Green Energy from Green Algae: Biofuel Production From

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Biofuel production from Chlamydomonas reinhardtii Green energy from green algae

Alexandra Dubini (National Renewable Energy Laboratory, USA)

Looking for alternative ‘green’ energy technologies? Don’t look too far! Microalgae are all around

us and are being used, processed and packaged for different applications, from food to pharma- Downloaded from http://portlandpress.com/biochemist/article-pdf/33/2/20/5135/bio033020020.pdf by guest on 02 October 2021 ceutical products and now to generate renewable green energy such as hydrogen, biodiesel and other biofuels. Microalgae in general and green algae in particular have been studied for decades with the objective of utilizing their photosynthetic capacity and their ability to adapt to changing environment and nutrient conditions as a source of a variety of products. A new era has arrived where these functions are now being examined and targeted to efficiently convert solar energy into useful carbon-based fuels and chemical precursors (alkane, ethylene), as well as gas (hydrogen) or lipid-based storage compound such as triacylglycerols (TAGs) for biodiesel application.

Chlamydomonas, the solitary cloak…. of a variety of genotypes. A wide range of laboratory wild-type and mutant Chlamydomonas strains have Microalgae are unicellular photosynthetic micro‑ been generated and can be used to deconvolute the organisms that are abundant everywhere on earth. metabolic pathways involved in biofuel production.

They are capable of utilizing CO2 and sunlight to Chlamydomonas is therefore a good working model generate end-products necessary for their survival. where a range of information is already available on Among those molecules are hydrogen, alcohols and hydrogen, ethanol and lipid production pathways. lipids, which can be processed into useful products for This article is a review of those metabolic pathways our society. Algae are also good candidates for bio- and report on the latest developments in the field. fuel production because of their high productivity per acre compared with typical terrestrial oil‑seed crops. Hydrogen metabolism They can be grown on non-productive non-arable land using a wide variety of water sources (fresh, brack‑ Photobiological pathway ish, saline and wastewater) and thus do not compete Green algae are capable of catalysing photobiological with food-based feedstock resources. Finally, algal hydrogen production using their photosynthetic ap- cultures can capture carbon dioxide from industrial flue paratus [Photosystem II (PSII) and I (PSI)], which can

gases and function as biological CO2 recyclers. Many convert solar energy into chemical energy, split water green algal strains are being studied and analysed and convey electrons to a hydrogenase (HYD) enzyme for their different capabilities; however, one strain (respectively), which in turn releases hydrogen gas. rises above the others as a model organism for biofuel This fascinating process has been studied for many production studies. years and the first report of hydrogen production in Chlamydomonas reinhardtii (Figure 1) is a uni- a green alga was released in 1942 by the pioneer Hans cellular alga, the name of which comes from the Gaffron3. Since then many researchers have worked on Greek: χλάμυς (chlamys) cloak or covering and μόνος photobiological hydrogen production4,5 and were able (monas) single1. Could Chlamydomonas be indeed the to dissect the pathways and characterize the enzymes only alga to allow us to uncloak the processes involved involved in this reaction (Figure 2)6,7. in biofuel production? This freshwater alga has re- Two distinct hydrogen-photoproduction path- cently emerged has a model organism for biofuel stud- ways have been described in Chlamydomonas. ies in green algae due to the availability of a sequenced The first pathway is dependent on PSII and PSI genome2, a wide array of sophisticated genetic tools to activities. Here water oxidation by PSII supplies manipulate its genome, the existence of bioinformatic reductants (electrons) for hydrogen production Key words: biofuel, green tools, such as an expressed sequence tag (EST) data- through PSI and ferredoxin (FDX), so-called direct alga, lipid, metabolic base (www.chlamy.org), and a simple sexual cycle that biophotolysis. Unfortunately, hydrogen production is pathway, photohydrogen allows classic genetic analysis and the generation/study rapidly discontinued due to the accumulation

