Research seeks to alter the optical characteristics of microalgae in order to improve solar-to-biofuels energy conversion efficiency in mass culture under bright sunlight conditions. This objective is achieved by genetically truncating the size of the light-harvesting chlorophyll arrays that serve to absorb sunlight in the photosynthetic apparatus.
© 2008 Optical Society of America
Unicellular microalgae hold promise as commercially viable vehicles in biofuels production. They are amenable to genetic engineering and exploitation in mass culture for biomass accumulation, carbon sequestration and biofuels generation. In any microalgal production system, the achievable solar-to-product conversion efficiency and photosynthetic productivity are the single most important factors in determining cost. The promise of microalgae is based on the measurement of a quantum yield of photosynthesis, which, under limiting intensity conditions, was reported to be equal to 0.103 oxygen evolved per photon absorbed . This quantum yield translates into a photon conversion efficiency of better than 82%, suggesting that the photosynthetic apparatus can efficiently utilize all absorbed photon if the rate of light absorption does not exceed that rate of photosynthesis (weak intensity of illumination). Based on a quantum yield of 0.103 O2 per hv, the productivity of microalgae under bright sunlight was estimated to be up to 75 g dry weight m-2 d-1 [2, and Melis, unpublished]. However, small-scale cultures of microalgae grown under full sunlight show maximal photosynthetic productivity of about 20–30 g dw m-2 d-1 [3, 4]. The reason for this discrepancy is that green algae assemble large arrays of light absorbing chlorophyll (Chl) antenna molecules in their photosystems. Up to 600 Chl a and Chl b molecules can be found in association with PSII and PSI [2, 5]. At high solar intensities, the rate of photon absorption by the large Chl antenna of the first few layers of cells in the mass culture far exceeds the rate at which photosynthesis can utilize them, resulting in dissipation and loss of excess photons as heat or fluorescence. Up to 80% of the absorbed photons could thus be wasted , minimizing solar-to-product conversion efficiencies and photosynthetic productivity to unacceptably low levels. In addition to the wasteful dissipation of excitation, and also due to the high rate of photon absorption by the photosynthetic apparatus, cells at the surface of the mass culture are subject to photoinhibition of photosynthesis [6, 7], a phenomenon that compounds losses in productivity. Meanwhile cells deeper in the culture are deprived of much needed solar energy, as this is strongly attenuated due to the filtering [2, 8, 9].
Biotechnological applications of microalgae in the field of biomass accumulation, carbon sequestration and biofuels production require utilization of strains that are not subject to the above-described optical pitfall and suboptimal utilization of sunlight in mass culture, and which operate with maximal light utilization efficiency and photosynthetic productivity under bright sunlight. To achieve these performance characteristics, it is necessary to minimize the absorption of sunlight by individual cells so as to permit greater transmittance of irradiance through the high-density green alga mass culture. This requirement was recognized long ago [10–12], but could not be satisfied because algae with a “truncated light-harvesting chlorophyll antenna size” are not encountered in nature. Until recently, this problem could not be addressed and solved in the laboratory either, due to the lack of the necessary technologies by which to approach it. The advent of molecular genetics in combination with sensitive absorbance-difference kinetic spectrophotometry for the precise measurement of the Chl antenna size in photosynthetic systems now offer a valid approach by which to pursue a genetic reduction in the number of Chl antenna molecules that service the chloroplast photosystems.
The main objective of the research in the field of bioengineering of the optical properties of microalgae is to minimize the Chl antenna size of the two photosystems to a combined low of 132 Chl molecules (37 for PSII and 95 for PSI). This is the smallest Chl antenna size that will permit assembly of the photosystems in chloroplasts . Such Chl antenna size configuration of the photosystems would compromise the competitive ability and survival of the cells in the wild. However, it would enable efficient solar-to-product conversion by the cells in mass culture, leading to high rates of biomass accumulation, and hydrogen (H2) or hydrocarbon (HC) production by these microorganisms.
