Journal of Applied Phycology 12: 441451, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Microalgal biotechnology at the turn of the millennium: A personal view

Amos Richmond
Miles and Lillian Cahn Professor of Economic Botany, Department of Dryland Biotechnologies, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Israel 84990 (fax +972-7-6596802; e-mail amosr@bgumail.bgu.ac.il)
Received 20 April 2000; in revised form 20 April 2000; accepted 20 April 2000

Key words: algal biotechnology, cost effectiveness, light curve, light regime, limits of productivity, photobioreactor efciency, photosynthetic efciency, strong light, tultrahigh cell density Introduction I was grateful and indeed honored to have had the opportunity to provide the opening presentation for the 1999 International Conference on Applied Algology based on my personal views. Microalgal biotechnology at the end of the millennium and after some fty years of much research, requires some soulsearching, for I cannot help a feeling of disappointment over the fact that Microalgal Biotechnology after so many years is still in its infancy, a sort of esoteric endeavor with a great future, which somehow does not get off the ground. One reason for the disappointment in the progress made in mass cultures is rooted in the reasons why I was originally so enthusiastically attracted to microalgaculture close to 30 years ago, in the early 1970s. The prospect of a novel method by to save the hungry and poor part of humanity excited me: Here was a worthwhile goal in life, a fantastic scientic challenge. After reading the works done in the early 1950s (Burlew, 1953), I adopted what I perceived as the Microalgae Doctrine according to which microalgalculture, in suitable geographic regions, can augment and even replace, cost effectively, conventional agricultural productivity. In spite of the many good reasons given for the superiority of algae over horticultural crops, by the early 1980s, it became very clear that mass production of microalgae as an agricultural commodity, providing e.g. protein, starch or oil, was a distant dream, not to be reached in the foreseeable future. Indeed, the false alarm of an impending food crisis, forecast by the United Nations Advisory Committee (Anonymous, 1967), which predicted an acute shortage of protein by the year 2000, only adds insult to injury. Hence the sober view held by some that microalgal biotechnology has a limited future, conned to specialized, relatively expensive products, which naturally have a rather limited market. Personally, I still believe that in the long run, phototrophic microorganisms will play a role similar to that which heterotrophic microorganisms have today. The basic idea of using solar energy to produce photoautotrophic or mixotrophic cell mass for food, feed and industrial chemicals, particularly on marginal agricultural resources such as sea water along coastal dry lands, is as valid as ever. It is nevertheless obvious that the coming of age of microalgal biotechnology has proved a very slow process. What is holding this technology back? Current weaknesses in algal biotechnology Four major elds in which algal biotechnology is presently inadequate may be identied. First, there is a need for a marked improvement in the growth performance as well as cell chemistry of economically promising species. For example, a cell content higher by an order of magnitude in astaxanthin, carotene or EPA over the present cell content of ca. 5%, would greatly alter the economic prospects of microalgaculture. So would a fast-growing starch-storing e.g. Chlorella, with a theoretically possible output rate of starch of ca. 30 g m2 day1 or 100 T ha1 yr1 (a good grain will yield ca. 1/10 of this output). It will take some time until such grossly improved algal strains will be available for common use; but there

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Figure 1. Idealized curve of specic photosynthetic rate (P) as a function of irradiance (Ed ), illustrating the maximum photosynthetic rate, Pm , and the saturation onset parameter, Ek . The variation of P/Ed (a measure of the efciency of utilization of incident light) with irradiance value is also indicated (. . .). (from Kirk, 1983).

is little doubt that eventually the fast progress taking place in gene technology will provide new strains with revolutionary performances. When gene splicing, in about a decade, greatly alters the practical reality of agricultural production, it will at the same time revolutionize microalgal biotechnology by providing much improved strains. This leaves for the immediate future three major subjects on which efforts in microalgal biotechnology should be focused. One concerns the effective utilization of solar or strong light, with the aim of increasing signicantly the light yield. Another concerns cost-effective modes for mass cultivation of microalgae, with special reference to species in demand for aquaculture. Finally, developing a wide range of products enriched with microalgae for human consumption and focusing on the vegetarian market would greatly stimulate industrial production. Let us turn our attention to the effective use of strong light. Progress in algal biotechnology entails appreciation of the distinction between research in photosynthesis and research in algal biotechnology. The former concerns basically the mechanisms of the photosynthetic reactions and the effect of light on these processes. Algal biotechnology in contrast deals principally with the effect of the light regime on costeffective productivity of cell mass or products. There is a key difference between the light source and the light regime, just as there is between the maximal rate of photosynthesis and the maximal rate of areal productivity.

