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Ling Xu1,2 Pamela J. Weathers3,4 Xue-Rong Xiong5 Chun-Zhao Liu1

1

Review

Microalgal bioreactors: Challenges and opportunities

Cultivating and harvesting of products from microalgae has led to increasing commercial interest in their use for producing valuable substances for food, feed, cosmetics, pharmaceuticals, and biodiesel, as well as for mitigation of pollution and rising CO2 in the environment. This review outlines different bioreactors and their current status, and points out their advantages and disadvantages. Compared with open-air systems, there are distinct advantages to using closed systems, but technical challenges still remain. In view of potential applications, development of a more controllable, economical, and efcient closed culturing system is needed. Further developments still depend on continued research in the design of photobioreactors and break-throughs in microalgal culturing technologies.
Keywords: Microalgae / Photobioreactor / Scale-up Received: December 8, 2008; revised: May 23, 2009; accepted: May 29, 2009 DOI: 10.1002/elsc.200800111

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China School of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China Department Biology/ Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609, USA Arkansas Bioscience Institute, Arkansas State University, State University, AR 72467, USA Division of Industrial Biotechnology, Bureau of Life Sciences and Biotechnology, Chinese Academy of Sciences, Beijing 100864, P. R. China

Introduction

Microalgae are unique and potentially valuable microorganisms because they are the light-harvesting cell factories that convert carbon dioxide into biomass or a variety of bioactive compounds. Although many can grow heterotrophically, all microalgae are photoautotrophs, requiring mainly sun, water, and inorganic nutrients for growth. Compared to higher plants, microalgae are simple in structure, being unicellular, lamentous or colonial, and energy is directed via photosynthesis into growth and reproduction; they do not need to establish and maintain complex tissues and organs [1]. Microalgae have the potential to produce valuable substances for the food, feed, cosmetic, pharmaceutical, and waste treatment industries [210]. More recently these photosynthetic microbes have also become the focus of considerable attention as a potential source of oils for biodiesel production [1114]. Indeed, the cultivation and harvest of products from microalgae has led to an increased commercial interest in their

Correspondence: Dr. Chun-Zhao Liu (czliu@home.ipe.ac.cn), National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China.

biotechnology because algae offer a number of advantages from an industry perspective. These include ease of culture and harvesting of products [1], greater photosynthetic efciency than terrestrial plants [15], higher biomass productivities, faster growth rates than higher plants, and higher rates of CO2 xation and O2 production [14]. Commercial culture of microalgae has440 years history with some of the main species grown being Spirulina for health food, Dunaliella salina and Haematococcus pluvialis for carotenoid production, and several species for aquaculture [10, 16, 17]. While in the past natural waters (lakes, lagoons, ponds) or articial ponds were used to grow algae, more recently closed photobioreactors have been employed [10, 18]. Open-culture systems have almost always been located outdoors and rely on natural light for illumination. Although they are inexpensive to install and run, open systems suffer from many problems: cultures are not axenic so contaminants may out compete the desired algal species; predators like rotifers can decimate the algal culture, and vagaries of weather can make proper control of nutrients, light intensity, and CO2 at best challenging [10]. Closed photobioreactors, on the other hand, have been used to axenically grow photosynthetic microorganisms such as microalgae, cyanobacteria, plant cells, and photosynthetic bacteria for various research and

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biotechnological applications. A comparison of open and closed systems for microalgae is shown in Table 1. Photobioreactors used for the cultivation of microalgae are either naturally or articially illuminated. Algal culture systems with naturally illuminated large surface areas include open ponds, at-plate, horizontal/serpentine tubular airlift, and inclined tubular photobioreactors [19]. Generally, laboratoryscale photobioreactors are usually articially illuminated with uorescent or other types of lamps. Closed reactors can be sited indoors or outdoors, and offer better control of culture conditions. Unfortunately they are also usually more expensive to install. As a result they are under intensive study in an effort to reduce costs to better facilitate their use especially for low value products like algal oils for biodiesel. Although cultivation of microalgae seems easy, there are many challenges including: (i) minimizing contamination (ii) efcient provision of carbon dioxide and light; (iii) controlling cultivation conditions; (iv) reducing capital and production costs; and (v) minimizing space requirements [20, 21]. For instance, although the high light intensity of full sunlight would seem desirable, it can lead to photoinhibition, yielding 1 . toxic photo products including H2O2, O2, O2 , OH, and triplet chlorophyll, and even damage the Photosystem II reaction center [22, 23]. Low light intensities, on the other hand may limit photosynthetic activity and thus, biomass productivity. Whereas many algae can become somewhat acclimated to different light intensities, the level of sensitivity to light varies with species [7]. Indeed we have observed that at very low light intensities Botryococcus braunii grows much better than Chlorella vulgaris which grows much better at the higher intensities that inhibit B. braunii (unpublished results). This variable light response has therefore required development of novel concepts in both design and operation of photobioreactors aimed at exploiting the full range of photosynthetic biochemistry [2426]. For example, transparent at angled culture systems have been developed and tested to maximize light incidence to the culture [24]. Another challenge to be met is optimal CO2 feeding of cultures. Algae are photoautotrophs using CO2 as a carbon source and this can aid in CO2 mitigation of the environment and reduction of global warming. On the other hand, maxiTable 1. A comparison of open and closed systems for microalgae [18, 97].
Open systems Contamination risk CO2 losses Evaporative losses Light use efciency Area/volume ratio Area required Process control Biomass productivities Investment costs Operation costs Harvesting costs Scale-up High High High Poor Low High Difcult Low Low Low High Easy Closed systems Low Low Low Excellent High Low Easy High High High Relatively low Difcult