2. Physiological state of the Chl antenna size and its molecular regulation in microalgae
Life on earth is sustained by oxygenic photosynthesis, a process that involves absorption and utilization of light energy from the sun. The chemical energy stored by this endergonic process results in the oxidation of water molecules, the release of oxygen and the generation of reductant (reduced ferredoxin) and ATP. Reduced ferredoxin and ATP are necessary and sufficient to support autotrophic growth by the organism, including the conversion of atmospheric carbon dioxide to sugars, and the generation of protein, lipid and other cellular matter. The absorption of sunlight and the conversion of excitation energy to chemical energy takes place in photosystem II (PSII) and photosystem I (PSI) in the thylakoid membrane of the chloroplast . In each photosystem, chlorophylls and other accessory pigments act cooperatively in the absorption of incoming solar radiation [15, 16]. The concept of the photosynthetic unit, first proposed by Gaffron and Wohl , stipulates that distinct assemblies, or arrays, of photosynthetic pigments serve as antennae for the collection of light energy and as a conducting medium for excitation migration toward a photochemical reaction center. Distinct pigment-protein complexes are contained within PSI and PSII and perform the functions of light absorption and excitation energy transfer to a photochemical reaction center [18, 19]. Up to 350 chlorophyll a (Chl a) and Chl b molecules can be found in association with PSII, whereas the Chl antenna size of PSI may contain up to 300 mainly Chl a molecules [5, 9, 20]. Most of these Chl molecules are organized within peripheral subunits of the socalled auxiliary chlorophyll a-b light-harvesting complex (LHC). In higher plants, there are six such subunits for PSII (LHC b1-b6) and four for PSI (LHC a1-a4) . Several isoforms of these LHC subunits have been identified in the model microalga Chlamydomonas reinhardtii . The amount of these LHCs in the peripheral Chl antenna determines the size of the functional Chl antenna of the photosystems.
The Chl antenna size is not fixed but can vary substantially depending on the developmental, genetic, physiological and even environmental conditions. In general, low light intensity during growth promotes a large Chl antenna size (Fig. 1). Growth under high irradiance induces the assembly of a smaller Chl antenna size [20, 23–25] (Fig.1). Such adjustments of the Chl antenna size in response to irradiance are a guided compensation reaction, as they are inversely related to the incident irradiance intensity [20, 26]. This molecular regulatory mechanism (Fig. 1) is highly conserved in nature and functions in all oxygenic and anoxygenic photosynthetic organisms [20, 26–29]. Accordingly, there are genetic determinants that define the size and permit adjustments in the Chl antenna size of photosynthetic organisms. The genes and processes that define Chl antenna size are only now beginning to be understood.
A genetic tendency of the algae to assemble large arrays of light absorbing chlorophyll antenna molecules in their photosystems is a survival strategy and a competitive advantage in the wild, where light is often limiting . Obviously, this property of the algae is detrimental to the yield and productivity in a mass culture. A truncated Chl antenna would inevitably compromise the ability of the strain to compete and survive in the wild. However, in a controlled mass culture in photo-bioreactors, it would help to diminish the over-absorption and wasteful dissipation of excitation energy by individual cells, and it will also diminish photoinhibition of photosynthesis at the surface, while permitting for greater transmittance of light deeper into the culture. Such altered optical properties of the cells would result in greater photosynthetic productivity and enhanced solar conversion efficiency by the mass culture. In support of this contention, early validation experiments [2, 9] confirmed that a smaller Chl antenna size would result in a relatively higher light intensity for the saturation of photosynthesis in individual cells, while permitting for an overall greater productivity by the mass culture [31–33]. Thus, elucidation of the molecular mechanism for the regulation of the Chl antenna size via genetic approaches is of fundamental importance to the field and of practical importance to the algal biotechnology industry.
3. Rationale and approach
The rationale for attempting a bioengineering approach to truncate the Chl antenna size in green microalgae is that such modification will prevent individual cells at the surface of a high-density culture from over-absorbing sunlight and wastefully dissipating most of it (Fig. 2). A truncated Chl antenna size would permit greater transmittance of sunlight deeper into the culture, thus enabling many more cells to contribute to useful photosynthesis and culture productivity (Fig. 3). It has been shown that a truncated Chl antenna size will enable a ~3-fold greater solar-energy conversion-efficiency and photosynthetic productivity than could be achieved with fully pigmented cells . Such bioengineering, therefore, would enhance photosynthetic productivity of the microalgae in mass culture.
A systematic approach to this problem is to identify genes that determine and/or regulate the Chl antenna size of photosynthesis (Fig. 1) and, further, to manipulate such genes so as to confer a permanently truncated Chl antenna size to the model green alga Chlamydomonas reinhardtii. Identification of such genes in Chlamydomonas would permit a subsequent transfer of this trait to other microalgae of interest to the alga biotechnology sector. This objective has been approached in the laboratory of the authors upon application of DNA insertional mutagenesis techniques [33–34] screening, biochemical/molecular/genetic and absorbance-difference kinetic spectrophotometry analyses of C. reinhardtii cells. This truncated light-harvesting chlorophyll antenna size (tla) property may find application in the commercial exploitation of microalgae for the generation of biomass, biofuel, chemical feedstocks, nutraceutical, and pharmaceutical products [35–42].
