical

stability

of the beads

System

Chl in the medium (%)

Gly in the medium (umol mg-’ Chl)

Mechanical stability

Immobilization technique

Ca-Alg Ba-AIg Agar

1 0 6

60 36 54

good very good medium

easy easy difficult

Gly, glycerol; Alg, alginate. Cells immobilized in various supports were resuspended in the corresponding liquid medium for glycerol photoproduction in the standard conditions described in Materials and methods, except 200 mM NaCl was used. After 24 h, chlorophyll and glycerol in the medium was determined. 100% chloroplyll represents the total immobilized biomass (about 4.5

R. Le&, F. Gal&n /Journal

ate provides good mechanical stability, but probably due to a toxic effect of Ba2+, the glycerol photoproduction is minor. The agar provides a good glycerol photoproduction system, but the mechanical stability is less than that for alginate and the immobilization technique is difficult. In all cases, liberation of the cells into the medium is not significant, according to previous results of Santos-Rosa et al. (1989b) for ammonium photoproduction. Other supports such as carrageenan and polyurethane foam have been tested without good results. The success of the alginate gel entrapment is mainly due to the gentle environment it provides for the entrapped material, its low price, mechanical stability of the beads, and the capacity to produce transparent beads ideal for entrapping whole photosynthetic cells (Smidsrod and Skjak-Braek, 1990). 3.2. Semicontinuous

glycerol photoproduction Ca-alginate cells of C. reinhardtii

I

8

I

2 ypII( (L

1

2

e_, ?5?4t 3 2-

-1600

w1

-500 -400

3 -I I-

E&

-300 $5 oh6 -200 a =t 100 g-

Time (day) Fig. 4. Continuous glycerol photoproduction by Ca-alginate entrapped cells of C. reinhardtii. Operating conditions: volume reaction, 150 ml; initial biomass, 3.4 mg Chl; biomass in continuous operation, 13.6 mg Chl; flow rate, 45 ml dd’ (arrow indicated); temperature, 25-30°C; pH, 7; 0, concentration, 100% saturation; aeration rate, 2 1 min-’ (5% CO*, v/v); illumination intensity with fluorescent lamps, 500 PE m-’ s-r. Other conditions for glycerol photoproduction as described in Materials and methods.

practically linear and reached between 6 and 8 g l- ‘. Such glycerol photoproduction was maintained for at least five succesive batches using the same immobilized biocatalysts; therefore, the semicontinuous process seems to have excellent possibilities. Similar results have been reported by Grizeau and Navarro (1986) using D. tertiolecta and by Bispin et al. (1990) using Pichia farinosa. Repeated batch cultures have shown the stability of C. reinhardtii in this culture system for long-term operations (data not shown). However, periodic changes of medium constitute a risk of contamination and a decrease in operational feasibility. 3.3. Continuous glycerol photoproduction by Ca-alginate entrapped cells of C. reinhardtii

6 4-

I

lo.; E aPii,.

65

by

Immobilization techniques allow the reuse of biomass by replacing the culture medium without loss of cells. This can be performed in a semicontinuous or contionuous manner. Both possibilities were studied for Ca-alginate entrapped C. reinhardtii cells. Semicontinuous glycerol photoproduction was performed in repeated batch-wise cultures (Fig. 3). In a 3-d batch period, glycerol excretion was

l"r

of Biotechnology 42 (1995) 61-67

3

4

5

6

Batch number Fig. 3. Semicontinuous glycerol photoproduction by Ca-alginate entrapped cells of C. reinhurdtii. The process was identical for each culture batch and the duration of each one was 3 d. Conditions for glycerol photoproduction as described in Materials and methods.

Using the continuous system with immobilized C. reinhardtii cells, the bioreactor stability was investigated under optimal operating conditions, similar to that used for the batch system (discontinuous or semicontinuous). The bubble column bioreactor was provided with reservoirs for influent and effluent media and a pump to supply the influent. The effluent came out through a lateral outlet when the liquid level was higher than 150

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66

of Biotechnology 42 (1995) 61-67

ml, as shown in Fig. 1. The maximum glycerol concentration obtained was about 7 g 1-l after 4 d (Fig. 4). After this period (batch system), performance of the continuous immobilized cell bioreactor was evaluated by measuring glycerol for a period of 27 d, using a flow rate of 45 ml d-l. The immobilized cells within matrix alginate grew from a cell loading of 150 to 600 pug Chl gg ’ beads during the batch period (about 4 d), in which a high cell density was reached on the periphery of the beads, since nutrients and light are more accessible to the cells in this area. From this period the peripheric cells of the beads go out into the medium, according to the model proposed by de Gooijer et al. (19901, but contribution of the free cells to glycerol photoproduction is practically insignificant because in the presence of 250 mM NaCl there is no cell viability outside the matrix alginate (Leon and Galvin, 1994). The gel strength made possible the performance of a prolonged continuous glycerol photoproduction in the bubble column biorreactor. The balance equation for the product formation in a bioreactor was given by Pirt (1975) as:

the conventional batch system, and the immobilized cell bioreactor appears to show promise of a continuous photoproduction of glycerol, which would be of biotechnological interest as a raw material for industrial activities. Although at this stage it is too early to do an economic analysis of the process, positive conclusions have been obtained from other similar studies previously made with different algal systems used also for glycerol production. This is the case of the systems developed by Koor Food Ltd. (Israel) or that proposed by Chen and Chi (1981) based on Dunalielfu sulina. This leads us to think that the described process using Chlamydomonas reinhardtii could also have possibilities of economic viability. In addition, this freshwater green alga excretes the glycerol out of the cell, while halotolerant green alga Dunaliella accumulates it inside, which is also an economic advantage of glycerol production with Chlamydomonas, due to the easier recovery of the product. Studies on a larger scale should be made to confirm this.

d( VZ’)/dt = I/r, - FP

Acknowledgements

dP/dt

= rr - FP/V=

D = F/V=

r,, - DP

l/T,

V = reaction volume (150 ml) P = product concentration in the effluent (7 g

1-l) rp = product formation rate F=flow rate=45 ml d-r D = dilution rate (0.3 d-‘) T, = residence time (3.3 d).

The change of medium culture term of synthesis outflow (- DP). dP/dt

product concentration in the for some time depends on the (or) and on the term of product In the equilibrium:

= 0

r p=DP=2.1

gl-‘d-’

corresponding to about 23.2 mg glycerol produced per mg Chl per d because in our case the biomass concentration was 90.7 mg Chl 1-l. Performance of the continuous bubble column bioreactor was found to be far superior to that of

This work was supported by set-up funds provide by Junta de Andalucia (Spain) to our group (32631, and by research project PB91-0613 from DGICYT (Spain). We also thank Dr. A. J. Marquez for critically reading the manuscript.

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