mizing cost efcient mass transfer of CO2 to cells in an aqueous environment is not a trivial task for large-scale liquid culture systems as is anticipated for outdoor algal cultures. Furthermore, many algae can also grow heterotrophically and although this is a benet in terms of remediation of liquid waste streams, the presence of any reduced carbon can also wreak havoc in nonaxenic algal systems because microbial contaminants with their faster growth rates can easily outpace algal growth. Consequently the supply of carbon to microalgal mass culture systems represents a principal difculty and limitation in obtaining cost effective production of algal biomass [2730]. The few commercial species that are currently being successfully cultured in large open ponds are extremophiles growing in a highly selective environment (high pH, salinity, or temperature). These conditions preclude the growth of most other algae and even many bacteria. For the future of microalgal biotechnology, however, it remains important to develop large-scale photobioreactors that can be operated under dened, optimal conditions with capability for sterilization, and with minimal contamination risks. Although it is difcult to compare open ponds with closed systems or with indoor photobioreactors, the general consensus suggests that open systems may predominate for mass cultivation of algae for low value products like biofuels, while photobioreactors will be more useful for production of high value products like therapeutics [31]. In this review, we have divided microalgal bioreactors described in the literature into classes according to their geometric features, indicated their advantages and disadvantages, and compared their overall performance. The purpose of this review is to outline the current status and recent developments in the technology used to culture microalgae while also noting challenges and opportunities.

Open-air cultivation systems

Open-air cultivation systems comprise natural or articial ponds, raceway ponds, and so-called inclined surface systems driven by paddle wheels, usually operating at water depths of 1530 cm [16]. They represent the classical processes used for production of algal biomass. Although different types of open reactors have been put into operation since the late fties by different groups, the most commonly used systems include shallow big ponds, tanks, circular ponds, and raceway ponds. Raceway-shaped culture ponds are used in Israel, the United States of America, China and other countries. A raceway pond consists of a closed loop recirculation channel that is typically about 0.3 m deep (Fig. 1) [32]. Nutrient fertilizer is added and the culture is agitated by a paddle wheel [7, 33]. The largest raceway-based biomass production facility located in Calipatria, CA, USA occupies an area of 440,000 m2 to grow Spirulina [34]. Compared to the classical at raceway pond, an inclined surface offers two advantages: better turbulent ow, and shallower culture depth (the mean depth of the culture in the inclined slope is 3.5 cm). This reduces the thermal inertia of the culture allowing a more rapid increase of its temperature

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Table 2. Comparison of different closed microalgal culture systems. Photon ux density (PFD), photosynthetic efciency (PE), light path or diameter (D), volume (V), biomass concentration (Cx), volumetric productivity (Pvolumetric), areal productivity (Pareal), supercial gas velocity (ug), overall mass transfer coefciency (kLa) , power supply (P).

P (w m3)

--42000 ----130 ---Articial Tubular reactor Articial Sun Articial Sun Bubble column Flat plate Airlift reactor Sun Articial 120 1289 1135 1000 1000 -72 500 -72 20 8.0 -13.4 6.8 a) 4.8 ---6.8 a) --1.2 6 3 3 3 10 79 8 21 79 10 5.5 200 75 3.4 5 200 170 1.9 64 170 3 3.26 2.38 3.03 -7.3 2.6 5.60 b) 0.91.0 0.41.4 6.77 b) 1.77 b) -20 --31.8 11.25 -----0.62 1.19 1.38 1.5 1.38 0.225 3.31 -0.03-0.20 4.09 0.80 -0.30 0.41 ---0.01 0.017 -0.01 0.4 -6 4 ----20-25 ----

Spirulina Phaeodactylum Phaeodactylum Dunaliella Phaeodactylum Nannochloropsis Chaetoceros Phaeodactylum Monodus Chaetoceros Haematococcus http://www.els-journal.com a) Calculated from biomass yield on light energy by using a value of 218.8 kJ mol1 photons [108] and a specic energy content of biomass of 25 kJ g-1 [109] b) Calculated from biomass concentration (cell mL1) by using a value of 2.228 g dry weight per 1 109 cells [75]

Figure 1. Aerial schematic view of a raceway pond [7].

