**3.1 Alterations in source strength**

The efficiency of solar energy conversion into chemical energy stored in biomass by plants and algae ranges from 3 to 5% of available solar energy. Theoretically, efficiencies as high as 11% for conversion of solar energy into the chemical energy in biomass can be achieved utilizing just the photosynthetically active radiation (400–700 nm) in the solar spectrum. Maximum efficiencies of energy conversion as high as 30% can be achieved using just red light (~650–700 nm) which is most efficiently harvested by the photosynthetic pigments [8, 37, 38]. Thus, it is conceivable that 2- to 4- fold increases in biomass yields are feasible through improvements in photosynthetic efficiency. It has long been recognized that the greatest potential for increasing photosynthetic efficiency is through enhanced light use efficiency by the photosynthetic apparatus (**Figure 2**) [39–41]. During photosynthesis, light saturates in all plants and algae at approximately one quarter of full sunlight intensity [38, 41]. Thus 75% of the energy captured by the photosynthetic pigments does no productive work leading to biomass production. Since the excess energy captured by the photosynthetic pigments does not drive electron transfer and carbon fixation processes it must dissipate through non-productive energy emission and/ or energy conversion pathways (heat, fluorescence, production of reactive oxygen species (ROS)) some of which (ROS) can lead to substantial damage to the photosynthetic apparatus further reducing biomass yields [42].

One approach to deal with the challenge of excess light absorption by the photosynthetic apparatus has been to reduce the optical cross section of the light-harvesting antenna complex to better couple the rate of light capture with rate-limiting electron transfer processes, i.e., plastohydroquinone oxidation by the

#### **Figure 2.**

*Organization of the peripheral light harvesting antenna complexes adjacent to the dimeric photosystem II reaction center in plants and green algae. Chlamydomonas transgenics having chlorophyll a/b ratios of 5 have lost the equivalent of one peripheral light-harvesting complex II trimer (LHC trimer). Figure modified from Dr. Jun Minagawa.*

cytochrome b6f complex and the development of an electron transport limiting trans-thylakoidal pH gradient [43, 44]. Various strategies have been developed to reduce the size of the light harvesting complex ranging from reducing the expression of the light harvesting complex proteins to targeted reductions in specific light harvesting pigment content often resulting in pleiotropic effects that indirectly affect photosynthetic efficiencies both negatively and positively [41, 45, 46]. Through the analysis of algae having a range in reduction in the light harvesting antenna size it has been empirically determined that the loss of approximately one third of the light harvesting apparatus (LHC2) results in maximum increases in photosynthetic efficiency of 20–30% and increases in biomass yield (40% greater) in both plants and green algae grown under outdoor cultivation conditions (**Figure 2**) [41, 45, 46]. A range in reductions of light harvesting antenna size were achieved by differential expression of the chlorophyllide a oxygenase gene (CAO) which produces chlorophyll b (Chl *b*). Chl *b* is present only in the light harvesting antenna complex proteins and not the photosynthetic reaction center. Since Chl *b* stabilizes the Chl *a/b* binding proteins, its reduction results in a corresponding loss in light harvesting antenna pigment-protein complexes. Significantly, a Chl *a/ b* ratio of 5 has been demonstrated both in plants and green algae to be optimal for achieving the greatest photosynthetic efficiency for plants and algae having altered light harvesting antenna sizes when grown at full sunlight intensity. Lesser or greater reductions in pigment (Chl *b*) content result in less than optimal photosynthetic performance due to indirect effects of Chl *b* reductions on the abundance of select light harvesting pigment-protein complexes, alterations in membrane architecture, reductions in energy transfer processes between the two photosystems, and increased susceptibility to photoinhibition [47, 48].

