Marine Microalgae

Abstract

Marine microalgae, the largest primary producer in the marine system, have been attracting wide attention as potential resources of new metabolites and biofuels. Whole genome sequencing and genetic modifications of microalgae have been rapidly advancing during the last few decades. In this chapter, the diversity of marine microalgae, the microalgal natural components, and the biotechnologies associated with microalgae are reviewed.

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1 Overview

Algae are the primary producers of oxygen in the aquatic environment. These microorganisms are widely distributed in marine systems and have a great diversity with respect to size, morphology, life cycle, pigments, and metabolism. About half of the global oxygen production is accomplished by marine microalgae. They play an important role in ${\mathrm{CO}}_2$ recycling through photosynthesis, which is similar to higher plants in ${\mathrm{O}}_2$-evolved systems (GlossaryTerm

PSI

(photosystem I) and GlossaryTerm

PSII

(photosystem II)). In addition to having a long history of use as food and as live feed in aquaculture, microalgae have also been considered as a promising source for high-value added products for pharmaceuticals, cosmetics, and other industrial applications, such as β-carotene, astaxanthin, polyunsaturated fatty acids (GlossaryTerm

PUFA

s). The utilization of microalgae as a suitable feedstock for sustainable biofuel production has gained worldwide attention over the last $20$ years. In general, microalgal triacylglycerols, hydrocarbons, and polysaccharides are considered as biofuel precursors. Alkanes and short chain fatty acid methyl esters (GlossaryTerm

FAME

s), ranging from C9 to C17, have been considered as the potential alternative jet fuel, while those ranging from C9 to C23 have been mainly recognized as biodiesel. GlossaryTerm

FAME

s are normally generated from the methyl esterification of triacylglycerols. On the other hand, with the degradation and fermentation of microalgal polysaccharides, the generated ethanol can be used as an alternative fuel to gasoline. Compared with higher plants, biofuel from microalgae has two advantages: 1) a relatively higher productivity, and 2) no competition to agriculture.

On the other hand, increased reports of the microalgal whole genome sequence data have been significantly facilitating the better understanding of their evolutionary lineage and the species specificity of the microalgal metabolic pathway. In addition, gene transformation has been achieved in $18$ microalgal genera.

In this chapter, we reviewed the fundamental characteristics of marine microalgae, the useful microalgal natural products, as well as the biotechnological aspects of marine microalgae.

2 Marine Microalgae

Algae are a very diverse group of photosynthetic organisms other than land plants, which have been classified into many classes, such as Cyanophyceae, Chlorophyceae, Rhodophyceae, Cryptophyceae, Dinophyceae, Bacillariophyceae, Haptophyceae, Euglenophyceae or Prasinophyceae. For convenience, they are referred to as blue-green algae, green algae, brown algae, or red algae due to the difference in composition of photosynthetic pigments. However, it is difficult to make a clear definition of algae because even multicellular eukaryotic microalgae (what is called seaweed) are also included. In this section, some representative marine microalgae are summarized to introduce their biotechnological applications.

Cyanophyceae (cyanobacteria, blue-green algae) are oxygenic photosynthetic prokaryotes that comprise a single taxonomic and phylogenetic group. Chloroplasts in eukaryotes evolved from endosymbiotic cyanobacteria. They show a large diversity in their morphology, physiology, ecology, biochemistry, and other characteristics. Typically, cyanobacteria contain chlorophyll a and phycocyanin. Three genera, i. e., Prochlorococcus, Prochloron, and Prochlorothrix, lack phycocyanin and possess chlorophyll a and b [1]. A unicellular cyanobacterium that synthesizes chlorophyll d has also been discovered [2]. Marine Synechococcus and Prochlorococcus contribute largely to global oxygen production. Cyanobacteria have gained attention as a source of bioactive compounds and biopolymers (polyhydroxyalkanoates (GlossaryTerm

PHA

)s) [3]. Bioactive compounds isolated from marine cyanobacteria were summarized by Burja etal [4] and Takeyama and Matsunaga [5]. Several strains of cyanobacteria (Synechococcus elongates and Anabaena variabilis) have been reported to produce long-chain alkanes and alkenes [6]. These findings make cyanobacterial alkane and alkenes a promising source of biofuels [7]. Two enzyme families that are responsible for straight-chain hydrocarbon production in cyanobacteria have recently been identified as an acyl–acyl carrier protein reductase (GlossaryTerm

AAR

) and an aldehyde-deformylating oxygenase (GlossaryTerm

ADO

). These enzymes convert fatty acid intermediates to alkanes and alkenes. This discovery of the cyanobacterial alkane biosynthesis indicates possibilities for optimizing the biodiesel production in cyanobacterial strains with modest gains in alkanes [8].