Closed cultivation systems

Unlike open-air systems, closed photobioreactors are aimed at mainly axenic single-species culture of microalgae. Photobioreactors have been used successfully for producing large quantities of microalgal biomass [7], and their design congurations include horizontal or serpentine tube, at-plate, bubble column, airlift column and stirred tank. A comparison of performance is shown in Table 2. As shown in the table, photosynthetic efciency in tubular reactors was higher than those in other systems. For the outdoor culture of

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Photobioreactor type

Light source

PFD `mol m2 s1) (m

during the morning. In order to better control temperature and use solar irradiance, an open pond was coupled with an appropriately sized and oriented at panel and thus the algal productivity increased in the outdoor culture [35]. Recent research showed that carbon dioxide absorption by algae could be further enhanced by 78% by installing a specially designed carbon dioxide sparging system in an outdoor raceway pond [36]. An open culture unit affords ready access to sunlight, and is easier to build and operate than most closed systems; open systems have been studied considerably over the past forty years [7, 37]. As a result, however, a number of major drawbacks have been identied [17, 35, 36, 38] including: (i) only a small number of algal species can be grown in large scale successfully; (ii) wild microbial predators are a problem; (iii) there are signicant evaporative losses and water conservation therefore becomes an issue; (iv) CO2 is not used efciently; (v) a large area of land is required, so only unproductive or waste land can be used; (vi) biomass productivity is lower than that in closed cultivation systems; and (vii) costs of harvesting algal biomass are still high. Although efforts have been made to improve open ponds with temperature control systems, supplies of appropriate nutrients, optimization of pond depth, CO2 injection systems, etc [39], productivity remains fairly low compared to closed systems. Because of these limitations, focus has shifted to development of cost effective closed cultivation systems which are described next.

PE (%)

D (cm)

V (L)

Cx (g L1)

Pareal (g m2 day1)

Pvolumetric (g L1 day1)

ug (m s1)

kLa ( 103s1)

Algal strain

[51] [41] [42] [98] [107] [63] [74] [67] [86] [74] [75]

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Phaeodactylum, better biomass concentration and areal productivity were obtained in the 5-L at plate reactor. Its volume, however, was smaller than that of the tubular reactor. For pilot-scale cultivation of Chaetoceros, high volumetric productivity was achieved in pneumatically agitated photobioreactors and the airlift system showed better performance.

3.1

Tubular photobioreactors

Fully closed tubular photobioreactors are potentially attractive for large-scale axenic culture of microalgae and is one of the more suitable types for outdoor mass culture [19, 40]. Tubular photobioreactors consist of an array of straight, coiled, or looped transparent tubes that are usually made of transparent plastic or glass [4143]. Algae are circulated through the tubes by a pump, or airlift technology [44]. Use of an airlift device seems to offer some advantages: it allows CO2 and O2 to be exchanged between the liquid medium and the aeration gas; potential cell damage associated with mechanical pumping may be minimized; and circulation is achieved without moving parts. In some cases the temperature of the tubular photobioreactor is reported to be better controlled by oating or submerging the tubes on or in a pool of water [31]. Gudin and Chaumont were the rst to develop a 100 m2 tubular reactor made of polyethylene tubes for the cultivation of the red alga, Porphyridium cruentum, and Torzillo et al. explored the development of a tubular photobioreactor for outdoor mass production of Spirulina platensis [45, 46]. Instead of being laid horizontally on the ground, the helical tubular system rises vertically as an arrangement of transparent, coiled polyethylene tubes arranged around an open circular framework [47, 48]. Other variants of tubular photobioreactors exist such the conical helical tubular photobioreactor (CHTP) [49, 50], which has been used to mass culture Chlorella sp. with the productivity per installation area of 26.6 g (m2 d)1 in a single basic unit and 31.0 g (m2 d)1 in a four-unit system [50]. Tube diameter is limited (generally 0.1 m). Increasing tube diameter results in a decrease in the surface/volume ratio, and this factor has a strong impact on the culture. As the algae grow and increase in density, they begin to shade one another and this translates to a volumetric reduction in biomass per unit of incident light [32]. The tube length, on the other hand, mainly inuences the circulation of the cultivation medium inside the reactor, i.e. the residence time [51]. Scaling up a tubular photobioreactor hypothetically can be achieved by either increasing the length or diameter of the tube [40, 44]. As mentioned above, increase in diameter is not desirable, however, there are also limitations to increasing the length of the tubes. Tubes that are too long allow the O2 produced by photosynthesis to accumulate, thereby possibly exceeding that of air saturation, and this in turn can inhibit photosynthesis via mechanisms previously described [22]. Indeed oxygen concentrations above 35 mg/L are toxic to most microalgae [10]. In long tubes, a decreasing CO2 gradient will also become established between gas entry and exit leading to depletion before exit, thereby starving some algae for carbon. Such a

