276 Marine Macro- and Microalgae: An Overview
Electroporation is a highly efficient method for transferring small amounts of DNA. This
transformation methodology was first utilized with a marine cyanobacterium Synechococcus sp.
(Matsunaga et al. 1990). In eukaryotic algae, efficient transformation mediated by electroporation was
firstly achieved with C. reinhardtti (Shimogawara et al. 1998). The maximum transformation frequency
was two orders of magnitude higher than with glass beads (Shimogawara et al. 1998). Recently,
transformation by electroporation was also successful with the diatoms Nannochloropsis sp. (Kilian et
al. 2011), Ostreococcus tauri (van Ooijen et al. 2012), and Phaeodactylum tricornutum (Niu et al. 2012).
Agrobacterium tumefaciens-mediated genetic transformation is a very popular method for
introducing gene in higher plants. The first report of an effective genetic transformation by A. tumefaciens
in algae was conducted in the marine red seaweed Porphyrayezoensis (Cheney et al. 2001). Only in 2004
transformation of C. reinhardtii by co-cultivation with A. tumefaciens was described holding promising
results—as the efficiency of transformation was higher than that of glass bead transformation (Kumar
et al. 2004), but this method has not yet been widely adopted for this alga. Recently, Agrobacteriummediated transformations of Nannochloropsis sp. (Cha et al. 2011) and Chlorella vulgaris (Cha et al.
2012) have been described. The microalgae commonly used as bioenergy sources and the most important
methods for their genetic transformation are shown in Table 1.
Achieving high biomass for biofuels production
Biofuels or their precursors are composed entirely of carbon, hydrogen, and oxygen – and their synthesis
results from the photosynthetic assimilation of CO 2 and water, not needing a net investment of other
nutrients into the final product. Hence, biofuel synthesis could in principle be uncoupled from the nutrient
expenses associated to cell proliferation. For these and other reasons, great hopes have been placed on
microalga cultures as a sustainable source of biofuel. However, it is recognized that, in order to achieve
an economically competitive status, the natural efficiency of the process has to be increased. Light into
chemical free energy conversion, under optimal conditions, is estimated as ca. 5% efficient in microalgae,
which compares favourably with 3% in higher plants but is still below 10% of current silicon-based
artificial solar cells producing hydrogen through water electrolysis (Blankenship et al. 2011). However,
algal biomass may still be a preferred source because of a higher versatility of extractable products,
including liquid fuels. Moreover, it is perceived that the energy gain of the natural photosynthetic process
taking place in microalgae could be further improved at several steps by biotechnological means. In this
regard, biotechnologically oriented genome manipulation has been encouraged by recent developments
in genetic engineering, including a specific adaptation of the CRISPR/Cas9 technology for editing genes
of marine algae (Nymark et al. 2016). Prospects, current limitations, and the research agenda for the
technological improvement of biomass and biofuel production have been recurrently reviewed in the
past years (Anemaet et al. 2010; Kumar et al. 2010; Stephens et al. 2010; Stephenson et al. 2011; Day et
al. 2012; Larkum et al. 2012; Rosgaard et al. 2012; Work et al. 2012; Ho et al. 2014; Hedge et al. 2015;
Gomaa et al. 2016; Banerjee et al. 2016; Ng et al. 2017). This section will focus on organic fuels resulting
from photosynthetic assimilation, while hydrogen production will be considered below in a separate
section.
Improving light capture
Sunlight delivers an average power of 170 W/m
2
on the Earth surface (Blankenship et al. 2011). However,
only a reduced range of wavelengths (roughly between 400 and 700 nm, with a narrow “green” window
of transmission around 555 nm) is photosynthetically active (the so-called PAR). Therefore, only a
fraction of light power is profitable through natural photosynthesis. The efficiency of radiation energy
capture may be improved through different strategies of artificial illumination (reviewed by Ramanna
et al. 2017), including wavelength selection and flashing. However, this section will focus on genetic
engineering approaches to enhance radiation capture under natural sunlight.
PAR matches the overlapping absorption spectra of the different pigments harboured by the
light-harvesting complexes that deliver the excitation energy to the photosynthetic reaction center.