Chlorophyceae, which are one of the classes of green algae, possess chlorophyll a and b, the same predominant photosynthetic pigments as those of land plants. Chlorophyceae form starch in the chloroplast as a storage product of photosynthesis. Especially, Chlamydomonas reinhardtii has been used as a representative eukaryotic microalgae for biology and molecular biology studies. Chloroplast transformation was firstly achieved in C. reinhardtii [9]. Some species of Chlorophyceae are found in the marine environment. A marine species of Chlorophyceae, Dunaliella has been cultivated commercially for food supplements and β-carotene production [10]. Chlorella, which is a genus of single-cell and chlorophyll a/b-containing algae, belongs to the phylum Chlorophyta. It has been known as a potential food resource because of its high content of protein and other nutrients. Miura and others [11] reported that Chlorella sp. NKG $042401$ contains $10\%$ γ-linolenic acid (C18:3), which is present in the cells mainly in the form of galactolipids. In Euglenophyceae, the genus Euglena is well known. The chloroplast of Euglena originated from the eukaryotic green algae and contains chlorophyll a and b. Although most species are found in freshwater environments, some species also occur in marine environments.

Bacillariophyceae (diatoms) possess chlorophyll a and c, and fucoxanthin as the major carotenoid. Diatoms are widely used as feed in mariculture/aquaculture [12, 13]. Chaetoceros calcitrans, Chaetoceros gracilis, Chaetoceros muelleri, Skeletonema costatum, and Thalassiosira pseudonana are commonly used as live feed for all growth stages of bivalve molluscs (e. g., oysters, scallops, clams, and mussels), for crustacean larvae, and for zooplankton used as feed for larvae. The genera Navicula, Nitzschia, Cocconeis, and Amphora also are used to feed juvenile abalone. They store energy either as lipids or as chrysolaminarin. Most diatoms have a high content of eicosapentaenoic acid (GlossaryTerm

EPA

) $20:5$ (n-3). Phaeodactylum tricornutum and Nitzschia laevis have been especially investigated for GlossaryTerm

EPA

production. In addition, GlossaryTerm

EPA

production by diatoms was reviewed recently by Lebeau and Robert [14, 15]. Recent advances in heterotrophic production of GlossaryTerm

EPA

by microalgae were also reviewed by Wen and Chen [16].

The cells of Haptophyceae are brownish or yellowish-green and contain chlorophylls a/c and carotenoids such as β-carotene, fucoxanthin, diadinoxanthin, and diatoxanthin. The cells are commonly covered with scales made mainly by carbohydrates or calcium bicarbonate. Many species known as coccolithophorids produce calcified scales called coccoliths. Most are primarily marine species inhabiting tropical seawater. Microalgal biomass of Haptophyceae is commonly used as living feed in aquaculture [17]. Isochrysis galbana and Pavlova lutheri, especially, are used as living feed for bivalve molluscs, crustacean larvae, and zooplanktons that in turn are used for crustacean and fish larvae. Some cells can produce GlossaryTerm

PUFA

s such as docosahexaenoic acid (GlossaryTerm

DHA

), or GlossaryTerm

EPA

. In addition, the DHA content in I. galbana has been shown to be enhanced by low temperature or incubation of the culture in the dark after reaching the plateau phase growth [18]. Furthermore, it was shown that these algae are useful for GlossaryTerm

DHA

enrichment of feed such as rotifers for the larvae of several marine fish species [19].

In Dinophyceae, a genus Symbiodinium (dinoflagellate) has been well described. Various marine invertebrates, such as reef-building corals, jellyfish, sea anemones, and bivalves form symbiotic associations with Symbiodinium, commonly known as zooxanthellae. Symbiodinium strains have been classified into more than three clades using restriction fragment length polymorphism based on 18S GlossaryTerm

rRNA

sequence analysis [20]. The composition of Symbiodinium populations may also play an important role in the tolerance or sensitivity of corals towards bleaching.