gradient in carbon xation can lead to pH gradients; a high rate of CO2 xation results in hydroxide ion build-up in the medium reaching a pH as high as 11. On the other hand, inadequate xation of CO2 sparged into the culture medium may result in acidication from carbonic acid formation. A well functioning photobioreactor will have a well balanced pH; the rising hydroxyl ion produced from photosynthesis is counterbalanced by increased carbonic acid production from CO2 sparging. Besides diameter and tube length, mixing is also problematic in extended tubes [40]. Ugwu et al., however, were able to improve mass transfer in tubular reactors by installing internal static mixers to enhance mixing [52, 53]. For the above reasons, therefore, tubular photobioreactors cannot be scaled up indenitely, and large-scale production plants would likely have to rely in part on an assemblage of modular reactor units [9].

3.2

Flat plate photobioreactors

When one considers the laminar morphology of plant leaves, they are well evolved solar collectors, and thus, plate type geometries seem to have been modeled similarly with a high surface/volume ratio. Compared to tubular bioreactors, however, there are some advantages with respect to compactness: their narrow U-turns may use less space than coiled tubes, and their wall thickness can be thinner than tubular bioreactor [31]. The cultures in these photobioreactors are also mixed with air introduced via a perforated tube at the bottom of the reactor. Usually, the panels are illuminated mainly on one side by direct sunlight and have the added advantage that they can be positioned vertically or inclined at an optimum angle facing the sun [54, 10]. Flat plate bioreactors, rst described by Samson and Leduy, were illuminated by uorescence lamps on both sides and agitated by aeration [55]. Tredici et al. further developed the idea by fabricating the photobioreactors from commercially available panels of transparent sheets partitioned to form a rigid alveolar panel [56]. The at inclined modular photobioreactor (FIMP), a tilted at plate photobioreactor, was angled to provide maximum exposure of the culture to solar irradiance as well as for substantial control of temperature. This type of at plate reactor was also

Figure 2. Scheme showing solar orientation of a at-plate photobioreactor system for outdoor culture [62].

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made of transparent glass glued with silicon rubber so that the light path of the at plate photobioreactor can be easily changed [57]. By maintaining a high optimum culture density of 4g L1, the FIMP enhanced the eicosapentaenoic acid productivity of Monodus subterraneus to 58.9 mg (L day)1 [58]. However, scale up was difcult. To achieve maximum productivity, a 35% enhancement compared to the vertical system, the tilt angle had to be adjusted four times a year [59] and this increased operation costs. Even if a xed tilt angle was instead used, the higher capital cost of the tilt design did not compensate for the 17% increase in productivity of Spirulina platensis [60]. For the above reasons, horizontal and vertical at plate photobioreactors were more commonly used for the cultivation of microalgae. A vertical system requires proper solar orientation to maximize irradiance. When compared to an east/west orientation, the decrease in irradiance from top to bottom of the channels was less than for a north/south orientated at plate reactor (Fig. 2). Algal cultures also grew better in the north/south orientated system [61]. As an apparent contradiction to this nding, however, Kurano and Miyachi obtained higher algal growth when they placed their at plate reactor in the east/west orientation [62]. These apparently conicting studies were recently explained by Sierra et al. Solar radiation increases with respect to the earths latitude. The east/west orientated at plates intercept more solar radiation than the north/south for latitudes above 351N, while for latitudes below 351N to the equator, the result is the reverse [60]. In vertical at plate photobioreactors there are some added benets with regard to light [57, 60, 61, 63]. For example, the proportion of microalgal productivity attributed to diffuse and/or reected radiation (62%) was larger than that of direct radiation (38%) [57]. The amount of light per unit volume is determined by the penetration depth of the incident light path of a plate system, but is variable with algal species. For example, volumetric (g L1 day1) productivity of Nannochloropsis sp. increased47 fold when the light path was decreased from 17 to 1.3 cm, with peak areal (g m2 day1) productivity at a light path of 10 cm