The very limited applicability of the light curve for understanding mass cultivation As an example of our ignorance to utilize light maximally, let us carefully examine the so called light response curve (Goldman, 1980) or P.I. curve (Figure 1) and observe how misleading it could be when applied without reservations to production physiology of mass cultures. The PI curve represents a typical growth response to substrate availability. First, the initial rate of response to increasing substrate availability is highest. Soon, the rate decreases signifying the initiation of a saturation process (in which an increasingly higher light ux is needed to affect a given response). Finally, there is no more net response, the photosynthetic machinery of all cells having become fully light saturated. And if the light ux increases much above saturation becoming greatly in excess, photoinhibition becomes often evident leading, if prolonged, to culture deterioration. This is where the terms saturating light and photoinhibitory light originate. Or consider another relationship, most important to mass cultures exposed to the strong light outdoors: the efciency (i.e. the fraction of chemical energy resulting from incident light energy) by which light is utilized in the cell (Figure 1). This efciency is highest, according to the light curve, at very low light: decreasing as soon as the light ux increases. At strong light, e.g. sunlight at midday, efciency has become ca. 20% or less of its peak obtained in low light.

443 I shall present evidence that adoption without scrutiny of these light curve concepts, to the production physiology of mass cultures outdoors is as a rule of little use in understanding strong light utilization in mass cultures. The falsication involved in the light curve is, of course, that it accurately depicts the response to light of only optically thin cultures, in which there is no mutual shading, all the cells receiving light continuously, in a manner all other inputs required for cell growth are received. This situation is non-existent in mass cultures, the major theme of which is maximal productivity either of cell mass or cell products per given rate of irradiance. Effective exploitation of high light requires relatively high cell densities in which mutual shading is most pronounced. Cells of mass cultures therefore receive light intermittently, which is the most practical mode by which to dilute strong light, ensuring effective use of light by the culture. Intermittent illumination may be expressed in endless combinations, and I believe one reason for the relatively limited progress obtained in understanding the production physiology of mass cultures outdoors relates to the complexity of the subject. Intermittent light entails exposure of culture cells in the reactor to cyclic periods of light (in the photic volume) and of darkness (the dark volume). These cycles as a reminder may take scores of milliseconds to a few seconds to complete and are reminiscent of the ashing light effect concept of past years (Myers, 1953). Light intermittence is thus associated with two basic parameters: rst, the ratio between the light and the dark period in the cycle and second, the frequency of the light-dark (L-D) cycle. Two other, dependent factors must also be considered, i.e. the actual lengths of the light-, and that of the dark periods in a cycle. Optimally, the photic volume occupies some 15% of the reactor volume. Thus, at any instant, ca. 85% of the cells are in the dark. Signicantly, this is generally true for an open raceway as well as for a narrow light path high cell density at-plate reactor. These factors, together with the light source, make the cells light regime or light climate which relates to the state of light available for the individual cell. The difculty with this parameter, the most important single factor in controlling productivity of mass cultures, is that it cannot be dened quantitatively in terms of a single parameter. Commercial failure The subtle conceptual fallacy imbued in the light curve (from the standpoint of mass cultivation) is that it places the light source as the major or only factor which affects photosynthetic rate and cell growth, thereby playing down the decisive impact on productivity exerted by the population density, the length of light path and the extent of culture turbulence affected by mixing. All these are major determinants in phototrophic productivity, not less important than the intensity of the light source. Gross mistakes in designing large-scale reactors have their roots in this background, as well as in the fact that many good researchers do not focus sufcient attention on the complexity of mass production physiology outdoors, which differs greatly from the performance of small scale laboratory cultures. An example is served in the strange disregard to the singular importance of turbulent ow in dense mass cultures: Laminar ow is detrimental to mass cultures of high cell densities for which turbulent streaming is strictly mandatory to eliminate diffusion gradients at the cells surface and more important, to distribute light evenly to all cells in the culture, cut down wall growth and decrease the cultures oxygen tension. The colossal design failures of industrial scale photobioreactors have been well described by Tredici (1999) reecting, sadly enough, ignorance of basic principles in mass cultivation. The plant designed for production of Dunaliella in Santa Ana, Murcia, Spain, comprised of a very intricate net works of very narrow plastic tubes. But if one intends to use a very narrow light path of 12 mm, one aims, in effect, at using relatively high cell densities to justify expensive reactor volume. In such a system, strong turbulent streaming is most essential, but to induce such turbulence in 50 m long, 12-mm tubes in a total of 125,000 m long networks requires very large pressures which are not practical. In such networks, there is no chance for control precipitation of salts from the medium, or prevent wall growth and biofouling, which must be strictly avoided in mass cultivation. In addition, the O2 path is too long for an actively photosynthesising system. The other gargantuan system described by Tredici (1999), the Hydrobiologia SA plant built at La Rioja, Argentina, which was designed to grow Spirulina, reects likewise disregard for the vital necessity of turbulent streaming in mass cultures of Spirulina. In the oblong plastic tubes which on the ground measured 35 cm wide and 9 cm high, the optimal population density