In nature, however, light intensities vary substantially over the course of the day, with depth in the canopy architecture or algal pond, and seasonally [48]. Theoretically, a light-harvesting apparatus that could be continuously adjusted in size to respond positively to differing light regimes would facilitate greater light use efficiency in dynamic light environments [47]. Recently, Negi et al. (2020) described a strategy for the continuous (daily) adjustment of the light-harvesting

**459**

*Recent Advances in Algal Biomass Production DOI: http://dx.doi.org/10.5772/intechopen.94218*

neered for improved photosynthetic efficiency.

antenna size in response to light intensity shifts in the green alga *Chlamydomonas reinhardtii* [47]. This dynamic antenna size regulation system is based on light regulated post transcriptional control of CAO activity. Protochlorophyllide a oxygenase (CAO) catalyzes the synthesis of Chl *b* which is found only in the peripheral, nuclear-encoded light-harvesting pigment-protein complexes. Light intensitydependent regulation of the light-harvesting complex size was achieved using as a host a CAO minus mutant which had been engineered to express a gene fusion product between the 5′ light regulated element (LRE) and the CAO gene [46]. A light regulated translational repressor, NAB1, binds to the LRE element and at high light represses translation of the modified CAO transcript reducing Chl *b* synthesis and decreasing the light harvesting antenna size. In low light such as occurs in dense cultures CAO translational repression by the NAB1 protein is reduced resulting in increased Chl *b* levels and increased light harvesting antenna size. Significantly, when the LRE-CAO transgenics were grown as monocultures under conditions mimicking those of a commercial production pond the transgenics had biomass yields that were more than two-fold higher than their wild-type parental strains. These are the greatest increases in biomass yield observed to date for algae engi-

Significantly, additional enhancements in photosynthetic rate are feasible in algae with optimized light harvesting antenna sizes. When the LRE-CAO transgenics were exposed to elevated bicarbonate concentrations there was an additional 20% increase in photosynthetic rates indicating that improvements in downstream carbon fixation processes could further enhance photosynthetic efficiency and biomass yield [46]. Obviously, elevated chloroplast CO2 concentrations could potentially suppress RubisCO oxygenase activity and photorespiration [49]. In addition to targeting single gene traits to enhance biomass productivity, engineering strategies based on altering the expression of master growth regulatory genes in algae has proven fruitful for increasing biomass yields. In *Chlamydomonas reinhardtii*, the blue light photoreceptor phototropin (Phot) plays a vital role in progression of the sexual life cycle [50, 51], the control of the eye spot size and light sensitivity and in the control of blue-light mediated changes in the expression of genes involved in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins [52]. Thus, it was anticipated that Phot expression could potentially play a role in regulating photosynthesis and biomass productivity. Negi et al., tested this hypothesis as well as identified downstream genes in the Phot regulatory pathway that were known to be master regulators of carbohydrate metabolism in plants

including analogues of the *Arabidopsis* KIN10 and KIN11 genes [53].

Based on a comparison of the photosynthetic attributes of two independent Phot mutants to their independent parental strains Negi et al., [50] demonstrated that the Chl a/b ratios were significantly greater in Phot mutants (2.9) than in wild type (2.0) grown at low light indicative of a smaller light harvesting antenna size in Phot mutants. When grown at high light intensities there was a further reduction in Chl a/b ratio (3.4) in Phot mutants indicating an ability to reduce the size of the light harvesting antenna grown resulting in increased light use efficiency [50]. The net result was that for Phot mutants photosynthetic rates were light-saturated at intensities 3-fold greater than for wild-type cells resulting in substantially accelerated cell division rates and biomass accumulation. RNAseq experiments indicated that these increases in productivity in Phot mutants were associated alterations in the patterns of expression for genes encoding enzymes involved photosynthesis, carbon metabolism, and those controlling cell division rates. Phot mutants had a 2- to 5-fold increase in the expression levels of multiple rate-limiting enzymes including; the Rieske Fe-S protein, ribulose-1,5-bisphosphate carboxylase/oxygenase, sedoheptulose 1,7 bisphosphatase glyceraldehyde-3- phosphate dehydrogenase, carbonic

