**2. Algal strains**

*Biotechnological Applications of Biomass*

and low biomass density [10].

that have a large global market demand (**Figure 1**).

reported for soybean [16]. Clearly, microalgae supersede traditional agriculture on multiple aspects, however, biomass harvesting is an area which is well established in case of crop plants but highly energy intensive in case of algae due to its small size

Regarding algal biomass harvesting systems the general objective has been to develop algae harvesting and concentrating systems that have parasitic energy consumption values of less than 10% of the total algal biomass energy content [6]. To reduce the costs of fuel production, recent efforts have focused on the direct conversion of harvested algal biomass into separate fuel and coproduct fractions in a continuous flow system while efficiently recycling water and nutrients. One of the more promising technology developments in this sector has been the development of two-stage HTL which allows for the separate recovery of coproducts and biocrude feedstock while recycling water and nutrients back to the pond thus avoiding the energy intensive step of drying the algal biomass before biomass to fuel conversion. The appropriate selection of what high value coproduct(s) to produce from algal biomass is critical for economic viability when coproduct production is coupled with fuel production. From this perspective the coproduct should have sufficient value based on biomass yields to be economically sustainable without saturating markets to the point of driving coproduct prices so low as to be economically untenable. As modeled by the US-DOE PACE algal biofuels consortium a fully integrated algal cultivation, harvesting, co-product and fuel production system with integrated water and nutrient recycling has the potential to recover over 60% of the energy content of the algae as biocrude while producing valuable coproducts

Optimizing algal biomass production and carbon sequestration also has the potential to address the existential threat of global climate change associated with greenhouse gas emissions. Currently, biological carbon capture and sequestration (BCCS) is one of the more feasible means to remediate the earth's atmosphere. As a BCCS system, algae are particularly attractive not only for their high areal

*PACE consortium working model for the integrated co-production of biofuels and co-products (green chemicals, polysaccharides (guar), and methane) from algae. Inorganic nutrients and wastewater are recycled. Algae are preloaded with nutrients (nutrient pulse) and grown in minimal media to reduce weedy species competition and continuously harvested at mid-log phase growth. HTL, hydrothermal liquefaction; CHG, catalytic* 

**454**

**Figure 1.**

*gasification.*

Substantial efforts have focused on the identification of algal strains having maximum biomass yields under cultivation. Ideal biomass production strains must not only have fast growth rates but also must be robust and tolerate well abiotic (temperature, salinity, light) and biotic (pathogen, herbivore and weedy algae) stress conditions to minimize pond crashes and downtime in algal cultivation. There have been several large-scale algal surveys of wild algal species to identify those strains that perform well in cultivation [18]. In addition, screening systems for identifying strains with elevated performance characteristics in high light environments among others have led to some success in the identification of high performing algal strains [19]. Given that there as many as 150,000 species of algae have been identified and that limited resources have been available to screen algae for high biomass production, there remains a significant number of algae that remain to be assessed for biomass productivity in select environments [7]. In addition, substantial potential to improve algal productivity may also be achieved in traditional and molecular assisted breeding practices. Algae breeding efforts, except for laboratory strains such as *Chlamydomonas*, have been limited, however. This is because the means to induce gametogenesis to identify sexual mating types in most algae is not well understood. If the increased yield achieved through plant breeding are to serve as a prognosticator of the potential to enhance algal productivity it can be anticipated that algal breeding programs may enhance yields in the field by as much as ten-fold.

### **2.1 Modulating cultivation conditions to impact oil and carbohydrate yields**

Given the fast rates of cell division and the absence of dedicated higher-order cellular structures including tissue and organs it is not unexpected that microalgae have an enhanced capability to metabolically remodel cellular functions under different growth conditions. Algae frequently live boom and busts cycles in the nutrient deserts of lakes and the open oceans. Thus, it is imperative that algae have flexible metabolic systems to survive in unpredictable and ever-changing environments and be unencumbered by programmed cell fates associated with the differentiation and organization of cells into higher order tissues and organs.

One of the manifestations of this metabolic flexibility is the ability to shift the biochemistry of the major cellular energy storage products from low energy density carbohydrates to high energy density hydrocarbons including triacylglycerol (TAG) and/ or polyterpenoids [20]. The metabolic shift from carbohydrate to hydrocarbon accumulation is typically induced by nutrient depravation. Upon shifting from a nitrogen-, sulfur- and/ or micronutrient-rich condition to a nutrient poor condition many algae will facultatively shift the metabolism of energy storage product accumulation from carbohydrates (starch) to hydrocarbons [21–25]. Hydrocarbons have more than 60% the energy density per fixed carbon of carbohydrates. Importantly, the facultative shift to hydrocarbon production allows algae to continue to generate and utilize reducing energy generated by the photosynthetic apparatus. Significantly, the accumulation of triacylglcerols may not only involve de novo synthesis but the remodeling of existing chloroplast membrane lipids into more fully reduced TAGs [26–29]. Given the desirability of hydrocarbons as a feedstock for biocrude production the ability to shift metabolism from carbohydrate to hydrocarbon production has been exploited to produce hydrocarbon rich biofuel feedstocks. The challenges with this strategy (nutrient deprivation) for facultative hydrocarbon production is that it can also lead to reduced rates of cell division and overall biomass accumulation. In a comprehensive empirical analysis of the impact of nitrogen deprivation on cell division rates, TAG accumulation, lipid remodeling, biomass accumulation and total caloric or biochemical energy accumulation in the green alga, *Chlorella sorokiniana*, it was demonstrated that upon shifting algae to a nitrogen-free growth medium there was a substantial increase in TAG accumulation and a redistribution of total cellular fatty acid profile to more energy dense saturated fatty acids [30]. Under the two-week nitrogen deprivation period employed in this study there was no statistically significant reduction in the rates of cell division or biomass (dry weight) accumulation. However, during the nutrient deprivation period the total chemical energy accumulated in biomass increased by greater than 60% associated with a 20-fold increase in TAG content. It is perhaps surprising that the two-week nitrogen deprivation period did not impair cell division and biomass accumulation suggesting that the alga had the capability to sequester nutrients and/ or catabolize and remodel existing nitrogen rich (proteins) molecules [30]. Not all algal species, however, exhibit similar responses to nutrient deprivation. For many algal species growth rates and biomass accumulation are substantially impaired during nutrient deprived growth conditions [31–33].

An additional practical application of nutrient deprivation for oil production is that growth in nutrient depleted media may reduce competition from weedy algal species [34]. This observation has led to the application of nutrient pulse technology to simultaneously induce oil accumulation during nutrient stress and inhibit the growth of weedy algal species. Under ideal growth conditions the limiting nutrients are withheld until there is an impairment in growth. At this transition point a pulse of the limiting nutrient is added to the growth media to support continued high growth rates [34]. Overall, the ability to induce oil production if managed well can lead to sustained high growth rates while enhancing the energy density of the biomass and the increased accumulation of biofuel feedstocks such as TAGs.
