**4. Microalgal biomass production systems**

Commercial culture of microalgae targeted at their metabolites has been taking place for over 40 years, and the main microalgal species grown are *Chlorella* and *Spirulina* for healthy foods, *Dunaliella salina* for β-carotene, *H. pluvialis* for astaxanthin and several species for aquaculture [44].

There are several reactor configurations that met with success in mass cultivation of microalgae – chosen according to such factors as physiology of the microalga, cost of layout land, intensity of labour, cost of energy, availability of water, cost of nutrients, suitability of climate (if the culture is implemented outdoors) and specification of final product(s). Largescale culture systems should be compared on the basis of such indicators as efficiency of light utilization, controllability of temperature, hydrodynamic stress allowable, ability to maintain unimicroalgal and/or axenic cultures and feasibility of scale-up.

A major decision to be made is whether to use closed photobioreactors (PBRs) or open ponds to cultivate a given microalga. The latter may entertain a large area, and are relatively cheap to build and easy to operate – but contamination is hard to control, stable environmental conditions (particularly temperature) are difficult to maintain, and the attainable cell density is relatively low because of mutual shading effects. On the other hand, extensive areas of land will be needed for commercial exploitation, besides substantial costs of harvesting afterwards [45,46]. The final choice of system is always a compromise between these parameters, aimed at achieving an economically acceptable outcome [44].

A common feature of most microalgal species produced commercially (i.e. *Chlorella*, *Spirulina* and *Dunaliella*) is that they grow in highly selective environments – which means that they can be grown in open air cultures and still remain relatively free of contamination by other microalgae and protozoa [47-49]. Species of microalgae that do not possess this selective advantage must be grown in closed systems; this includes most marine algae grown as aquaculture feeds (e.g. *Skeletonema*, *Chaetoceros*, *Thalassiosira*, *Tetraselmis* and *Isochrysis*), as well as the dinoflagellate *Crypthecodinium cohnii* [44].

Typical systems used indoors for microalgal mass culture include carboys (10 to 20 L), polythene bags (100 to 500 L) and tubs (1000 to 5000 L); these are usually operated batch- or continuouswise [44]. For larger volumes, outdoor tanks or ponds are preferred, which are operated semicontinuously; depending on their scale, hatcheries may produce between several hundreds to tens of thousands of liters of microalgal biomass per day. However, the culture systems employed at present are still fairly unsophisticated: e.g. *D. salina* is cultured in large (up to ca. 250 ha) shallow open-air ponds with no artificial mixing; and *Chlorella* and *Spirulina* are also grown outdoors, in either paddle-wheel mixed ponds or circular ponds (up to 1 ha each) with a rotating mixing arm. The production of microalgae for aquaculture occurs generally on a much lower scale. Other commercial large-scale systems include tanks used in aquaculture, the cascade system developed in the Czech Republic [50] and heterotrophic fermenter devices used for culture of *Chlorella* in Japan and Taiwan [47,51], and for culture of *C. cohnii* in USA [52,53].

The choice of which configuration is preferable depends obviously on the objective function; e.g. wastewater treatment might preclude open systems, owing to the unacceptably high costs arising from the large volumes to be processed and the low added value of the resulting products [54]. There has been a major effort directed at examining alternatives for the production of fresh microalgae, and also at more costefficient production systems.

#### **4.1 Open cultivation systems**

Microalgae cultivation in open ponds has been in current used since the 1950s [44]; these systems have been categorized as natural waters (lakes, lagoons and ponds), and artificial ponds or containers – with raceway ponds being the most frequently used artificial system [55]. The four major types of open-air systems currently in use (i.e. shallow big ponds, tanks, circular ponds and raceway ponds) have all advantages and disadvantages. This type of system usually consists of either circular ponds with a rotating arm to mix the culture, or long channels in a single or multiple loop configuration stirred by paddle wheels [56] – although simpler configurations have also met with sucess [54]. Raceway ponds are usually built in concrete, but compacted earth-lined ponds with (white) plastic have also been proposed. In a continuous production cycle, broth and nutrients required by microalgal growth are introduced in front of the paddlewheel, and circulated through the loop to the harvest extraction point; said paddlewheel undergoes a continuous motion to prevent sedimentation. The CO2 requirement is usually satisfied using the open atmosphere as source – yet submerged aerators may be installed to enhance CO2 supply, and thus absorption yield [57].

Compared to closed photobioreactors, open ponds represent a less expensive investment for large-scale production of microalgal biomass. On the other hand, open pond production does not necessarily compete with agricultural crops for land, since it can be implemented in areas with marginal crop production potential [58]. Open ponds also have low energy input requirements [59], and regular maintenance and cleaning are easier [60].

Open ponds and raceways were the first large-scale designs implemented, and are still the most widely applied in industrial processing. The main constraints related to their operation are the difficulty to control contamination and to keep the culture environment

Typical systems used indoors for microalgal mass culture include carboys (10 to 20 L), polythene bags (100 to 500 L) and tubs (1000 to 5000 L); these are usually operated batch- or continuouswise [44]. For larger volumes, outdoor tanks or ponds are preferred, which are operated semicontinuously; depending on their scale, hatcheries may produce between several hundreds to tens of thousands of liters of microalgal biomass per day. However, the culture systems employed at present are still fairly unsophisticated: e.g. *D. salina* is cultured in large (up to ca. 250 ha) shallow open-air ponds with no artificial mixing; and *Chlorella* and *Spirulina* are also grown outdoors, in either paddle-wheel mixed ponds or circular ponds (up to 1 ha each) with a rotating mixing arm. The production of microalgae for aquaculture occurs generally on a much lower scale. Other commercial large-scale systems include tanks used in aquaculture, the cascade system developed in the Czech Republic [50] and heterotrophic fermenter devices used for culture of *Chlorella* in Japan and Taiwan [47,51],