3 Microalgal Genomes

Sequencing of microbial genomes has become a routine procedure for gene discovery and genetic engineering of microalgae. Synechocystis sp. PCC $6803$ was the first photosynthetic organism for which the entire genome sequence was determined. Currently, $72$ finished cyanobacterial genome sequences are listed in GenBank, and many additional genome analyses are currently in progress. Most cyanobacteria possess a circular chromosome and a small number of additional plasmids. Genome sizes range from a minimum of $1.44\mathrm{Mb}$ for the marine cyanobacterium UCYN-A [21] to a maximum of $11.58\mathrm{Mb}$ for the Calothrix sp. PCC7103 [22]. Prokaryotes typically contain a single copy of their chromosome such as Escherichia coli, while large differences between cyanobacteria and other prokaryotes have been reported for chromosomal copy numbers. Some cyanobacteria are oligoploid, for example, Synechocystis sp. PCC $6803$ are highly polyploid, and the motile wild-type strain contains $218$ genome copies in exponential phase and $58$ genome copies in linear and stationary phases [23].

Recently, a comparative genomics-based approach was used to screen cyanobacteria for the direct production of alkanes, the primary hydrocarbon components of gasoline, diesel, and jet fuel [8]. Eleven different cyanobacteria with available genome sequences were grown, and their culture extracts were evaluated for hydrocarbon production. Indeed, ten of these strains produced alkanes. The comparison of predicted proteins from these ten genomes against the eleventh finally led to the discovery of two hypothetical proteins as candidates for alkane biosynthesis. This discovery is the first description of genes responsible for alkane biosynthesis and the first example of a single-step conversion of sugar to fuel-grade alkanes by an engineered microorganism. A comparison of the genome sequences of producing and non-producing organisms led to the identification of the responsible genes.

In eukaryotic microalgae genomics, large-scale sequencing has been demonstrated by next-generation sequencing technologies. These have drastically increased the number of bases obtained per sequencing run while at the same time decreasing the costs per base. The first whole genome sequence of C. merolae was determined in 2004 [26]; this was the first identified eukaryotic microalgal genome. Up until November 2012, the whole genome sequence of $11$ strains of microalgae had been sequenced, including $2$ diatoms (Thalassiosira pseudonana [24] and Phaeodactylum tricornutum [25]), a red alga (Cyanidioschyzon merolae [26]), and $8$ green algae (Chlamydomonas reinhardtii [27], Ostreococcus lucimarinus [31], Ostreococcus tauri [32], Chlorella variabilis [28], Volvox carteri [30], Coccomyxa subellipsoidea [33], Micromonas pusilla [29], and Micromonas sp. [29]), see Table 5.1. In addition, the draft genome sequences of $17$ strains of microalgae can been found in the GlossaryTerm

NCBI

GenBank databases [34]. With next generation technology, the draft genome sequence of the biodiesel producing microalga Nannochloropsis gaditana strain CCMP526 were also identified recently [35]. The identified microalgal whole genome sequences provide a powerful tool for the discovery of genes and metabolic pathways. Even though most of the predicted microalgal pathways have been proved to be similar to corresponding pathways in higher plants, the urea cycle identified from genomes of diatoms is absent in higher plants but present in animals [24]. The existence of an animal metabolic pathway in microalgal cells further highlights the importance of genome analysis for microalgae.

Tab. 5.1 Sequenced whole genomes of microalgal strains

4 Genetic Engineering of Microalgae

4.1 Genetic Transformation Methods

Genetic studies on microalgae have been redirected mainly toward analysis of photosynthesis and metabolic pathways. A limited number of microalgae such as cyanobacteria have been used in biotechnological applications. The development of molecular techniques for physiological analysis and enhancement of biotechnological applications is necessary. Many attempts at gene transfer have been made in eukaryotic and prokaryotic microalgae. Genetic manipulation in prokaryotic microalgae cyanobacteria was studied extensively after several transformable unicellular strains were discovered. First, the freshwater cyanobacterium Synechococcus PCC7942 was reported to have the ability to take up GlossaryTerm

DNA

. Subsequently, several other naturally transformable freshwater strains were found. Gene transfer has been developed mainly in the freshwater strains Synechococcus, Synechocystis, Anabaeba, and Nostoc [69]. Several marine cyanobacterial strains of the genus Synechococcus have been also used for heterogeneous gene expression and other genetic applications [70, 71]. There are two commonly used gene transfer procedures: transformation using naturally occurring or artificially competent cells, e. g., conjugation with Escherichia coli, or physical methods for gene introduction, e. g., electroporation and particle bombardment. Natural transformation has been reported for Synechococcus sp. PCC7002 [72]. Other strains have been transformed successfully by electroporation or conjugation. Further, plasmids harvested from several marine microalgal species have been used as vector GlossaryTerm

