High cell density culture of microalgae in heterotrophic growth

421

High cell density culture of microalgae in heterotrophic growth Feng Chen Microalgae are a great source of many highly valuable products such as polyunsaturated fatty acids, astaxanthin and bioactive compounds. Large-scale production of these products, however, has been hindered by an inability to obtain high cell densities and productivities in conventional photoautotrophic systems. High cell density processes suitable for heterotrophic cultures of microalgae may provide an alternative means for the large-scale production of algal products of high value. This paper reviews recent studies on the formation of algal products in various cultivation systems, with emphasis on the use of heterotrophic techniques. The potential employment of heterotrophic high cell density strategies for commercial production is discussed.

The uses of algae have been recognized for centuries and include human food from blue-green and green algae, soda ash, iodine and alginic acid from brown algae, and agar and carrageenans from red algae. In recent decades, microalgae have been mainly produced and sold as health food, because of their high content of protein, vitanfins and other nutrient supplements. Microalgae have also found application in agriculture, waste treatment and aquaculture. More recently, attention has turned to high value chemicals and pharmaceuticals such as phycobiliproteins from red algae and cyanobacteria, anticancer agents from cyanobacteria, [3-carotene from Dunaliella, xanthophylls from green algae and diatoms, polyunsaturated fatty acids (i.e. eicosapentaenoic acid, docosahexaenoic acid and arachidonic acid) from diatoms and cryptophyta and astaxanthin from Haematococcus 1 s. Nevertheless, microalgae constitute a large resource of genetic and metabolic diversity which has been largely untapped for products of commercial interest. Although there may be over 50 000 microalgal species in habitats almost everywhere on earth, less than ten species have been commercially exploited4. One of the few commercially successful examples is the production of [3-carotene from DunalMla in Australia and a few other countries 3,('. Many other microalgal products with commercial potential, particularly high value chemical and pharmaceutical compounds, have been discovered and summarized in several recent Chen (sfchen@l~kuxa.hku.hk) is at the Departmentqf Botan),, Tire University of Hong Kong, Pokfulam Road, Hong Kong.

F,

reviewss,v,s. However, there has been little progress in bringing these products to the market. This article briefly examines the present cultivation technologies, and discusses potential cultivation methods, with emphasis on heterotrophic culture that may be used for the future commercial production of microalgal products, particularly those of high value. Existing photosynthetic mass culture the p o n d systems

The idea of photosynthetic mass production of microalgae was first developed in Germany in the early 1940s, for the production of lipids from diatoms, which were found to accumulate fats under nitrogenstarved conditions9. More systematic research into photoautotrophic mass culture ofmicroalgae began in the early 1950s by workers at the Carnegie Institute, Washington, USA m. It is well known that photosynthesis is the most abundant energy-storing and lifesupporting process on earth. Microalgae are usually photoautotrophs, which use light as an ener~" source and carbon dioxide as a carbon source. Therefore, it is not surprising that the photoautotrophic growth mode is still the most common technique employed in microalgal industries. Open ponds are the oldest and simplest systems for algal cultivation, in which algae are cultivated under conditions identical to the external environment. Open ponds can be operated as either extensive or intensive systems. An extensive open pond usually comprises a large pond without special modifications such as C O 2 addition and stirring. Extensive open

Copyright © 1996, Elsevier Science Ltd. All rights reserved. 0167 - 7799/96/$15.00. PII: S0167 7799(96)10060 3