[63]. In contrast Spirulina plufensis grown in a at plate photobioreactor decreased from 51.1 g m2 day1 to 33.6 g m2 day1 in areal productivity as the light path increased from 1.3 cm to 10.4 cm [57]. Each microalgal culture appears to have an optimum light path that is a compromise between the growth inhibition in deep layers due to insufcient illumination and the light inhibition or saturation effect in supercial layers. Furthermore, there is a conict between biomass concentration and light penetration and thus the light path is also limited by biomass concentration. Flat plate photobioreactors are used for mass production of microalgae in outdoor and indoor culture systems because of advantages including high illumination surface area, low accumulation of dissolved oxygen compared to horizontal tubular photobioreactors and due to their modular design convenience in scale-up. Doucha et al. [64] described an optimized large-scale at plate photobioreactor module of 1000 m2, and one commercially available at plate photobioreactor has a reported capacity of 6000 L [31, 61]. Although high biomass concentrations (480 g L1) can be reached in narrow light path at panels [65], there are some limitations. There is some degree of algal wall adhesion; there is potential for high stress damage associated with aeration; systems are not amenable to sterilization; and they are incompatible with off the shelf industrial fermentation equipment [19, 60, 61]. Temperature control is also a problem and sprinkler systems are often used for evaporative cooling [57]. A recent study provided an alternative remedy for temperature control by using a heat exchanger consisting of stainless steel tubes located inside the at plate reactor [60]. The overall heat transfer coefcient for the internal heat exchanger was much higher than that between the culture and the external environment, with maximal values of 505 and 37 Wm2 K1, respectively [60]. To attain the same mass transfer capacity, the power supply of 53 Wm3 for the at plate reactor [60] was much lower than the 20003000 Wm3 [41, 42, 66] required for tubular photobioreactors. Unfortunately it was still higher than the 40 W m3 needed in bubble columns [67].

A
PFD Liquid flow

Air / CO2

Figure 3. Schematic of airlift photobioreactor (A) and growing microalgal cultures in these airlift reactors (B). PFD, photon ux density.

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3.3

Airlift and bubble-column photobioreactors

Airlift and bubble-column bioreactors are simple devices that have been used in bioprocessing, wastewater treatment, and the chemical process industry. These vertical column photobioreactors are compact, low-cost, and easy to operate axenically [68, 69]. Pneumatically agitated bubble columns and airlift devices attain the requisite mass transfer coefcient of 0.006 s1 and liquid circulation velocity at a relatively low power input for practicable culture of microalgae [69, 70]. Camacho et al. reported that the maximum biomass volumetric productivity of Phaeodactylum tricornutum obtained in vertically oriented concentric-tube airlift photobioreactors (ALP) was about half of that obtained by a horizontal-loop tubular photobioreactor (HLTP) [71]. A similar result was obtained in a bubble column, where the volumetric biomass productivity was about 60% of that in HLTP for Phaeodactylum tricornutum grown under identical conditions [43]. Compared to bubble column photobioreactors, air-lift photobioreactors (Fig. 3) showed superior growth of microalgae. At a low aeration rate of 1 L min1, cultures of Undaria pinnatida and Porphyridium Sp. grown in an airlift reactor attained 3350% higher growth rates than when grown in a bubble column [72, 73]. Diatom yields were also about double in an airlift device than in a bubble column [74]. It appears that, in contrast to a bubble column where cell ow patterns are more random, the airlift system produces a more homogeneous ow pattern that moves cells from dark (riser) to light (downcomer) zones [75]. Thus, cells in a bubble column may reside in high or in low light intensities for a long time without circulation. Furthermore, cell sedimentation occurred in the bubble column while cells remained more uniformly suspended in the medium of the airlift photobioreactor [72, 75]. When large-scale diatom cultivation was compared in both bubble column and airlift photobioreactors, there was no signicant different in the specic growth rate, which was 2.46 102 h1 and 2.58 102 h1 for bubble column and airlift reactor, respectively [74]. This may be the result of the non-ideal ow pattern in the airlift system and the internal circulation within the riser itself. One of the costs of growing algal cultures in airlift and bubble column reactors is that of the added gases. Merchuk et al. compared Porphyridium culture in an airlift photobioreactor with that in a bubble column reactor. By adding a helical ow promoter to the airlift system, the cost of gases for the production per kg microalgal biomass was 50% of that in the bubble column to achieve the same specic growth rate [76]. Bubble column and airlift photobioreactors are also used for microalgal production in aquaculture. A new bubble column design using PVC bristles to help dislodge the adhesive diatom being cultivated resulted in algal biomass increasing 20% compared to growth in a bubble column without bristles [77]. This modication will likely prove useful for the mass production of other microalgae that are highly adhesive. Another photobioreactor design that uses an external loop airlift with a swirl ow has been reported for continuous microalgal production. This system consists of a succession of modules, each one being composed of two vertical interconnected columns (riser column