DNA

for gene transfer. Marine plasmids have been found in Synechococcus sp. NKBG042902, which has a high phycocyanin content and a rapid growth rate. This strain contains more than three cryptic endogenous plasmids, and one of these, the plasmid pSY10 has the unique replication characteristic that its copy number increases under high salinity conditions [73]. Plasmids are maintained at a high copy number in cyanobacteria, which suggests the possibility that they act as a shuttle vector between cyanobacteria and E. coli. In fact, a shuttle vector with E. coli has been constructed using pSY10. Conjugative gene transfer using a broad-host range vector pKT230 was successful for the marine cyanobacterium Synechococcus sp. NKBG 15041C [74]. It has been demonstrated that this plasmid is stably maintained in cyanobacterial cells [75]. In marine cyanobacteria, in addition to the plasmid vector system, the construction of a phage vector system is also required to enable the cloning of large GlossaryTerm

DNA

fragments in specific cyanobacterial hosts. Since cyanophages were first reported by Safferman and Morris [76], various types of cyanophages have been found in seawater [77, 78] and characterized according to their genetic diversity and phylogenetic affiliations [79].

Due to the advance of genome, proteome, and metabolome analyses of microalgae, many attempts at gene transfer to eukaryotic microalgae have been made to enhance the production of useful compounds and biomass. However, because of the stiff cell wall of microalgae, the introduction of exogenous genes into microalgal cells could be challenging. The additional frustules and coccoliths surrounding some species of microalgae cells further increases the difficulty. Thus, the optimization of the gene transformation method for each specific species turns out to be important. Depending on the physiological characteristics of microalgal cells, electroporation, glass beads-mediated transformation, agrobacterium-mediated transformation, and biolistics have frequently been used. Moreover, the level of target protein varied due to multiple insertion, random integration, and (or) gene silencing [80]. Stable transformants that have already been reported are summarized in Table 5.2.

Tab. 5.2 Microalgal strains achieved for the stable transformation

Biolistics, also referred to as a gene gun that was originally designed for the delivery of nucleic acid through the cell wall of intact plant cells, has been mostly applied for microalgae gene transformation. The payload in this system is a plasmid GlossaryTerm

DNA

-coated tungsten particle (particle size: $0.6$–$1.6\mathrm{\mu }\mathrm{m}$), which can be shot with helium gas. After bombardment, the tungsten particles were shot down to the plant organism or the cell culture on the petri dish. Some cells that are not disrupted by the firing may envelope the GlossaryTerm

DNA

-coated tungsten particles and the GlossaryTerm

DNA

can then migrate to and integrate into the plant chromosome [81]. The transformation efficiency of this methodology is not related to the physical property of the host cell but is highly controlled by the gas pressure at the point of firing. Therefore, theoretically, despite the hard cell wall and frustules, gene transformation can be achieved when the gas pressure is high enough.

Electroporation is a phenomenon when the electrical conductivity and permeability of the cell membrane increase by the externally applied electrical field. If the host cells and plasmids are mixed together, the plasmids can be transferred into the host cells through the transient holes in the cell membrane generated by the electronic shock. Electroporation-based gene transformation methodology has been commonly used to transform mammalian cells with plasmids. The transformation efficiency was significantly decreased for plant cells due to their thick cell walls. Electroporation-based gene transformation has only been achieved with the Chlamydomonas reinhardtii cell wall-deficient mutant and Dunaliella salina cells, which have no cell wall [46, 57, 82]. However, in the studies mentioned above, the transformation efficiency was tenfold higher than the gene gun method applied to the corresponding strains [83].

The glass beads method is a relatively simple transformation procedure that has a higher transformation efficiency than biolistics but it is only capable of transforming cells without cell walls. Both the cell-wall deficient C. reinhardtii mutant and D. salina have been reported to have been successfully transformed by the glass bead method with a higher efficiency than with the gene gun method [56].

Agrobacterium-mediated transformation is based on the characteristic of the soil bacterium Agrobacterium tumefaciens that it naturally transfers and inserts its genes into plant chromosomes. Exogenous genes can be transferred into plant cells through Agrobacterium transformation using target gene inserted agrobacterium transfer GlossaryTerm

DNA

(GlossaryTerm

T-DNA

). Although reports of microalgae transferred by agrobacterium transformation are few, Kathiresan etal achieved a twofold transformation efficiency with Haematococcus pluvialis over the gene gun method [60].