4. Mutations in pigment biosynthesis and genetic manipulation of LHC proteins
Earlier efforts contributed with the identification of pigment biosynthesis mutants, and identification of the corresponding genes, conferring a partially truncated Chl antenna size in the model microalga C. reinhardtii. A Chl b-less mutant was identified and examined, which lacked a functional chlorophyllide a oxygenase (CAO) gene  and which failed to synthesize Chl b. This strain was derived upon DNA insertional mutagenesis, where the chlorophyllide a oxygenase gene was interrupted by the transforming plasmid . The advantage of such tagged genetic transformation for the generation of mutants is that genes responsible for a given property could then be isolated.
The Chl b-less mutant (termed cbs3) of C. reinhardtii had elevated QA/total Chl and P700/total Chl ratios relative to the wild type, signifying a smaller Chl antenna for the photosystems. Spectrophotometric-kinetic analysis revealed a significantly truncated Chl antenna size for PSII and a slightly increased Chl antenna size for PSI (Table 1, ). The PSII Chl antenna size in the Chl b-less mutant (Chl-PSII=~90 Chl a molecules) was substantially smaller than that in the wild type (Chl-PSII=230 a and b molecules). Nevertheless, the PSII Chl antenna size in the cbs3 strain remained significantly larger than that of the minimal PSII-core antenna (Chl-PSII=~37 Chl molecules, ). In contrast to PSII, the Chl antenna size of PSI in the Chl b-less mutant (Chl-PSI=290 Chl a molecules) was similar to, or even slightly larger than that of the wild type (Chl-PSI=240 Chl a and b molecules). It was concluded that the PSI auxiliary light-harvesting Chl antenna can fully assemble and be functionally connected with the PSI photochemical reaction center, even in the absence of Chl b. Additional studies were conducted to determine the role of the CAO gene in the function of the Chl antenna regulatory mechanism (Fig. 1) It was determined that CAO gene expression is highly regulated during Chl antenna size adjustments by the photosynthetic organism [29, 45]. Modulation of CAO gene expression exerted a significant effect on the Chl antenna size of PSII, but not so much on that of PSI. The CAO gene expression may thus be a target for generating a truncated PSII Chl antenna size.
Comparative measurements of the light-saturation curve of wild type and cbs3 photosynthesis revealed identical initial slopes (light-limiting conditions) indicating similar quantum yields of photosynthesis for the two strains. However, a significantly greater light-intensity was required for the saturation of photosynthesis in the cbs3 than in the wild type, consistent with the smaller functional Chl antenna size in the cbs3 strain (Fig. 4).
A C. reinhardtii double mutant npq2 lor1  lacked the β,ε-carotenoids lutein and loroxanthin as well as all β,β-epoxy-carotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). The only carotenoids present in the thylakoid membranes of the npq2 lor1 cells were β-carotene and zeaxanthin. The effect of these pigment mutations on the Chl antenna size of photosynthesis was investigated . Table 1 shows that the npq2 lor1 mutant had elevated QA/total Chl and P700/total Chl ratios relative to the wild type, signifying a smaller Chl antenna for the photosystems. Spectrophotometric-kinetic analysis revealed a smaller PSII light-harvesting Chl antenna size and a slightly larger PSI Chl antenna size for the mutant compared to the wild type.
Comparative measurements of the light-saturation curve of wild type and npq2 lor1 photosynthesis revealed identical initial slopes under light-limiting conditions, indicating similar quantum yields of photosynthesis for the two strains. However, a significantly greater light-intensity was required for the saturation of photosynthesis in the npq2 lor1 than in the wild type, consistent with the smaller functional Chl antenna size in the npq2 lor1 strain (Fig. 5). It was concluded that a lesion in the lycopene ε-cyclase gene prevents synthesis of lutein in green microalgae and this specific mutation affects the functional Chl antenna size of PSII but not that of PSI . Xanthophyll-biosynthesis genes in general, and the lycopene ε-cyclase gene in particular, may be suitable targets for a truncated PSII Chl antenna size.