TIBTECH NOVEMBER1996 (VOL ]4}

422

fOCUS pond systems have a number of advantages, including: minimal cost of construction and operation, the utilization of arid and/or salty lands (otherwise unsuitable for agriculture) and the possibility of integration with wastewater treatment processes and/or aquaculture. These systems, however, also possess problems: the difficulty of maintaining monocultures; the control of environmental parameters (particularly solar irradiance and temperature); and the high cost for cell recovery owing to low cell density (typically 0.5g 1 1)3,7,11, An intensive open pond is a smaller pond which has been modified to improve algal biomass productivity by, for example, the installation of facilities for stirring and carbon dioxide supplement. In this system, sufficient CO 2 can be provided for photosynthesis, and better mixing ensures more efficient light utilization. Furthermore, better control of contamination may be achieved. Using open ponds for mass cultivation ofmicroalgae, in general, has been adopted for only a few species. These algae can be grown only in a selective and specialized environment, which is hostile to most other competitors such as unwanted algae, bacteria, fungi and predators. For instance, Dunalielta salina grows in brines with a high salinity (NaC1 concentrations >20% w/v) and Spirulina platensis grows in highly alkaline waters 3 (pH >9.2). Other algae, which may be grown in open ponds include some fast-growing species, such as Chlorella, Scenedesmus and Phaeodactylum. Under optimal growth conditions these algae may outgrow most competitors 3. It is worth noting that many commercially desired algal species are unlikely to be successfully cultivated in outdoor systems. For example, Porphyridium cruenturn, which produces a valuable polysaccharide, and Bot~TOCOCCUsbmunii, which produces hydrocarbons, have been reported to be readily contaminated in open pond mass culture v. Since large-scale open pond culture of microalgae for valuable products is unlikely to be sustainable or economic, attempts have been made to overcome some of their limitations (i.e. insufficiency of C O 2, lack of temperature control, evaporation and contamination) through the use of closed pond or enclosed photobioreactor systems. The simplest type of closed system involves covering the raceway with a polyethylene sheet 12. This modification, however, cannot entirely overcome the problems mentioned above 7. For example, contamination cannot be prevented, unless the whole system, including the nutrient media, is sterilized, which is neither practical nor economically justified. Furthermore, commercially available materials for covering ponds are not entirely transparent, and because of condensed water droplets and dust settling on the inner and outer surfaces, respectively, the available light energy" is significantly reduced.

Enclosed photobioreactor systems Enclosed photobioreactors are more sophisticated reactors than closed pond systems. They generally possess lighting, CO 2 addition, stirring and cooling facilTIBTECHNOVEMBER1996 (VOL 14)

ities. Enclosed photobioreactors offer many advantages over pond systems, including (1) better control of culture environment; (2) a larger surface-to-volume ratio; (3) better control of gas transfer; (4) prevention of evaporation; (5) a better thermal profile; (6) easier installation in any open space; (7) better protection from ambient contamination; and (8) higher cell productivities. The most commonly used photobioreactors are tubular for the following reasons: (1) effective sterilization; (2) improved control of gas transfer; (3) high efficiency of light utilization owing to a large surfaceto-volume ratio; and (4) ease of installation in any open space. Generally, tubular photobioreactors can be used to produce higher cell productivity than that in open ponds. Tredici and Materassi 1~ have reported that the productivity of Spirulina in a tubular photobioreactor averaged 30-33 t ha I y 1 in five years of experimentation. Under the same climatic conditions, Spirulina in open ponds seldom achieved more than 20 t h ~ 1 y< (Ref. 13). Two new photobioreactor designs have recently been reported. These are the vertical alveolar panel (VAP; Ref. 14) and fibre optic photobioreactors is. The purpose of the VAP reactor was to maximize the surface-to-volume ratio (i.e. 80 m 2 m -3 for the system). A cell productivity o f 2 4 g m 2 d< was achieved in the reactor with Spirulina plateusis M2 (IKef. 14). The fibre optic photobioreactor system consists of six major components: (1) a light source; (2) an optical transmission system; (3) a reactor; (4) a gas exchange unit; (5) an ultrafiltration unit; and (6) an on-line sensor for monitoring the culture condition. This system can effectively improve the light distribution, which resulted in a very high cell concentration (109 cells nf1-1 = 30 g 1<). A similar optical fibre photobioreactor has also been reported by Takano et al. 1~'to culture a marine cyanobacterium Synechococcussp. for CO 2 removal. This system was connected to a ceramic membrane module to enhance the cell density, and a final cell concentration of l l . 2 g l 1 was achieved. However, it proved difficult to scale-up these two systems, and their capital cost may be high owing to their complexity. Moreover, since the distance light penetrates is inversely proportional to the cell concentration w, the light limitation problem can be reduced but not entirely overcome by using these enclosed photobioreactors. It has been estimated that light penetration is only 2 mm at a cell dry weight concentration of 30 g E1 (tLet: 15). In addition, the high concentration of oxygen accumulation in the culture of the photobioreactor is an unsolved problem.