In the transformants generated by the methods mentioned above, it is not rare to find the continuous expression of the target genes in the chloroplast and (or) mitochondria due to the insertion of the target genes into their organelle genome. By using a specific vector containing a homologous sequence in the organelle genome, stable chloroplast and (or) mitochondria transformation can be expected. On the other hand, the target genes are usually found to be randomly inserted into the nucleic genome and even homologous recombination occurs. Thus, it is hardly possible to control the insertion site and the number of the target genes inserted into the nucleic genome, which has made gene functional analysis via gene knock-out difficult. With further consideration of the dual nature of the microalgal life cycle as either haploid or diploid, the possibility of complete knock-out dwindled significantly in diploid cells. The homologous recombination has been applied to the transformation of C. reinhardtii and Volvox carteri, which maintain an asexual haploid zoospore during the life cycle; their recombination efficiency, however, was inferior [84, 85]. Recently, highly efficient homologous recombination was reported in Nannochloropsis sp., which suggested the possible use in microalgal gene functional analysis [68]. For those diploid microalgae, the knock-down of the target gene via GlossaryTerm

RNAi

has been reported and considered as the substitute for knock-out [40, 44, 55, 64].

So far, six microalgae including Phaeodactylum tricornutum, Thalassiosira pseudonana, Chlamydomonas reinhardtii, Chlorella vulgaris, Volvox carteri, and Cyanidioschyzon merolae have not only obtained stable transformants but also the whole genome sequence. P. tricornutum has been widely used for the studies of metabolic engineering towards enhanced lipid production. Yet, most research in this field has focused on the established stable transformant rather than on high oil-producing strains whose transformation method have not been determined.

4.2 Metabolic Engineering

Enhanced production of valuable primary or secondary metabolites in microalgae can be rendered possible by high density cultivation and/or application of genetic manipulation. Recent pharmaceutical interest in unsaturated fatty acids has triggered the search for sources of these valuable compounds. Several eukaryotic microalgae are known to produce highly unsaturated fatty acids such as GlossaryTerm

EPA

and GlossaryTerm

DHA

, which are valuable dietary components [16, 19]. Genetic engineering has been applied to produce GlossaryTerm

EPA

in the marine cyanobacterium Synechococcus sp. [71]. Cyanobacteria do not have the biosynthetic pathway to produce them. The GlossaryTerm

EPA

synthesis gene cluster (ca. $38\mathrm{kbp}$) isolated from a marine bacterium Shewanella putrefaciens SCRC-2738 was cloned to the marine cyanobacterium using a broad-host cosmid vector. The content of GlossaryTerm

EPA

grown at $2\text{°C}$ increased to $0.64\mathrm{mg}{\mathrm{g}}^{-1}$ dry cells after $24\mathrm{h}$ incubation at $17\text{°C}$. Furthermore, GlossaryTerm

EPA

production was improved by partial deletion of the GlossaryTerm

EPA

gene cluster to stabilize its expression and maintenance in host cyanobacterial cells [86].

Genetic engineering of microalgae for industrial purposes has also been performed in freshwater cyanobacteria where the ketocarotenoid astaxanthin, an extremely efficient antioxidant, was synthesized by the introduction of the β-c-4-oxygenase gene (crtO) from the green alga Haematococcus [87]. Ethylene production was also demonstrated in the cyanobacterium Synechococcus elongates PCC7942 by chromosomal insertion of an ethylene forming enzyme [88]. However, the reaction catalyzed by the ethylene forming enzyme induced metabolic stress, which was detrimental to the host cell.

Microalgal biodiesel production is expected to be improved though metabolic engineering. Several transformants have been established for the increased oil content of the microalgal cell, enhanced biomass productivity, and improved quality of the lipids. Acetyl GlossaryTerm

CoA

carboxylase (GlossaryTerm

ACCase

), which catalyzes the carboxylation of acetyl-GlossaryTerm

CoA

to form malonyl-GlossaryTerm

CoA

, the primary substrate of fatty acids synthesis, has been overexpressed in diatom cells to elevate the cell lipid content [36]. The vector containing the GlossaryTerm

ACCase

gene and its 5${}^{\prime }$ GlossaryTerm

UTR

(untranslated region) from the diatom Cyclotella cryptica was constructed and introduced into the diatoms C. cryptica and Navicula saprophila. Stable high GlossaryTerm

ACCase

expression transformants were obtained successfully, yet the predicted increase of neutral lipid content was not achieved, which indicates that GlossaryTerm