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Figure 2. Output rate in a at plate reactor as a function of photon ux density (PFD; mol m2 s1 ) and culture density. Reactors were irradiated on both sides, each at the indicated total PFD, as follows: , 270; , 740; , 1200; , 2000; , 6000; and, , 8000 mol m2 s1 . (From Hu et al., 1996b).

would be ca. 1 g L1 and the streaming velocity cannot be less than 50 or 60 cm s1 . A circulation speed of 6 to 10 cm1 for Spirulina cultures, as was reportedly exercised in the system, is bound to fail, producing O2 inhibition of growth, low output rates, the culture quickly deteriorating due to wall growth and biofouling. It is worth noting, however, that other species of algae, e.g. Nannochloropsis sp., which require a much lower rate of streaming compared with Spirulina, may have fared better in this system. Intermittent illumination which results in diluted light for the average cell, is without exception mandatory for effective utilization of strong light, bringing up a salient point: Maximal light utilization by the culture requires that the light regime be optimal. This is ensured by optimizing the population density and the length of the light-path for the intensity of the light source. A basic generalization to ensure maximal utilization of incident light becomes clear: the optimal light-path for each species must be identied, as well as the optimal cell density and rate of stirring. Ultrahigh cell density A key to high yields This is the background for the promising approach of utilizing effectively strong light by diluting it (for the average single cell) through greatly increasing the

population density of photoautotrophic cultures, e.g. 50 g dry wt L1 Spirulina sp. or 1 1010 cells mL1 of Nannochloropsis sp. (ca. 50 g dry wt L1 ). Such ultra high cell density cultures (UHCD readily obtained by using a vertical reactor with a narrow light-path, vigorous stirring and continuous removal of autoinhibitory substances; Hu et al., 1996a), do not exhibit photoinhibition or saturating light at high PFD. A good example is seen in the work of Hu et al. (1998b). As long as cell density is optimized in relation to each elevation in incident light, the culture does not become fully light saturated, evidenced in that as the photon ux density increases, so does the output rate (Figure 2). Plotting the output rate at optimal cell density as a function of the optimal cell density reveals a gradual emergence of light saturation, as increasingly higher light quanta are affecting progressively lower increments of cell mass output. Full saturation however, is not obtained, even at 8000 mol photon m2 s1 (Figures 2, 3). The same effect was obtained for Chlorococcum litorale (Figure 4) (Hu et al., 1998a). Photoinhibition is also essentially nonexistent under these circumstances, for excess light essentially implies a population density which has not been adjusted to the intensity of the light source, or to low temperature. The effect of population density on

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Figure 3. Output rate of Spirulina cell mass as affected by the optimal culture density at a wide range of PFD. Figure notations as for Figure 2 (from Hu et al., 1996b).

Figure 4. Output rate of cell mass of Chlorococcum littorale as affected by culture density and incident light. (From Hu et al., 1998a).

the light regime (and hence on cell-response to light) may be well seen in its effect on photoinhibitory damage (Figure 5a). Clearly, a large rise in cell density, which is associated with a marked increase in mutual shading, reduces sharply the photon ux density per cell (PFD cell1 , i.e. photons available to the average cell in the culture). What represents in effect, excess light for a culture with a cell density of 1.8 g L1 , becomes useful light for a culture of 16 g L1 , in which

each cell receives a greatly reduced share of the incident radiation outdoors, the areal cell concentration having been increased by about an order of magnitude (Figure 5b). Under these circumstances, only a slight photoinhibitory effect was measured (Figure 5a) for a short while at noon at the optimal cell density of 8 g L1 (Hu et al., 1996b). Light saturation and photoinhibition must not necessarily be therefore evident in high-density mass cultures exposed to strong light.

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Figure 5. Effect of culture density on (a) Fv/Fm and (b) output rate of biomass in outdoor cultures of S. platensis in the at plate bioreactor during June-July. Cultures were operated on semi-continuous modes. Population density (g L1 ): = 1.8; = 8.5; = 15.2. (from Hu, 1995).

