**3.2 Alterations in carbon sink strength**

Given the primary role of starch metabolism as carbon reserve and an intermediate in the production of hydrocarbons it is not unanticipated that alterations in starch metabolism may impact hydrocarbon and biomass yields [58]. For example, *Chlamydomonas* sta6 [ADP-glucose pyrophosphorylase] and sta7–10 [isoamylase] mutants having reduced capacity to synthesize starch had substantial increases in lipid accumulation during nitrogen deprivation relative to the wild-type controls but suppressed total biomass accumulation [58]. In addition, suppression of starch metabolism has been shown to impair upstream CBBC activity resulting in the dissipation of excess photosynthetically produced electrons through non-productive reduction of oxygen [54]. These results point to the central role of starch metabolism and accumulation in overall cellular homeostasis and biomass accumulation in algae and its impact on the thermodynamic efficiency of light energy conversion into chemical energy (biomass) [8, 58]. It has been estimated that carbohydrate metabolism can accounts for as great as 20% reductions in thermodynamic efficiency of photosynthesis [39]. These efficiencies can be further reduced by partitioning carbon into hydrocarbon storage products instead of starch. This is due to the central role of pyruvate (3C) metabolism in hydrocarbon (lipids, terpenes, and waxes) production. The production of acetyl CoA (2C) via the decarboxylation of pyruvate for hydrocarbon production results in the loss of 1/3 of the previously fixed carbon. In contrast, starch production from photosynthetically derived sugars has no associated decarboxylation steps. Hydrocarbons, however, have nearly twice the energy density of carbohydrates due to their more reduced state. Modeling studies indicate that the production of carbohydrates using solar photons is potentially 10–20% more efficient for solar energy conversion than hydrocarbon production [8]. Furthermore, the kinetics of lipid production are substantially slower than starch synthesis. Thus, algae that primarily store starch may accumulate biomass

**461**

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

) while lipids have a density of 0.91 g/cm3

tors versus lipid accumulators remains to be assessed.

**3.3 Product storage and metabolism**

cm3

faster than algae that store hydrocarbons as energy reserves. The ecological downside of starch storage, however, is that starch has high volumetric density (1.56 g/

algae that store starch must invest energy in motility devices and associated energy expenditures to avoid sinking to depths where light availability may be limiting for photosynthetic growth. It might then be predicted that algae that store starch, e.g., *Chlamydomonas*, predominantly inhabit soil environments that provide physical support whereas lipid accumulating algae, e.g., *Nanochloropsis*, tend to occupy aquatic environments where they are less dense or near the density of water and can remain at levels in the water column where light is not limiting for photosynthesis. To date, the relative energetic costs needed to support motility in starch accumula-

Following the metabolic engineering paradigm for increasing product yield, i.e., push, pull, sequester and block storage product turnover, less attention has been directed towards the metabolic engineering of storage and product turnover in microalgae. As stated previously, energy reserves in algae fall into two classes, carbohydrates, and lipids. The genetic manipulation of starch accumulation in algae has received much attention. The chloroplast is the site of starch synthesis and storage in plants and algae. In contrast to plant cells, however, microalgae typically have only a single chloroplast per cell since chloroplast division must be synchronized with cell division to ensure that each progeny has a chloroplast [59]. Thus, there is no differentiation of plastids in single-celled microalgae into specialized starch storing amyloplasts as occurs in plants. As a result, increasing starch storage sites is not a viable strategy for increasing starch accumulation. Starch accumulation in a plastid can be genetically manipulated, however. Structurally, starch is composed of two types of glucose polymers, amylose and amylopectin, that differ in their degree of branching. The glucose density of starch granules and their size is controlled by the levels of starch branching and debranching enzyme activities. Genetic manipulations of enzymes controlling starch branching has been shown to substantially impact biomass production [58]. Enhanced lipid storage in microalgae has been achieved by over-expression of enzymes implicated in fatty acid and TAG biosynthesis [60–63], or by repression of lipid catabolism [62, 63]. Additionally, genetic manipulations to decrease starch accumulation also leads to substantial increases in storage lipid accumulation per cell. A *C. reinhardtii* mutant blocked in starch accumulation nearly doubled the amount of lipids accumulated under nitrogen deprivation relative to the control strain, indicating that TAG can act as an alternate sink for excess carbon and photosynthetic reducing equivalents [62]. High energy dense hydrocarbons are primarily stored as TAGs in microalgae and contained in membrane bound lipid droplets. Lipid droplet size and numbers are regulated in part by the production of lipid droplet proteins which are present in the membranes surrounding lipid droplets. Reductions in the expression of major lipid droplet proteins using RNA silencing techniques has been shown to significantly decrease the size of lipid droplets [63]. However, genetic manipulations to increase TAG accumulation by enhancing lipid droplet protein production to our knowledge has not been reported to date. Overall, genetic manipulation of genes controlling select aspects of source, sink, storage, metabolism, and cell growth rates have all proven to enhance biomass yields. Integration of multiple aspects of carbon metabolism, storage and growth leading to enhanced biomass yields have been achieved by alterations in mastery regulatory genes. But much remains to be characterized to achieve maximum thermodynamic