The choice of which configuration is preferable depends obviously on the objective function; e.g. wastewater treatment might preclude open systems, owing to the unacceptably high costs arising from the large volumes to be processed and the low added value of the resulting products [54]. There has been a major effort directed at examining alternatives for the production of fresh microalgae, and also at more cost-

Microalgae cultivation in open ponds has been in current used since the 1950s [44]; these systems have been categorized as natural waters (lakes, lagoons and ponds), and artificial ponds or containers – with raceway ponds being the most frequently used artificial system [55]. The four major types of open-air systems currently in use (i.e. shallow big ponds, tanks, circular ponds and raceway ponds) have all advantages and disadvantages. This type of system usually consists of either circular ponds with a rotating arm to mix the culture, or long channels in a single or multiple loop configuration stirred by paddle wheels [56] – although simpler configurations have also met with sucess [54]. Raceway ponds are usually built in concrete, but compacted earth-lined ponds with (white) plastic have also been proposed. In a continuous production cycle, broth and nutrients required by microalgal growth are introduced in front of the paddlewheel, and circulated through the loop to the harvest extraction point; said paddlewheel undergoes a continuous motion to prevent sedimentation. The CO2 requirement is usually satisfied using the open atmosphere as source – yet submerged aerators may be installed to enhance CO2 supply, and thus

Compared to closed photobioreactors, open ponds represent a less expensive investment for large-scale production of microalgal biomass. On the other hand, open pond production does not necessarily compete with agricultural crops for land, since it can be implemented in areas with marginal crop production potential [58]. Open ponds also have low energy

Open ponds and raceways were the first large-scale designs implemented, and are still the most widely applied in industrial processing. The main constraints related to their operation are the difficulty to control contamination and to keep the culture environment

input requirements [59], and regular maintenance and cleaning are easier [60].

and for culture of *C. cohnii* in USA [52,53].

efficient production systems.

**4.1 Open cultivation systems** 

absorption yield [57].

steady, and the cost associated with harvesting. Furthermore, the open character of the system makes it possible for naturally occurring microalgae or their predators to infiltrate, and thus compete with microalgae intended for cultivation. Therefore, a monoculture can only be maintained under extreme conditions of pH, salinity or temperature that guarantee dominance by the desired strain (e.g. *D. salina* dominance requires highly salted media, whereas *Spirulina platensis* demands high pH values). Unfortunately, high pH, temperature and salt concentration are not compatible with most microalgal species of interest.

Regarding biomass productivity, however, open pond systems are less efficient than closed photobioreactors [61]. This can be attributed to such parameters as evaporation losses, temperature variation, CO2 deficiency, inefficient mixing and light limitation. Although evaporation losses make a net contribution to cooling, they may also cause significant changes in the ionic composition of the medium – with detrimental effects upon microalga growth [62]. Although this type of reactor is extensively used in industrial microalgal production – e.g. to produce *Spirulina* and *Dunaliella* spp. up to worldwide totals of 5000 and 1200 ton/yr, respectively [24], open systems have apparently reached their upper limit – with little room for further technological improvement.

#### **4.2 Closed cultivation systems**

Despite the success of open systems, future advances in microalgal mass culture will require improved closed systems, as the most interesting microalgal species cannot grow in highly selective environments [44]. Hence, photobioreactor technology is on the rise, which is designed to overcome the major constraints associated with open pond production systems [63]; recall that both pollution and contamination risks preclude use of open ponds to prepare high-value products for eventual use as active ingredients in aquaculture feed formulation [60].

Closed systems include tubular, flat plate and fermenter types, among other possibilities. The former two are specifically designed for efficient recovery of sunlight, whereas the latter may require artificial illumination. Owing to the higher cell mass productivities attained, harvesting costs can be significantly reduced. Closed photobioreactors also provide reproducible cultivation conditions, good heat transfer, better biomass yield, higher product quality and opportunity for flexible technical design [44,60]. Note, however, that the costs of closed systems are substantially higher than their open pond counterparts [54]. A variety of closed photobioreactors have been tested (or at least proposed) for industrial microalgal biomass production [64,65], but engineering and economic analyses of such reactors still lag behind the open ponds [66-70].

A typical photobioreactor is essentially a four-phase system, consisting of solid microalgal cells, a liquid growth medium, a gaseous phase and a light radiation field [71]. Its productivity is limited by various design features – but, most importantly, the reactor is to be operated under favorable illumination conditions, with optimized surface-to-volume ratio and light/dark cycle, coupled with adequate mass transfer features [72].

The current consensus is that commercial (photoautotrophic) production of metabolites with interest for aquaculture by microalgae should resort to outdoor enclosed photobioreactors [56,62,65,73,74]. Tredici [65] reviewed the development of those type of reactors over the last decade; while many types of experimental PBRs have been considered, built and tested, very few have actually succeeded on a commercial level. Commercial application of photobioreactor technology remains indeed restricted to the production of two Chlorophyte microalgae: *Chlorella* and *Haematococcus* [62,75].

Scale up of photobioreactors from bench to commercial scale is not trivial – since it needs changes in illumination, gas transfer and temperature to be taken into account, all of which are severely affected by turbulence in the reactor, and consequently require a tight control. Therefore, scale up appears to be much more of an engineering problem than a biological one; and general recommendations as to possible maximum scales have accordingly been produced [75,76].