Finally, let us consider another light curve concept, the efciency of incident light utilization, which declines fast with the rise in photon ux density (Figure 1). This concept is sound in general and yet may not depict the reality involved in high cell density cultures. When cell density in Spirulina cultures is carefully optimized, a very signicant surge in incident light, up to ca. 2000 mol photon m2 s1 , is not associated with the expected decline in the efciency of light utilization (Figure 6). Only when the photon ux further increases to values which are above the solar ux, the expected progressive decline in efciency of light utilization takes place. Hence indication that ultrahigh density cultures, in which cell concentration may be higher by some two orders of magnitude than in open raceways, may utilize light falling on a given area at a signicantly higher efciency than conventional normal cultures. Finally, it

is well to note that the similarity, in the range of optimal cell density, between UHCD cultures and heterotrophic cultures (an alternative to photoautotrophy), tends to reduce the advantages of heterotrophic over phototrophic microalgaculture. The essential promise ingrained in ultrahigh cell density cultures concerns the high productivity, both volumetric and areal, of cell mass or products which results from efcient utilization of strong light: using small areal volumes and high cell densities, ultrahigh density cultures represent a rich eld to be explored. Nevertheless, a severe difculty with these cultures is autoinhibitory substances. Not much information is available concerning these substances, which are released by phototrophic microorganisms grown in unusually high concentrations (Javanmardian & Palsson, 1991). In Nannochloropsis sp. growth inhibitory activity may be bioassayed in the culture medium as

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Figure 6. Areal output rate ( ) and photosynthetic efciency (%) as affected by PFD. The indicated rate of PFD represents the total applied on both sides of the at plate reactor. (From Hu et al., 1996b).

Figure 7. Inhibitory activity assayed in ethyl acetate extracts of Nannochloropsis salina supernatant along the culture period. Inhibition unit = affecting 50% growth inhibition (11.2 g CEA mL1 of bioassay). From: Zhang C-W and Richmond A (unpublished data).

soon as cell concentrations rises above ca. 2 billion cells mL1 , the inhibitors activity rising signicantly as cell density further increases (Figure 7). In Skeletonema costatum, the inhibitor was identied as 15-hydroxyeicosapentaenoic acid (Imada et al., 1992). Preliminary evidence resulting from the work of Zang Cheng-Wu in my laboratory reveals that the Nanno-

chloropsis sp. inhibitor is not species-specic, but is more potent in arresting growth of Nannochloropsis than in the other species tested. Table 1 has been inspired by a prominent citizen of Firenze, Dante Alighieri. Dante described the descent into hell, the seven levels through which the sinner goes deeper and deeper in hell. The scheme (Table 1) showing the in-

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creasing complexity of the limitations to cell growth, ascending (in only six levels) as culture density increases until no net growth is possible: Assuming all other growth conditions are optimal, the sole limitation to photosynthetic activity and cell growth in optically thin cultures is the intensity of the light source. As cell concentration rises and the cells receive light intermittently, cell density becomes the major factor in light per cell. Further up the cell concentration scale, and provided nutrient limitation is barred by maintaining nutrient-sufciency, the rate of mixing becomes ever more effective in controlling culture growth and thereafter, the length of the light path becomes most important in determining the light regime for the individual cells, facilitating maintenance of ultra high cell concentrations by decreasing the length of the light path. As cell concentration rises up further, autoinhibitory substances and nally perhaps sheer crowdiness become most signicant in limiting growth. A novel concept is thus introduced in algaculture UHCD based; shifting the limitation to growth from light, the major growth limitation in mass cultures, to an altogether different mode of growth limitation. Limits of phototrophic productivity The surge in productivity exhibited by high cell density cultures exposed to strong light rekindled, in my mind, an old controversy (with which I was personally associated) i.e. the limits of phototrophic productivity. Raven (1988) suggested the usual quantum demand for photosynthesis to rest between 10 and 16, eight photons at least being required per mole O2 released or a mole of CO2 assimilated according to the conventional Z scheme. Pirt (1983) believed that under conditions of severe light limitation, photosystems 1