Heterotrophic growth - potential high cell density mass culture Heterotrophic culture may provide a cost-effective, large-scale alternative method of cultivation for some microalgae that utilize organic carbon substances as their sole carbon and energy source 18-2°. This mode of growth eliminates the requirement for light and, therefore, offers the possibility of greatly increasing

423

fOCbl5

microalgal cell concentration and, hence, volumetric productivity in batch systems. This may be further improved by using high cell density techniques, such as fed-batch culture, chemostat culture and membrane cell recycle systems, which have been used to produce cell densities above 130 g1-1 in yeast systems2>23. These systems may find use in the production of high value, low-volume products (e.g. pharmaceutical proteins) because the high cell concentrations obtained should greatly reduce the cost of down-stream processing, a cost which may be a major proportion of the total production expense. However, to date, the very few reports of such processes for microalgal cultivation have mostly been on lab-scale work. These include batch cultures of Chlorella sorokiniana24, Chlorella saccharophia2s, Chlorella pi, mnoidosa2~, Crypthecodinium cohnii27, NTtzschia alba27 and Tetraselmis suecica28, fedbatch cultures of Chlorella sorokiniana29, Chlamydomonas reinhardtii3° and Spon2iococcum exetriccium31, chemostat cultures of Chlorella regularis32 and Chlamydomonas reinhardtii33,34 and perfusion culture of Chlamydomonas reinhardtii35. It is noteworthy that several attempts have been made to develop industrial-scale processes for the heterotrophic cultivation of microalgae. For example, in the late 1970s in Japan and Taiwan, two Chlorella species were cultivated heterotrophically in stainless steel tanks using glucose and acetic acid as carbon and energy sources for the production of health foods36. More recently, Cell Systems (Cambridge, UK) has developed a process for the heterotrophic cultivation of TetraseImis suecica CSL/61 in 5 1 fermentors in order to produce a mollusc feed using glucose; the process was subsequently successfully scaled-up to 500001 (tLef. 28). In Japan, several heterotrophic processes for producing docosahexaenoic acid (DHA) by Cr},pthecodinium cohnii have recently been patented 37. Martek Biosciences (Columbia, MD, USA) has set up an industrial heterotrophic cultivation process (150 000 1) for the production of a DHA-containing oil by Crypthecodinium cohnii according to general fermentation industry norms 38. In addition to these heterotrophic batch processes, an optimal heterotrophic fed-batch process has also been developed using a 450 1 fermentor for the production of Spony,iococcum exetriccium at Coors Biotech Products (Fort Collins, CO, USA) 31. In heterotrophic cultures, optimal growth and production conditions can be easily maintained, and contanfinating or predatory organisms can be eliminated by sterilization of the medium and aseptic operation, which is necessary for heterotrophic culture. This requirement, and the higher cost of the medium compared with photosynthetic culture, requires heterotrophic cultures to obtain a higher cell density to be economically competitive. This mode of growth is particularly suitable for the production of high value chemicals and pharmaceuticals, where Good Manufacture Practice codes must be met. Heterotrophic culture of microalgae is not without significant problems, including: (1) the limited num-