#### *Recent Advances in Algal Biomass Production DOI: http://dx.doi.org/10.5772/intechopen.94218*

*Biotechnological Applications of Biomass*

cytochrome b6f complex and the development of an electron transport limiting trans-thylakoidal pH gradient [43, 44]. Various strategies have been developed to reduce the size of the light harvesting complex ranging from reducing the expression of the light harvesting complex proteins to targeted reductions in specific light harvesting pigment content often resulting in pleiotropic effects that indirectly affect photosynthetic efficiencies both negatively and positively [41, 45, 46]. Through the analysis of algae having a range in reduction in the light harvesting antenna size it has been empirically determined that the loss of approximately one third of the light harvesting apparatus (LHC2) results in maximum increases in photosynthetic efficiency of 20–30% and increases in biomass yield (40% greater) in both plants and green algae grown under outdoor cultivation conditions (**Figure 2**) [41, 45, 46]. A range in reductions of light harvesting antenna size were achieved by differential expression of the chlorophyllide a oxygenase gene (CAO) which produces chlorophyll b (Chl *b*). Chl *b* is present only in the light harvesting antenna complex proteins and not the photosynthetic reaction center. Since Chl *b* stabilizes the Chl *a/b* binding proteins, its reduction results in a corresponding loss in light harvesting antenna pigment-protein complexes. Significantly, a Chl *a/ b* ratio of 5 has been demonstrated both in plants and green algae to be optimal for achieving the greatest photosynthetic efficiency for plants and algae having altered light harvesting antenna sizes when grown at full sunlight intensity. Lesser or greater reductions in pigment (Chl *b*) content result in less than optimal photosynthetic performance due to indirect effects of Chl *b* reductions on the abundance of select light harvesting pigment-protein complexes, alterations in membrane architecture, reductions in energy transfer processes between the two photosystems, and

*Organization of the peripheral light harvesting antenna complexes adjacent to the dimeric photosystem II reaction center in plants and green algae. Chlamydomonas transgenics having chlorophyll a/b ratios of 5 have lost the equivalent of one peripheral light-harvesting complex II trimer (LHC trimer). Figure modified from*

In nature, however, light intensities vary substantially over the course of the day, with depth in the canopy architecture or algal pond, and seasonally [48]. Theoretically, a light-harvesting apparatus that could be continuously adjusted in size to respond positively to differing light regimes would facilitate greater light use efficiency in dynamic light environments [47]. Recently, Negi et al. (2020) described a strategy for the continuous (daily) adjustment of the light-harvesting

increased susceptibility to photoinhibition [47, 48].

**458**

**Figure 2.**

*Dr. Jun Minagawa.*

antenna size in response to light intensity shifts in the green alga *Chlamydomonas reinhardtii* [47]. This dynamic antenna size regulation system is based on light regulated post transcriptional control of CAO activity. Protochlorophyllide a oxygenase (CAO) catalyzes the synthesis of Chl *b* which is found only in the peripheral, nuclear-encoded light-harvesting pigment-protein complexes. Light intensitydependent regulation of the light-harvesting complex size was achieved using as a host a CAO minus mutant which had been engineered to express a gene fusion product between the 5′ light regulated element (LRE) and the CAO gene [46]. A light regulated translational repressor, NAB1, binds to the LRE element and at high light represses translation of the modified CAO transcript reducing Chl *b* synthesis and decreasing the light harvesting antenna size. In low light such as occurs in dense cultures CAO translational repression by the NAB1 protein is reduced resulting in increased Chl *b* levels and increased light harvesting antenna size. Significantly, when the LRE-CAO transgenics were grown as monocultures under conditions mimicking those of a commercial production pond the transgenics had biomass yields that were more than two-fold higher than their wild-type parental strains. These are the greatest increases in biomass yield observed to date for algae engineered for improved photosynthetic efficiency.

Significantly, additional enhancements in photosynthetic rate are feasible in algae with optimized light harvesting antenna sizes. When the LRE-CAO transgenics were exposed to elevated bicarbonate concentrations there was an additional 20% increase in photosynthetic rates indicating that improvements in downstream carbon fixation processes could further enhance photosynthetic efficiency and biomass yield [46]. Obviously, elevated chloroplast CO2 concentrations could potentially suppress RubisCO oxygenase activity and photorespiration [49].