and 2 may work in parallel, promoting the view for the existence of an additional pathway, requiring a lower quantum demand than mandated by the Z-scheme. Experimental data obtained in his laboratory at Kings College (Yuan Kun Lee and I conducted the experiments) showed a conversion efciency of PAR to cell mass up to 47% (Pirt et al., 1980). Such high efciency is not possible if the minimal quantum demand is 8 and Pirt (1983) suggested therefore that alternate routes must exist at low incident light. Pirt et al. (1980) estimated the maximum possible conversion of total solar energy to biomass free energy at 18.1%, placing the maximal productivity potential in the sunny parts of the world higher by an order of magnitude than the conventional. Indirect support for the possible existence of auxiliary routes in photosynthetic electron transport came from an unexpected source: Otto Pulz introduced a novel principle for mass production of photoautotrophic microorganisms by placing at plate reactors a short (e.g. 20 cm) distance apart, obtaining thereby a large culture surface on a small ground area (Pulz, 1992; Pulz and Broneske, 1995). He reported extraordinary high yields of cell mass per ground area and although some of those high gures have most probably not been corrected for peripheral effects, (which may introduce large errors in computing areal productivity), preliminary results from my lab, obtained by Hu Qiang, prompt me to believe that using the Pulzs reactor arrangement, record photosynthetic efciencies are obtainable, and Spirulina may yield as high as 120 or 130 g d1 dry wt per m2 of ground area. If one converts this gure to an annual yield, a familiar gure is obtained i.e. ca 500 t ha1 yr1 , which is reminiscent of Pirt et al. (1980), authors whose views were met 20 years ago with utter disbelief.

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Figure 8. Meandering plate cultivator 100 to 6000 L. IGV Institut fr Getreideverarbreitung.

Cost-effective photobioreactors In addition to shedding new light on the controversy concerning maximal photosynthetic productivity and efciency, Pulzs work directs attention to light utilisation efciency in photobioreactor design. The large enhancement in ground area productivity obtained by a close setting of plate reactors stems from a most effective dilution of strong light. The greatly reduced incident light per illuminated culture area, facilit-

ates a great surge in photosynthetic efciency (PE), which approaches its maximal potential. PE in itself is nevertheless not pivotal in reactor design because of the high toll paid for the high PE in the form of greatly increased investment cost in reactor hardware, as well as increased operational costs involved in manipulating large culture volumes of relatively low cell concentrations, a regression to the open raceways concept.

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Figure 9. A biocoil photoreactor. Commercial continuous algal biomass production system incorporating photobioreactor technology. ADDAVITA LIMITED, Derbyshire UK.

Figure 10. A 500-L at plate glass photobioreactor built at the authors laboratory in theThe Jacob Blaustein Institute for Desert Research.

Clearly, progress in development of cost-effective photobioreactors requires establishment of criteria for reactor efciency. An efcient photoautotrophic production system entails high areal as well as high volume-productivity. Volumetric productivity however is to a great extent a function of the length of the light path, the smaller the latter, the higher the former. A high volume-yield therefore does not necessarily entail efcient utilization of incident light, a measure of which can be obtained only from the areal-yield, for

which there is an optimal areal cell density as well as optimal cell concentration. Both high volume and areal yields coupled with simple, effective reactor maintenance must be considered in determining reactor efciency in relation to cost effectiveness. Progress in microalgal biotechnology will take place when this most important practical issue receives much more attention than given hitherto. This is evidenced upon examination of available industrial-size vertical reactors. The three vertical

451 photobioreactors shown in Figures 8, 9 and 10 have been designed along different principles and differ in price. Prior to investing considerable sums of capital, some objective criteria should be available and several parameters must be addressed: one concerns the photobioreactor as a biological instrument aiming to yield high productivity of high quality products. Specically, which is likely best to sustain continuous monoalgal cultures? In which reactor biofouling would be readily controllable, reducing the likelihood of culture deterioration? In which reactor would the oversaturated oxygen tension remain smallest? What is the reactor cost per unit reactor volume and what are the amortisation and maintenance costs? In summary I believe the future of microalgaculture, particularly in suitable geographic regions, is in the long term as bright as ever. In the immediate future, intensive efforts should be devoted to developing new microalgal products for human consumption, as well as screening of species for altogether new products. Attention should be paid to the rapidly growing aquaculture industry, i.e. the farming of sh, shrimp and shellsh, which would create a growing demand for microalgae, several species of which being an essential component for the early life stages of many aquatic organisms. To be effective as a feed additive for aquacultural purposes as well a for human food consumption, the approach to phototrophy should attempt to emulate the general features of the successful heterotrophic systems. This entails fast growing cultures of very high cell densities, responding efciently to strong light and producing cells with a high content of desired products. For this purpose, species for mass cultivation should be very greatly improved and effective utilization of strong light should be much better understood. Reactor efciency and cost-effectiveness of industrial size reactors should deserve full attention by researchers of mass culture physiology, in a genuine effort to contribute to one of the most important themes for the future of microalgal biotechnology. In this context, development of suitable mixotrophic cultures may be very useful in greatly increasing the yields and reducing production costs. In the long run i.e. in one to two decades, efcient production modes of phototrophic cell mass, coupled with algal gene platforms to produce commercial products, will nally transform algal biotechnology into a signicant industrial endeavor. References
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