ber of available heterotrophic algal species; (2) potential contamination by bacteria; (3) inhibition of growth by soluble organic substrates at low concentrations; and (4) the inability to produce some light-induced products, such as pigments. The first problem may be overcome by carrying out extensive screening programmes. For instance, BioTechnical Resources (Manitowoc, WI, USA) successfully selected a heterotrophic microalga, Chlorella pyrenoidosa, suitable for ascorbic acid production after extensive screening ofmicroalgae and yeasts26. Martek Biosciences (Columbia, MD, USA) and OmegaTech (Boulder, CO, USA) has also conducted active research and development projects to screen microalgal strains suitable for production of omega-3 polyunsaturated fatty acids in heterotrophic culture 27. As a result, two heterotrophic strains, Nitzschia alba and Crypthecodinium cohnii, were identified as good producers of eicosapentaenoic acid (EPA) and DHA, respectively27. The second problem is mainly due to the relatively low growth rate ofmicroalgae compared with that of other fast growing microorganisms such as bacteria. This problem can be effectively overcome by rigorous sterilization and aseptic operation. In addition, optimization of growth conditions for the microalgae may help to minimize this problem. Contamination has rarely occurred in our heterotrophic experiments24,33 3s, although they have all been lab-scale cultivations. Problem (3) has hindered the use of batch culture as a commercial production technique since high initial substrate concentrations are essential if high cell densities are to be achieved. For example, the highest glucose concentration was found to be 20gl q for growing Chlorella sorokiniana, above this concentration the cell growth was severely inhibited (F. Chen, unpublished). Acetate concentrations above only 0.4g1-1 inhibited Chlamydomonas reinhardtii33. Inhibition not only resulted in a decrease in specific growth rate, but also resulted in a decrease in cell growth yield, which caused an increase in the maintenance energy39. These problems have attracted nmch work recently in which fed-batch, chemostat and membrane cell recycle systems were investigated3°,33-35. The fourth point represents a most difficult problem which may be partially solved by using mixotrophic culture 4°. We have been able to use mixotrophic culture to grow Spimlina platensis on glucose at high cell densities (10.24gl -~) for the production of phycocyanin41, although it can be foreseen that any production-scale process will be eventually limited by the availability of light. The term fed-batch (or semi-batch) culture was first introduced by Yoshida et al.42 to refer to a batch culture fed continuously or intermittently with nutrient medium. The significant advantages of fed-batch systems for mass culture ofheterotrophic microorganisms include high cell density, the mininfization ofsubstrate inhibition effects by feeding of a concentrated substrate stream to the fermentor and simplicity of operation. For these reasons, fed-batch systems are widely TIBTECHNOVEMBER1996 (VOL14)

424

fOClAS

b .0

2.7 V

2.4

B.5

"-~ 2.1 ~

8.0

1.8

"~ 1.5

{

7.5

1.2

-~-

,

-

0.9, 0.6

7.0 6.5

O 0.3 0.

12

24

36

48 60 Time (h)

72

6.0 96

84

Figure 1 Fed-batch culture of Chlamydomonas reinhardtii on acetate medium in a 3.7-1 fermentor (BioengineeringAG, Switzerland)under heterotrophic conditions of growth at 35°C. (a) An uncovered view (for heterotrophic culture, the vessel is shieldedwith a light-excludingmetal sleeve).(b) Time course of the fed-batch culture. Data: (0) OD; (@) acetate; (/~) pH. Reproduced, with permission, from Ref. 30.

3.0 2.7 2.4

.

___

. . . . .

.

2.1 T

1.8

_~-z~--a

1.5 1.2 0.9 0.6 0.3

a \

' /

_,'// ,, I

0.0 ' 0.00

/

D

[]

r13

Iq

I~

I

I

t

I

I

I

I

I

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Dilution rate (I h -~) Figure 2 Comparison between the simulations and the steady-state cell concentrations at various feed acetate concentrations. Model: (--) 1.7gl<; ( - - ) 2.55gi-1; ( - - - ) 3.4g1-1; (.,..) 5.1 gl-1; Data: (Z) 1.7g1-~; (11) 2.55g1-1; (/~) 3.4gl 1; (A) 5.1 g I-L Reproduced, with permission,from Ref. 34. TIBTECH NOVEMBER1996 (VOL 14)