and downcomer column) [70]. In this system, a capillary sparger was used to provide a swirling motion to minimize cell adhesion to the wall with an effect similar to the previously described helical ow promoter in an airlift reactor [70, 76]. This modular design facilitated easy, long term operation and a 120-L prototype has been implemented for microalgal production in a commercial hatchery. Considering the high mass transfer, low energy costs, and extremely low physical stress, some bubble column photobioreactors are equipped with a rubber membrane diffuser or dual spargers to improve the mass transfer of gases: provision of CO2 and removal of O2 [78, 79]. In the case of dual sparging, the efciency of CO2 transfer increased vefold relative to that of conventional sparging [79]. Bubble size, however, apparently is also crucial for minimizing shear damage to cells. When Rocha et al. grew Nannochloropsis gaditana using small vs. large bubbles, they found better algal growth with larger bubbles and as air ow rate (vvm) was increased the cells suffered more shear with smaller vs. larger bubbles [80]. Berberoglu et al. recently used Mie theory and experimental data to show that bubbles may also offer an advantage. Bubbles produced via sparging of Anabaena variabilis appeared to increase light penetration into a culture by up to 20% as a result of light scattering [81]. It is, therefore, probably wise to measure the effect of bubbles on each species of alga being grown in air sparged reactors. Aeration rate is restricted by considerations of shear sensitivity. There is an upper limit on the acceptable level of turbulence, because algal species are affected differently by hydrodynamic stress [69, 82]. Generally, increasing aeration rate increases mixing, liquid circulation, and gas-liquid mass transfer in bubble column and airlift reactors. While a high supercial gas velocity increases mixing, it also aids in preventing oxygen accumulation and provides good gas-liquid mass transfer for efcient use of CO2 [69]. Not surprisingly some microalgal species also suffer negative effects from an increase in supercial gas velocity due to the high shear stress caused by high aeration rate. For example, when Haematococcus pluvialis was cultivated in an airlift bioreactor with different supercial gas velocities, results indicated that the maximum cell density declined to about 10% of the optimum level when the velocity was increased from 0.4 cm s1 to 3 cm s1 [75]. This was likely from the higher shear stress on H. pluvialis as the gas ow rate was increased, and probably resulted in cell damage. It was determined that shear stress varied in the airlift with the radius of the culture column and increased with respect to the supercial gas velocity in the riser; in the downcomer, however, shear was quite uniform and much lower. High shear stress also was observed in the top and bottom regions of an airlift bioreactor, and changing the size of the top and bottom clearance greatly altered the ow structure and shear stress in these regions [83]. These changes may signicantly affect airlift bioreactor performance, particularly in applications involving stress sensitive microalgae. Vertical column photobioreactors, especially the airlift photobioreactor, possess some advantages for microalgal cultivation: no moving parts, low power consumption, high mass transfer rate, good solids suspension, homogeneous shear, rapid mixing, and with the vertical orientation, less land is required [71, 84, 85]. With scale-up, however, penetration of light into the reactor decreases exponentially with distance

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from the light source [86] and may pose a problem when scaling-up these bioreactors. Like tubular reactors, increasing the diameter of the column has limitations. Large-scale outdoor culture of Phaeodactylum tricornutum in bubble columns showed that the optimal dimensions of vertical column photobioreactors are about 0.2 m diameter and 4 m column height [43]. In addition to considerations of space demands and ease of cleaning, an optimum column diameter must be determined as a compromise between productivity and capital cost [70].