efficiency for conversion of photons to the chemical energy of biomass.

or less than that of water. Thus,

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

*Biotechnological Applications of Biomass*

(CBBC) reactions of photosynthesis.

**3.2 Alterations in carbon sink strength**

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

Given the primary role of starch metabolism as carbon reserve and an intermediate in the production of hydrocarbons it is not unanticipated that alterations in starch metabolism may impact hydrocarbon and biomass yields [58]. For example, *Chlamydomonas* sta6 [ADP-glucose pyrophosphorylase] and sta7–10 [isoamylase] mutants having reduced capacity to synthesize starch had substantial increases in lipid accumulation during nitrogen deprivation relative to the wild-type controls but suppressed total biomass accumulation [58]. In addition, suppression of starch metabolism has been shown to impair upstream CBBC activity resulting in the dissipation of excess photosynthetically produced electrons through non-productive reduction of oxygen [54]. These results point to the central role of starch metabolism and accumulation in overall cellular homeostasis and biomass accumulation in algae and its impact on the thermodynamic efficiency of light energy conversion into chemical energy (biomass) [8, 58]. It has been estimated that carbohydrate metabolism can accounts for as great as 20% reductions in thermodynamic efficiency of photosynthesis [39]. These efficiencies can be further reduced by partitioning carbon into hydrocarbon storage products instead of starch. This is due to the central role of pyruvate (3C) metabolism in hydrocarbon (lipids, terpenes, and waxes) production. The production of acetyl CoA (2C) via the decarboxylation of pyruvate for hydrocarbon production results in the loss of 1/3 of the previously fixed carbon. In contrast, starch production from photosynthetically derived sugars has no associated decarboxylation steps. Hydrocarbons, however, have nearly twice the energy density of carbohydrates due to their more reduced state. Modeling studies indicate that the production of carbohydrates using solar photons is potentially 10–20% more efficient for solar energy conversion than hydrocarbon production [8]. Furthermore, the kinetics of lipid production are substantially slower than starch synthesis. Thus, algae that primarily store starch may accumulate biomass

**460**

faster than algae that store hydrocarbons as energy reserves. The ecological downside of starch storage, however, is that starch has high volumetric density (1.56 g/ cm3 ) while lipids have a density of 0.91 g/cm3 or less than that of water. Thus, algae that store starch must invest energy in motility devices and associated energy expenditures to avoid sinking to depths where light availability may be limiting for photosynthetic growth. It might then be predicted that algae that store starch, e.g., *Chlamydomonas*, predominantly inhabit soil environments that provide physical support whereas lipid accumulating algae, e.g., *Nanochloropsis*, tend to occupy aquatic environments where they are less dense or near the density of water and can remain at levels in the water column where light is not limiting for photosynthesis. To date, the relative energetic costs needed to support motility in starch accumulators versus lipid accumulators remains to be assessed.