adopted in biotechnology industries 43. It is obvious that fed-batch culture can be used to produce high cell concentrations and productivity while maintaining a good cell growth yield. Johns 29 has used fed-batch culture to grow Chlorella sorokiniana on glucose in heterotrophic culture. The concentrated glucose solution was supplemented continuously to the fermentor via a peristaltic pump. The glucose concentration in the culture broth was mainrained below an inhibition concentration by controlling the flow rate. The final cell concentration obtained was 9 g b 1, which was twice as much as that in the batch culture, although the control of nutrient concentrations during feeding was not as good as desired. Fed-batch culture was also successfully employed for growing Chlamydomonas reinhardtii on acetate medium (Fig. 1)3". Despite this organism being very susceptible to acetate [the optimal acetate concentration was only 0.4 gl 1 (Ref. 33)], the final cell concentration obtained was 1.0 g p l , which was twice that in the batch culture 3". As with fed-batch culture, a continuous culture may be used to cultivate heterotrophic microorganisms at high cell densities• The growth inhibition by the substrate can be nfinimized by controlling the dilution rate since, in a continuous culture, the substrate concentration in the culture broth is dependent on the dilution rate, but not on the feed substrate concentration34• There are several types of continuous culture system 44. Among them, the chemostat is the most common system. Chemostat has been used to culture Chlamydomonas reinhardtii using acetate as the sole carbon and energy source 33,34. The cell concentration increased with increasing acetate concentration except for the 5. I gb* culture in which the cell growth was inhibited by sodium associated with the acetate in the feed (Fig. 2) 34 . The highest cell concentration achieved was 1.5gl -1 at a feed acetate concentration of 3.4gl 1, which was threefold that in the batch culture system (Fig. 2) 31. The conventional chemostat culture technique has not been widely used as a production process. The only continuous culture processes of production scale are glucose isomerase, single cell protein, beer, buttermilk souring and yoghurt 4s. One of the major problems which prevents conventional chemostat culture from achieving a very high cell density is the instability of the process. To solve this problem, a chemostat with cell recycle has to be developed, in which cells from the recycle stream continuously inoculate the culture vessel, which will increase the stability of the system and minimize the effect of process perturbations. Membrane bioreactors possess an in situ cell separation capacity, which permits cell retention (i.e. re-inoculation) and prevention of cell washout at high dilution rates while inhibitory compounds (e.g. sodium) and soluble products can selectively pass through the membrane and be removed (Fig. 3) 35 . This unique property makes high cell density in such systems possible.

425

fOCUS Dialysis (a typical membrane bioreactor technique) has been employed to cultivate the marine pennate diatom Phaeodact},lum tricornutum by continuously dialysing the culture with NO3, N O 2 and PO 4 under photosynthetic conditions4% This culture system allows for the maintenance of prolonged growth and high densities depending on the light intensity and dilution rate. The main reason for employing a membrane bioreactor is to achieve high cell concentrations and selective separation of products and inhibitory substrates from cells at the same time. The major limitations of employing a membrane bioreactor are the complexity of the system and the difficulty of process control and scaling-up as compared with fed-batch systems47,4s. Chen and Johns ss have employed a membrane bioreactor system for the heterotrophic cultivation of Chlamydomonas reit~hardtiiat high cell densities. The experiment was performed continuously for 540 h and the highest cell concentration of approximately 9 g l 1 (dry weight) was obtained at a dilution rate of 0.1 h I with no cell bleed (Fig. 4) 3s. This cell density is much higher than any so far reported in the literature.

BF

~,

~, S,X,P

~

~ (1->B)F S,X=O,

So, F •

@ ~X,~Sv, P~ ,

/0,, Fermentor

,

P

Pump

Hollow fibre filter

Figure 3 Schematic diagram of a hollow fibre cell recycle system used for heterotrophic cultivation of Reproduced, with permission, from Ref. 35.

Chlamydomonas reinhardtii.

25

I

a

I

b

I

c

I

d

I

e

I

c

'7 (1.)

15

Conclusions Heterotrophic growth of microalgae eliminates the requirement for light, and thus offers the possibility of greatly increasing the algal cell concentration and productivity on a large-scale but, to date, only a few industrial heterotrophic processes have been attempted. This is probably because of a lack of understanding of the mechanisms of heterotrophic growth and product formation of the microalgae. More extensive screening of microalgae for their heterotrophic production potential is needed as well as studies of algal biochemistry and physiology. Since microalgae can only tolerate relatively low concentrations of organic carbon substrates as compared with other microorganisms, such as bacteria and yeasts, more systematic investigations into the development of high cell density processes, in which the medium substrate concentration can be maintained at low levels, is also required.

Acknowledgements The author gratefully acknowledges the support of the Hong Kong Research Grants Council and the University of Hong Kong Committee on Research and Conference Grants for this work,

References 1 Cohen, Z. (1986) in C R C Handbook if Miova{,~al :vlass Cuhuw (Richmond, A., ed.), pp. 421-454, CRC Press 2 Metting, B. and Pyne, J. W. (1986) Euz},mc Microb. Trchnol. 8, 386 394 3 Borowitzka, k.J. and Borowitzka, M. A. (1989) in A(,dal amt C},anobacterial Biotechnology (Cresswell, 1C C., l
10 5 Acetate 0