3.4

Stirred tank reactor

Stirred tank reactors (STR) were originally proposed to grow microalgae photoautotrophically using articial light sources or sun light mainly because the STR was an industry and laboratory standard [31, 87, 88]. As an example, a Hydraulically Integrated Serial Turbidostat Algal Reactor (HISTAR) system, which had a total volume of 3.6 m3, was used to grow Selenastrum capricornutum Printz. HISTAR consisted of two sealed turbidostats and a series of open, hydraulically connected, continuous ow stirred-tank reactors (CFSTRs). The CFSTRs functioned as a biomass amplier of the inoculated culture [89]. This system was recently used to establish a deterministic model to predict microalgal productivity in order to establish practical feasibility for large scale application; however, subsequent implementation has not yet been reported.

these reactor designs and this depends in part on developments in material science. The idea of placing a light source inside the cultivation vessels has been repeatedly exploited by researchers. A photobioreactor using three concentric glass cylinders, with a light source mounted on the axis within them was described by Tsygankov [91], where Rhodobacter capsulatus was grown in the device at biomass concentrations up to 550 mg L-1 without light limitation. Patino et al. further investigated the growth of Chlorella vulgaris using an internal light source from either a 150-W xenon lamp or a uorescent at lamp tted into a special housing (Oriel, 60100) [92]. A cooling system and easy orientation of the light was provided by an F/4.4 ellipsoidal AlMgF2 reector. As mentioned previously, optical bers have also been used to collect and distribute solar and articial light into stirred tank photobioreactors [93] and have been used for mass cultivation of microalgae including Chlorella sorokiniana, C. vulgaris, and Phaeodactylum tricornutum [9496]; a cell density of l09 cells mL1 (30 g L1) for C. vulgaris was achieved [96]. Although optical ber assisted internal-illumination is a promising technology for providing light in closed photobioreactors, low efciencies in light delivery via optical bers has been reported;450% of the light was lost in transport through the bers [94]. The major advantages of using optical bers for internally-illuminated photobioreactors are that they can be heat sterilized and are stable to the mechanical agitation methods used in a conventional stir tank. Although adding considerably to capital costs, use of optical bers for illumination allows for scale-up while maintaining an efcient light supply and constant productivity [94, 99].

Approaches for lighting photobioreactors 5 Scaling up

The outermost layer of algae in the reactor can at times have so much light that growth is photo inhibited. On the other hand, the innermost layer of algae might not have enough light because of self-shading and, this reduces the volumetric biomass productivity per unit of incident light. Thus, efcient use of light in the culture is a major constraint in photobioreactors and makes their scale-up difcult. A number of different options have been studied to improve light penetration and control in photobioreactors. It is possible to enhance light intensity into a reactor by using an irradiance oriented (tilted) type of culture system or by using optical bers for added internal-illumination. The tilt at plate photobioreactor has succeeded in some microalgal cultivations with higher cell densities than that of a vertical at plate [5759]. However, the tilt system seems difcult to scaleup due to its complex operation and higher cost, which do not compensate for increases in productivity [60]. Recently, the Green Solar Collector (GSC), a light-and areaefcient photobioreactor with light redistributing plates and external light collection, was introduced for microalgal production [90]. It increased light capture efciency by 57% except in winter. However, the production costs of the GSC are expected to be higher than those of conventional outdoor photobioreactors, such as horizontal and vertical tubes or vertical panels, even despite ease of construction and maintenance and the use of cheap materials (PMMA) in GSC. Therefore, economy of scales may signicantly reduce costs of

Biomass productivities per area in reported photobioreactors are limited by suboptimal circumstances in the reactor, limiting biological efciency, or by a suboptimal design limiting light supply into the reactor. Design and scale-up methodologies for photobioreactors still have to be improved to achieve efcient light provision, minimize carbon dioxide losses, and ensuring efcient mixing and removal of photosynthetically generated oxygen [21, 100, 101]. Among the closed photobioreactors, the tubular and at plate designs have been applied in large-scale microalgal cultivation while the bubble column is more widely used in aquaculture. Although the airlift photobioreactor showed prior performance in algal culture than the bubble column, no signicant difference was observed in large-scale culture. Thus, the challenge is to also develop effective scale-up methods to enable large scale photobioreactors to function as well as at the laboratory scale. Computational uid dynamics (CFD) can be used to optimize the structural conguration of photobioreactors for scale-up. By using principles of uid dynamics the cost, workload, and design period can all probably be reduced. Yu et al. recently applied CFD methods to the inner structure parameter optimization of an airlift at-panel photobioreactor through a volume increase from 15 L to 300 L [102]. A model was developed to predict algal cell growth and then experimentally conrmed.

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Modularization is another option for scale-up. The advantage to this approach is the use of a relatively small scale reactor where light limitations, carbon dioxide losses, and inefcient mixing can be minimized or even eliminated. Furthermore, a modular system provides redundancy; if one module becomes contaminated or suffers a mechanical problem, the entire process is not lost. Recently, a 120-L multimodule photobioreactor, consisting of 18 elementary modules each with a 6.1 L working volume was commercialized [70]. Although this is much too small a capacity for some products like biofuels, it is reasonable for high value products like therapeutics. Further developments in scaling to large, reliable systems depend on continued research in photobioreactors and a break-through in the design of culture systems, where high photosynthetic efciencies and effective mass transfer are possessed and maintained in large scale and at high light intensities during long term operation.