5. Novel genes for the regulation of the Chl antenna size of photosynthesis
The ability of the photosynthetic apparatus to regulate the size of the functional Chl antenna was first recognized in pioneering work by Bjorkman, more than 30-years ago . In spite of the substantial number of physiological and biochemical studies on this phenomenon since then [5, 20, 26], genes that either determine or regulate the Chl antenna size of photosynthesis remained unknown. Recent bioengineering efforts by which to truncate the Chl antenna size of photosynthesis contributed to the first-time cloning of a Chl antenna size regulatory gene. This was achieved through the application of DNA insertional mutagenesis with the green alga C. reinhardtii. Based on the screening protocol applied , a mutant having a truncated light-harvesting chlorophyll antenna size (tla1) was identified. Genetic crosses and mapping of the DNA around the insertion site showed that the exogenous plasmid interfered with a novel gene, termed by us Tla1 . DNA, mRNA and protein sequences of the Tla1 gene were elucidated and deposited in the GenBank (Accession No. AF534570 and AF534571). Evidence was presented that the Tla1 gene is responsible for defining the Chl antenna size in green microalgae [33, 49].
Sensitive absorbance-difference spectrophotometry showed that the tla1 mutant had elevated QA/total Chl and P700/total Chl ratios relative to the wild type (Table 2). This effect of the tla1 mutation was more pronounced than that of the Chl b-less and lutein-less mutations (compare with Table 1). These results signified a smaller Chl antenna for the photosystems in the tla1 mutant. Spectrophotometric-kinetic analysis revealed that the tla1 mutant had a truncated PSII Chl antenna size, down to 50% of the wild type, and a truncated PSI Chl antenna size, down to 67% of that in the wild type (Table 2). Thus, in the tla1 strain, both PSII and PSI had a smaller Chl antenna size relative to the wild type, which is an improvement over the result of pigment mutations on the Chl antenna size of photosynthesis (Table 1).
Comparative measurements of the light-saturation curve of wild type and tla1 photosynthesis revealed identical initial slopes under limiting intensity of illumination, indicating similar quantum yields of photosynthesis for wild type and tla1 strains. However, a significantly greater light-intensity was required for the saturation of photosynthesis in the tla1 mutant than in the wild type, consistent with the smaller functional Chl antenna size in the tla1 strain (Fig. 6).
When plants are grown under low light intensity, the photsystems contain relatively high amounts of Chl b, have a large Chl a-b light harvesting complex (LHC) and a high LHC-to-PS core ratio [20, 26, 29]. As stated above, LHCs are divided into two distinct components, the so-called LHCI (for PSI) and LHCII (for PSII) protein families, based on their predominant association with PSI or PSII, respectively. These peripheral LHC subunits are not essential for the process of photosynthesis  and ideally, the corresponding genes (Lhcb and Lhca) could be deleted from the genome of the organism in order to limit the size of the Chl antenna. In practice, this gene deletion approach may be difficult because of the possible existence of multiple copies for each of these genes, all of which would have to be deleted. An additional difficulty is that, in the absence of one of the LHC subunits, the algae can recruit another protein subunit for the assembly of the fully pigmented Chl antenna [52, 53]. Recently, however, powerful gene silencing RNA interference technologies were applied in the model microalga C. reinhardtii to generate a mutant, termed stm3LR3, which had a significantly reduced content in both LHCII and LHCI proteins . The stm3LR3 strain exhibited lower levels of in vivo Chl fluorescence, a higher photosynthetic quantum yield, and a reduced sensitivity to photoinhibition, resulting in an elevated efficiency of cell cultivation under high irradiance conditions .
The above results [2, 31–33, 54] clearly suggest that a truncated Chl antenna size improves solar conversion efficiency and photosynthetic productivity in mass culture under bright sunlight conditions. However, a recent report by Huesemann et al.  addressed the performance of two algal strains with truncated Chl antenna and reported that, despite the theoretical prediction and expectation of a significant increase in light utilization efficiency and thus biomass productivity under bright sunlight, actual improvements in biomass yield were not observed in either semi-continuous laboratory cultures or outdoor ponds. However, truncated Chl antenna mutants by Huesemann et al.  were generated by ethylmethylsulfonate or UV mutagenesis. It is well known that chemical or UV mutagenesis will cause dozens, if not hundreds, of mutations in any single cell. Some of these mutations will interfere with the metabolic robustness of the organism, translating into loss of fitness and slower growth. This is a well know shortcoming of the chemical or UV mutagenesis approach, and explains the compromised performance of their “truncated antenna” mutants.