-51oo

---~-~22CC~2--CC$---T.C-C~2ZCC"2.~CC$22

I

I

I

I

I

I

I

0

100

200

300

400

500

600

Time (h) Figure 4 Overall experimental results of in the hollow fibre cell recycle system (the 11 fermentor was connected with a hollow fibre cartridge with a membrane pore size of 0.2 >m and a total membrane surface area of 2800 cm2; the culture broth was recirculated through the hollow fibre cartridge by a peristaltic pump at 60 ml min<; feed to the fermentor and the filtrate flow was transported by two peristaltic pumps). (a) Dilution rate = 0.065 h4 and bleed ratio = O; (b) dilution rate = 0.1 hq and bleed ratio = 0.1; (c) dilution rate = 0.1 h< and bleed ratio = O; (d) dilution rate = 0.1 h-1 and bleed ratio = 0.3; (e) dilution rate = 0.1 h-~ and bleed ratio = 0.5. Reproduced, with permission, from Ref. 35.

Chlamydomonas reinhardt#

6 13orowitzka, L.J. (1991) Bioresource Tedmol. 38, 251-252 7 Benemann, J. 1~.. (1989) in A!~jal and Cyanoba~tcrial Biotedmolo£y (Cresswell, R. C., Rees. T. A. V. and Shah, N., cds), pp. 317-337, Longman Group 8 Borowitzka, M. A. (1995)J. Appl. PIwol. 7, 3-15 9 Harder, 1(. and yon Witsch, H. (1942) Bet. Dr:oh. Bot. Ges. 60, 146152 10 Burlew, J. S., ed. (1953) A!~al Culture- Fwm Laborator), > Pilot Platlt, Carnegie Institution of Washington 1l Richmond, A. (1992)j. Appl. Ptty,d. 4, 281-286 12 Richmond, A. (1986) CRC Cnt. Rev. Biotechuol. 4, 368-438 13 Tredici, M. tC and Materassi, R. (1992)J. Appl. Phyad. 4, 221-231 14 Tredici, M. R., Carlozzi, P., Chini Zittelli, G. and Materassi, R. (1991) Bion,source Tedmol. 38, 153-159 15 Javanmardian, M. and Palsson, B. O. (1991) Biotcdmol. Bioenfl. 3B, 1182 1189 TIBTECHNOVEMBER1996 {VOL14)

426

fOCHS 16 Takano, H. el a/. (1992) Appl. Biodlem. Biotedmol. 34/35, 449-458 17 Oswald, W.J. (1988) in Micro-a(wl Biowchnolo,w (Borowitzka, M. A. and Borowitzka, L.J., eds), pp. 305 328, Cambridge University" Press, Cambridge 18 Neilson, A. H. and Lewm, R. A. 119741 Ph),cologia 13, 227 264 19 Droop, M. R. (19741 in A(~,a/Physiology amt Biochemistry (Stewart, W. 1). P., ed.), pp. 5311 559, Biackwell Scientific Publications 20 Johns, M. IC, Tan, C. K. and Chen, F. (1989) in lSoa'edings o[-the E(~,hth Australian Biotedmoh,y.), Cot!fiwme, pp. 542-545, Universig' of New South Wales 21 Yee, L. and Blanch, H. W. 11993) Biotechnol. Bioet(~. 41,781 790 22 Shay, L. K., Hunt, H. R. and Wegner, G. H. (1987)J. hid. Microbial. 2, 79-85 23 Chang, H. N., Lee, W. G. and Kim, B. S. (19931 Biotedlnol. Bioeng. 41,677-681 24 Chen, F. and Johns, M. R. (1991).I. App1. Phycol. 3,203-9/!9 25 Tan, C. K. and Johns, M. I~,. (1991) H},drobiologia 215, 13 19 26 Running, J. A., Huss, R. J. and Olson, P. T. 11994)./. Appl. Phl,col. 6, 99-104 27 Barclay, W. K., Meager, K. M. and Abril, J. R. (1994)J. Appl. Ph)~ad. 6, 123 129 28 1)ay, J. 1)., Edwards, A. P. and Rogers, G. A. 11991) BioresourceTechnol. 38, 245-249 29 Johns, M. R. (1994) in A!~al Biotechnolog), in the Asia-Pac!~icRq~io, (Phang, S. M., Lee, Y. K., Borowitzka, M. A. and Whitton, B. A., eds), pp. 1511-154, University of Malaya, Kuala Lumpur 30 Chen, F. and Johns, M. R. (19961 Bion'source Technol. 55, 103-110