The role of transgenic algae in photobioreactor operation

Until recently the potential for using transgenic algae for over production of useful compounds has not been realistic. Recently, however, methods for obtaining stable transformed algal lines are now much better developed [103] and have led to speculation that a number of interesting algal species can now be metabolically engineered to produce nutraceuticals, pharmaceutical proteins, and lipids and hydrocarbons for biofuels [1, 104, 105]. Consequently, it is important to consider how genetically altered microalgae might impact photobioreactor design and operation. Some interesting recent results provide insight on how reactor design might have to be changed. As described earlier, one of the drawbacks to growing microalgae in outdoor systems is their photoinhibition by high light intensity. Recent work by Mussgnug et al. (2007) has focused on altering the light harvesting complex (LCHI and LHCII) of photosystems I and II (PSI and PSII), respectively, to minimize this problem [106]. Using Chlamydomonas reinhardtii, they employed RNAi technology to down regulate the expression of the light harvesting complex (LHC) proteins. Compared to wild type, the genetically altered algal strain, Stm3LR3, showed about a 30% improvement in photosynthetic efciency. Although photon conversion into chemical energy can be inefcient, of primary concern here is that at high light intensities, the cells nearest the wall of the reactor receive such high light intensity that they become photoinhibited and can waste up to 90% of the incident light energy as uorescence and heat [106]. Cells behind the surface cells, therefore, receive decreased amounts of light not only from this wasted energy, but also from shading. While mixing helps to establish more homogeneity to obviate the latter, the overall efciency of the reactor is greatly impaired. By reducing the sensitivity of PSII to high light intensity, less of the incident light energy is wasted, so there is better light penetration into the reactor. With greater light penetration, it would be possible to increase the depth of the culture and this could result in use of larger reactor volumes, e.g.

wider tubular reactors, deeper ponds, etc., with an anticipated reduction in cost. Metabolically engineered algae are also likely candidates for culture in photobioreactors with some potential for impacting reactor design, but this mainly depends on the product being produced. For example, Spoehr and Milner rst showed that nitrogen starved Chlorella were able to produce up to 85% of their biomass as lipids, and many other algae show a similar response [7]. Using this type of algae for biofuel production might thus, require a two step process: rapid growth in medium replete with nitrogen, followed by a second step in nitrogen decient medium where growth essentially stops, but algae shift into lipid metabolism. One remedy would be to use two reactors in series to accommodate fuel production. On the other hand, a metabolically engineered alga that constitutively produces high levels of lipids would require only 1 reactor, and if coupled with a modied LHCII the reactor could also be designed with a longer light path. Clearly one of the nontechnical implications of using transgenic algae is that of public and environmental concerns about the safety of growing huge volumes of genetically modied cells especially in outdoor reactors. Given the current at best tepid public acceptance of genetically modied crops, one must assume that similar resistance may be encountered towards large outdoor algal cultivation systems. Use of transgenic algae, therefore, may preclude use of open ponds and instead mandate focus on either outdoor or indoor fully enclosed photobioreactors. Indeed use of closed systems would help to defuse public concerns, but such systems would likely be expected to follow the guidelines expected of any released genetically modied species. Although it would not impact actual photobioreactor design, the overall system would probably have to also include spill containment technology to preclude escape of any transgenic alga into the environment and water systems.

Conclusions

Of the many types of culture systems proposed for microalgal culturing, open pond systems have dominated. However, during the past decades, great progress has been made in bioengineering and biotechnology for efcient microalgal mass production. Thus, several types of closed culture systems, such as tubular or at plate photobioreactors show promise for application in large scale microalgal culture for bioenergy, aqua- and agricultural uses. Difculties arise with scale-up because relative volumes of light and dark zones change as the tube diameter or the plate thickness increases and the large area of land required. New developments in lighting and its control and in scale-up are needed to make closed culture systems cheaper, controllable and more efcient. The future of microalgal biotechnology appears promising, and innovative processes and products are expected to emerge over the next few years.

Acknowledgements
This work was nancially supported in part by the National Basic Research Program (973 Program) of China

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(No.2007CB714301) and the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KGCX2-YW-337). PJ Weathers is partially supported by a US Department of Energy grant (No. DE-FG36-08GO88025).

Conict of interest
The authors have declared no conict of interest.

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