Further, Mussgnug et al.  reported on a state transitions mutant (stm3), which had increased levels of LHCII subunits and a lower Chl a/b ratio, indicating a larger than wild type Chl antenna size in the mutant. The affected nuclear gene in the stm3 strains encodes the RNA binding protein NAB1 (nucleic acid binding protein). NAB1 binds and stabilizes the LHCII mRNA at the pre-initiation stage via sequestration and thereby represses translation of the LHCII protein. Thus, over-expression of NAB1 gene in C. reinhardtii can apparently lead to generation of mutant strains with a smaller chlorophyll antenna size for PSII. Taken together, these results show progress toward identification of genes and elucidation of processes that define the chlorophyll antenna size of oxygenic photosynthesis.
6. Microalgal productivity in mass culture
The productivity of microalgae with a truncated Chl antenna size was tested under mass culture and high cell-density conditions. The tla1 strain had enhanced photosynthetic productivity and improved light utilization efficiency under mass culture conditions in the greenhouse , consistent with earlier mini-scale up studies [31, 32]. Fig. 7 shows such a mini-scale up experiment conducted in the greenhouse under ambient sunlight conditions, measuring the photosynthetic productivity of wild type and tla1 mutant . Fig. 8 presents a systematic plot of the results from such productivity experiments in the greenhouse. It is seen that the rate of photosynthetic O2-accumulation is a function of the Chl concentration (biomass amount) in the respective culture at the time of measurement. Results from this detailed analysis showed that productivity of green microalgae in a mass culture increases linearly as a function of cell density and Chl concentration in both the wild type and the tla1 mutant. The linear increase is observed under conditions when the amount of the biomass, but not irradiance, is the yield-limiting factor. This initially linear increase in the yield of the culture levels-off, as the green alga biomass reaches a certain density. The ‘saturation’ occurs because, at a threshold Chl concentration, cells in the culture would absorb all incoming irradiance. From that point on, light utilization efficiency would define yield [2, 33]. In the wild type, rate and yield saturated at a Chl concentration of about 2–3 µM, whereas rate in the tla1 mutant continued to increase, surpassing that of the wild type and reaching saturation at about 5 µM Chl. The Chl saturated rate of the tla1 mutant (~44 ml O2 per h) was about 2-fold greater than that of the wild type (~23 ml O2 per h). The slower rate and lower yield of the wild type in this experiment is attributed to the greater fraction of photons that are absorbed but not utilized , resulting in dissipation and loss of the excess photons as heat or fluorescence . This is apparently alleviated to some extent by the smaller Chl antenna size in the tla1 mutant.
It is an objective of this work to minimize, or truncate, the chlorophyll antenna size in green microalgae in order to maximize solar-to-product conversion efficiency and photosynthetic productivity in mass culture. Further, the work seeks to identify currently unknown genes that determine the Chl antenna size in photosynthetic organisms, and to demonstrate that a truncated Chl antenna size would minimize absorption and wasteful dissipation of sunlight by individual cells, resulting in better light utilization efficiency and greater photosynthetic productivity under mass culture conditions. To achieve these objectives, a promising approach was employed, based on DNA insertional mutagenesis, screening, biochemical and molecular analyses for the isolation of “truncated Chl antenna size” strains in the green alga C. reinhardtii. In addition to the Tla1 gene, efforts are currently under way to identify and characterize other novel gene(s) that affect or define the “Chl antenna size” in the model microalga C. reinhardtii. Eventually, the work seeks to genetically manipulate these genes to generate “truncated Chl antenna size” strains in C. reinhardtii and in other green algae of interest to the alga biotechnology sector.
Current progress suggests that a partially truncated chlorophyll antenna size of the microalgae alleviates the over-absorption of incident sunlight by individual cells in a high-density culture, and minimizes the wasteful dissipation of irradiance. A truncated light-harvesting chlorophyll antenna size in such mutants diminishes the severe cell shading that occurs with normally pigmented wild type, permitting a more uniform illumination of the cells in a mass culture, and resulting in a greater solar-to-product conversion efficiency and photosynthetic productivity of the algae under high cell density and bright sunlight conditions. Accordingly, the truncated light-harvesting chlorophyll antenna size (tla) property may find application in the commercial exploitation of microalgae for the generation of biomass, biofuel, chemical feedstock, as well as nutraceutical and pharmaceutical products.
The work was supported by the DOE Hydrogen, Fuel Cells and Infrastructure Technologies Program.
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