Electroporation is a highly efficient method for transferring small amounts of DNA. This
transformation methodology was first utilized with a marine cyanobacterium Synechococcus sp.
(Matsunaga et al. 1990). In eukaryotic algae, efficient transformation mediated by electroporation was
firstly achieved with C. reinhardtti (Shimogawara et al. 1998). The maximum transformation frequency
was two orders of magnitude higher than with glass beads (Shimogawara et al. 1998). Recently,
transformation by electroporation was also successful with the diatoms Nannochloropsis sp. (Kilian et
al. 2011), Ostreococcus tauri (van Ooijen et al. 2012), and Phaeodactylum tricornutum (Niu et al. 2012).
Agrobacterium tumefaciens-mediated genetic transformation is a very popular method for
introducing gene in higher plants. The first report of an effective genetic transformation by A. tumefaciens
in algae was conducted in the marine red seaweed Porphyrayezoensis (Cheney et al. 2001). Only in 2004
transformation of C. reinhardtii by co-cultivation with A. tumefaciens was described holding promising
results—as the efficiency of transformation was higher than that of glass bead transformation (Kumar
et al. 2004), but this method has not yet been widely adopted for this alga. Recently, Agrobacteriummediated transformations of Nannochloropsis sp. (Cha et al. 2011) and Chlorella vulgaris (Cha et al.
2012) have been described. The microalgae commonly used as bioenergy sources and the most important
methods for their genetic transformation are shown in Table 1.
Achieving high biomass for biofuels production
Biofuels or their precursors are composed entirely of carbon, hydrogen, and oxygen – and their synthesis
results from the photosynthetic assimilation of CO 2 and water, not needing a net investment of other
nutrients into the final product. Hence, biofuel synthesis could in principle be uncoupled from the nutrient
expenses associated to cell proliferation. For these and other reasons, great hopes have been placed on
microalga cultures as a sustainable source of biofuel. However, it is recognized that, in order to achieve
an economically competitive status, the natural efficiency of the process has to be increased. Light into
chemical free energy conversion, under optimal conditions, is estimated as ca. 5% efficient in microalgae,
which compares favourably with 3% in higher plants but is still below 10% of current silicon-based
artificial solar cells producing hydrogen through water electrolysis (Blankenship et al. 2011). However,
algal biomass may still be a preferred source because of a higher versatility of extractable products,
including liquid fuels. Moreover, it is perceived that the energy gain of the natural photosynthetic process
taking place in microalgae could be further improved at several steps by biotechnological means. In this
regard, biotechnologically oriented genome manipulation has been encouraged by recent developments
in genetic engineering, including a specific adaptation of the CRISPR/Cas9 technology for editing genes
of marine algae (Nymark et al. 2016). Prospects, current limitations, and the research agenda for the
technological improvement of biomass and biofuel production have been recurrently reviewed in the
past years (Anemaet et al. 2010; Kumar et al. 2010; Stephens et al. 2010; Stephenson et al. 2011; Day et
al. 2012; Larkum et al. 2012; Rosgaard et al. 2012; Work et al. 2012; Ho et al. 2014; Hedge et al. 2015;
Gomaa et al. 2016; Banerjee et al. 2016; Ng et al. 2017). This section will focus on organic fuels resulting
from photosynthetic assimilation, while hydrogen production will be considered below in a separate
section.
Improving light capture
Sunlight delivers an average power of 170 W/m
2
on the Earth surface (Blankenship et al. 2011). However,
only a reduced range of wavelengths (roughly between 400 and 700 nm, with a narrow “green” window
of transmission around 555 nm) is photosynthetically active (the so-called PAR). Therefore, only a
fraction of light power is profitable through natural photosynthesis. The efficiency of radiation energy
capture may be improved through different strategies of artificial illumination (reviewed by Ramanna
et al. 2017), including wavelength selection and flashing. However, this section will focus on genetic
engineering approaches to enhance radiation capture under natural sunlight.
PAR matches the overlapping absorption spectra of the different pigments harboured by the
light-harvesting complexes that deliver the excitation energy to the photosynthetic reaction center.