In addition to targeting single gene traits to enhance biomass productivity, engineering strategies based on altering the expression of master growth regulatory genes in algae has proven fruitful for increasing biomass yields. In *Chlamydomonas reinhardtii*, the blue light photoreceptor phototropin (Phot) plays a vital role in progression of the sexual life cycle [50, 51], the control of the eye spot size and light sensitivity and in the control of blue-light mediated changes in the expression of genes involved in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins [52]. Thus, it was anticipated that Phot expression could potentially play a role in regulating photosynthesis and biomass productivity. Negi et al., tested this hypothesis as well as identified downstream genes in the Phot regulatory pathway that were known to be master regulators of carbohydrate metabolism in plants including analogues of the *Arabidopsis* KIN10 and KIN11 genes [53].

Based on a comparison of the photosynthetic attributes of two independent Phot mutants to their independent parental strains Negi et al., [50] demonstrated that the Chl a/b ratios were significantly greater in Phot mutants (2.9) than in wild type (2.0) grown at low light indicative of a smaller light harvesting antenna size in Phot mutants. When grown at high light intensities there was a further reduction in Chl a/b ratio (3.4) in Phot mutants indicating an ability to reduce the size of the light harvesting antenna grown resulting in increased light use efficiency [50]. The net result was that for Phot mutants photosynthetic rates were light-saturated at intensities 3-fold greater than for wild-type cells resulting in substantially accelerated cell division rates and biomass accumulation. RNAseq experiments indicated that these increases in productivity in Phot mutants were associated alterations in the patterns of expression for genes encoding enzymes involved photosynthesis, carbon metabolism, and those controlling cell division rates. Phot mutants had a 2- to 5-fold increase in the expression levels of multiple rate-limiting enzymes including; the Rieske Fe-S protein, ribulose-1,5-bisphosphate carboxylase/oxygenase, sedoheptulose 1,7 bisphosphatase glyceraldehyde-3- phosphate dehydrogenase, carbonic anhydrase, ADP glucose pyrophosphorylase, starch synthase, and genes involved in respiration and fatty acid biosynthesis. Additionally, genes involved in cell cycle control including; NIMA (never in mitosis), NEK2, NEK6 (NIMA related kinases), RCC1 (regulator of chromosome condensation, cyclin and cyclin-dependent kinases (CDK): Cyclin-dependent kinases, and MAT3 a homolog of retinoblastoma protein (MAT3/RB) were upregulated 2–15-fold in Phot mutants relative to their parental wild-type strains. The net result of this global alteration in gene expression was a two-fold increase in biomass productivity in Phot mutants relative to wild type [50].

Additional improvements in photosynthetic efficiencies have also been achieved by reducing apparent rate limitations in the Calvin–Benson–Bassham cycle (CBBC). Previous studies have demonstrated that the CBBC enzymes, fructose 1,6-bisphosphate aldolase (aldolase), sedoheptulose1,7-bisphosphatase (SBPase), and transketolase (TK), have the highest metabolic flux control coefficient values (maximum 0.55, 0.75, and 1.0, respectively) of any CBBC enzymes and thus have been targets for metabolic engineering to enhance carbon flux and accumulation in engineered plants and algae [54, 55]. Overexpression of the cyanobacterial dual functional fructose 1,6−/sedoheptulose 1,7-bisphosphatase (FBP/SBPase) and/ or plant SBPase was shown to significantly increase photosynthetic rates and growth in transgenic plants or algae [55, 56]. Similar to plants, mutagenesis studies in algae have demonstrated that hexokinase globally regulates genes involved in photosynthesis and hydrocarbon production and similar to Phot mutants can be manipulated to control biomass accumulation [57]. Thus, substantial gains in biomass productivity are feasible through targeted manipulations in both the light reactions and dark (CBBC) reactions of photosynthesis.