31 Hilaly, A. K., Kanm, M. N. and Guyre, 1). 11994) BiotedmoL Bioeng. 43, 314 32O 32 Endo, H., Hosoya, H. and Koibucbi, T. (1977)J. bi'rmen~. Tedmol. 55,369-379 33 Chen, F. and Johns, M. R. (1994) ProcessBiochem. 29, 245_952 34 Chen, F. and Johns, M. R. 11996) ProcessBiochem. 31,601-604 35 Chen, F. and Johns, M. R. 119951.I. AppI. Phycol. 7, 43-46 36 Kawaguchi, K. 1/980) in Algae Biomass (Shelef, G. and Soeder, C. J., e&), pp. 25-33, North-Holland Biomedical Press 37 Borov¢itzka, M. A. 119951J. Appl. Phyad. 7, 509-520 38 Radmer, R. J. and Fisher, T. C. 11996) in Abstracts of the 7th

hltemational Co~!fi'rence, lrltematiotlal Association of Applied Al~oology, p. 60, Knysna, South Africa. 39 Chen, F. and Johns, M. R. 11996)_I. Appl. Phycol. 8, 15 19 40 Chen, F., Zhang, Y. and Guo, S. (19961 Biotechnol. La,tt. 18,603-608 41 (;hen, F. and Zhang, Y. E¢~zyme Microb. Technol. (in press) 42 Yoshida, F., Yamane, T. and Nakamoto, K. 11973) Biotedmol. Bioeng. 15, 257-2711 43 Shioya, S. (1992) Adv. Biochem. E~(¢./Biotechnol. 46, 111-142 44 Pirt, S. J. 11975) Pmlciph's of Microbe and Cell Cultivatio,, Blackwell Scientific Publications 45 Heijnen, J. J., van Scheltinga, A. H. T. and Straathof, A.J. 11992) J. Biotechnol. 22, 3-9/I 46 Marsot, P., Cerebella, A. and Loule, L. (1991).J. Appl. Phycol. 3, 1-1/) 47 Ramasubramanyan, K. and Venkatasubramanian, K. 119901 Adv. Biodwm. Eng./Biotechnol. 42, 13 26 48 Chang, H. N. and Furusaki, S. (1991) Adv. Biochem. E~(~j./Biotedlnol. 44, 27-64

Heterologous expression of G-protein-coupled receptors Christopher G. Tate and Reinhard Grisshammer G-protein-coupled receptors (GPCRs) are integral membrane proteins of great pharmacological importance owing to their central role in the regulation of cellular responses to external stimuli. Heterologous expression systems have been used to explore ligand binding, G protein and effector coupling, and structural aspects of the receptors. GPCRs can be expressed in a functional form in all expression systems, but with varying degrees of success because of differences in receptor and host cell characteristics. This article will discuss aspects related to the choice and suitability of expression systems for the intended analysis of GPCR properties.

G-protein-coupled receptors (GPCRs) are a superfamily of integral membrane proteins containing seven transmembrane o~ helices 1, which play a crucial role in transmembrane signal transduction by binding extracellular ligands (e.g. neurotransmitters and peptide hormones). Ligand binding is thought to trigger a conformational change, resulting in the activation of C. G. Tare (q~,[email protected]) and R. Grisshammer (rkg,@ mrc-lmb.cam.ac.uk) are at the M R C Centre, Hills Road, Cambridge, U K CB2 2QH. TIBTECH NOVEMBER 1996 (VOL 14]

heterotrimeric guanine nucleotide-binding proteins (G proteins) on the intracellular face of the plasma membrane. The central role of GPCRs in the response of cells to external stimuli, and the number of available drugs that bind to GPCRs, has led to the investigation of newly identified GPCRs as potential therapeutic targets. Unfortunately, the analysis of a particular GPCR in native tissues can be complicated by the presence of distinct, but pharmacologically similar, receptors. In most cases, heterologous expression allows the analysis of single receptor subtypes in a

Copyright © 1996, Elsevier Science Ltd. All rights reserved. 0167

7799/96/$15.00. PII: S0167 7799(96)10059-7