TAG

(triacylglycerol) accumulation in the microalgal cell is much more complex than previously assumed. With the expression of the hexose transporter that transfers the monohexose from the culture medium into the cell, the transformants of green algae C. reinhardtii and V. carteri as well as diatom P. tricornutum have been demonstrated to be capable to grow in the dark in a medium containing glucose [89, 90, 91]. Especially, the glucose transporter (Glut1) gene transformant of P. tricornutum cultured in dark conditions showed an almost threefold higher biomass production than in light conditions [91]. On the other hand, the de-regulation of the light-harvesting proteins in C. reinhardtii has been demonstrated to be able to elevate the solar energy conversion efficiencies in photosynthesis when the light-harvesting chlorophyll antenna size is minimized [92]. This permits a greater photosynthetic productivity under high cell density conditions as well as the possibility of culturing cells under high sunlight conditions. The transformation of Cinnamomum camphora (GlossaryTerm

C12-TE

) and Umbellularia californica (GlossaryTerm

C14-TE

) Acyl-GlossaryTerm

ACP

thioesterases genes into diatom P. tricornutum resulted in an increased lauric (C12:0) and myristic acid (C14:0) accumulation mutant [93]. Levels of lauric acid of up to $6.2\%$ of total fatty acids and myristic acid of up to $15\%$ by weight were achieved. Moreover, $75$–$90\%$ of the shorter chain length fatty acids produced were demonstrated to be incorporated into triacylglycerols.

5 Photobioreactors for Marine Microalgae

Microalgae mass cultivation for the production of useful compounds has been widely discussed since the 1950s. Even though large-scale production of astaxanthin, GlossaryTerm

DHA

, and GlossaryTerm

EPA

from microalgae have been achieved, the industrial production of microalgal biofuel is still under development. Lower cost and higher productivity and efficiency than current bioreactors are necessary due to the extremely low final price of biodiesel ($1{\mathrm{dollarL}}^{-1}$) compared with those high value-added microalgal products.

Both the biology and the economics of microalgae mass cultivation are strongly influenced by photobioreactor design. Photosynthetic microalgae can be cultured in photobioreactors as either an open culture system or a closed system. Based on their localization, these photobioreactors can be divided into outdoor culture systems or indoor culture systems.

Outdoor open culture systems are the simplest method of algal cultivation due to the low construction cost and effortless operation. However, the productivity of these systems can be easily affected by several environmental factors such as contamination of other microorganisms, changes of weather conditions, and the disability of transgenic microalgae cultivation. The need to achieve higher productivity and to maintain monoculture of algae led to the development of closed photobioreactors. Despite higher biomass concentration and better control of culture parameters, ${\mathrm{CO}}_2$ recycling efficiency, energy profit ratio, energy payback time, and cost of production in these enclosed photobioreactors are not better than those achievable in open systems.

The growth rate and maximum biomass yield of microalgal strains are affected by culture parameters (light, temperature, and pH) and nutritional status (${\mathrm{CO}}_2$, nitrogen, and phosphate concentration). On the other hand, increasing the density of cultures decreases photon availability to individual cells. Light penetration of microalgal cultures is poor, especially at high cell densities, and such poor photon availability decreases specific growth rates. Higher biomass yields can be expected if sufficient photons are provided in high density cultures of microalgae. Two major types of bioreactors (tubular [94, 95] or flat plate [96, 97]) are generally applied for the enclosed system (Fig. 5.1).

Fig. 5.1 a,b

Schematics of various photobioreactors. (a) After [95], (b) after [96]

Currently these enclosed photobioreactors are industrially applied for biodiesel production, for instance, the outdoor enclosed culture system used in Solix Biofuels Inc. and the indoor enclosed culture system used in Solazyme Inc. In addition, the use of intermittent light instead of continuous illumination can reduce the photoinhibition effect and improve the efficiency of light utilization through the flashing-light effect. On the other hand, the culture medium inside the photobioreactors needs to be stay flowing for mixing the algae and other nutrients. A newly developed flat plate photobioreactor achieved a $1.7$-fold increased biomass production for Chlorella vulgaris [96] by applying the intermittent light from one side of the bioreactor, and the introduction of the air bubbling from the bottom of the bioreactor which generates turbulence inside the container. Recently, based on the flashing-light effect, a triangle bioreactor constructed by three tubular bioreactors with the air bubbling from the hypotenuse has achieved the currently highest volumetric productivity ($365\mathrm{t}{\mathrm{ha}}^{-1}{\mathrm{a}}^{-1}$) [95, 98].