of oxygen released from water splitting which inhibits the HYD enzymes. In the second pathway, only NADP–plastoquinone oxidoreductase (NPQR) 5,8

and PSI activities are required . NPQR transfers Downloaded from http://portlandpress.com/biochemist/article-pdf/33/2/20/5135/bio033020020.pdf by guest on 02 October 2021 reductants released by the glycolytic degradation of glucose or other organic compounds in the chloroplast to the photosynthetic electron transport chain at the level of plastoquinone (PQ) pool. Upon illu- mination, the reductants are re-energized at the level of PSI and reduce hydrogenases through FDX5,8. The two hydrogen-photoproduction pathways have been observed to contribute at different levels depending on growth conditions1. In the laboratory, sustained photobiological hydrogen production is only observed under conditions that continuously remove9 or limit photosyn- 4 thetic O2 evolution, such as sulfur deprivation . This method allows for continuous hydrogen production over 4 days. By depriving the cultures of sulfate, this Figure 1. C. reinhardtii (Yuuji Tsukii, Laboratory of Biology, Science Research Center, Hosei system circumvents the O2‑sensitivity of the HYD enzymes. In the absence of sulfate, the D1 protein of University: Protist Information Server, http://protist.i.hosei.ac.jp) PSII (which is essential for photosynthesis, but turns over rapidly due to continuous photodamage) cannot

be replaced quickly, leading to the gradual loss of O2 evolution. Simultaneously, as a response to nutrient stress, sulfur-deprived algae over-accumulate starch to amounts 8–20‑fold higher than under non-stressed 4 conditions . When the rate of O2 evolution becomes + lower than the rate of respiratory O2 consumption, the cultures undergo a rapid shift to anaerobiosis and, after a few hours, large amounts of hydrogen become detectable4. From that point forward, starch starts being degraded, which contributes to maintaining anaerobiosis and leads to increased levels of reductant for hydrogen production, as well as pyruvate, which is dissimilated into acetate, formate and ethanol10.

Dark fermentative pathway Green algae are also able of producing hydrogen Figure 2. Hydrogen-production pathways in Chlamydomonas. Red represents the PSII-de- through dark fermentation (Figure 2)10. Under anaer- pendent photobiological pathway. Yellow symbolizes the NPQR-dependent pathway. Orange obic fermentative conditions, pyruvate is oxidized via corresponds to the end reaction of red and yellow pathways. Green denotes the fermenta- the pyruvate–ferredoxin oxidoreductase (PFR), gen- tive production pathway. See text for more details. Most of those reactions happen in the chloroplast (green box) and the rest outside of this organelle in the cytosol. PFR1 might have erating CO2, acetyl-CoA and reduced FDX, which in

turn transfers electron to the hydrogenase enzyme to a dual location (chloroplast and cytosol). Chl, chlorophyll; Cyt, cytochrome; H2ases, hydroge- produce hydrogen7. During dark fermentation, cellular nases; ox, oxidized; PC, plastocyanin; Pheo, pheophytin; red, reduced. Figure adapted from The starch reserves are metabolized to generate ATP, while Chlamydomonas Sourcebook: Organellar and Metabolic Processes1 and Orr et al.11.

Figure 3. Figure 3. Simplified overview of metabolic pathway involved in biofuel production in Chlamydomonas. Ethanol is produced through fermentation via two routes: (i) through the alcohol dehydrogenase (ADH1) pathway; (ii) in a two-step reaction through pyruvate decarboxylation (PDC3) and ADH2. The three hydrogen-production pathways are displayed, but are reported in more detail in Figure 2. TAGs are proposed to be synthesized via the direct glycerol pathway14. De novo fatty acids biosynthesized in the chloroplast are sequentially transferred from CoA to positions 1 and 2 of glycerol 3-phosphate, resulting in the formation of the central metabolite, phosphatidic acid (PA). Dephosphorylation of PA, catalysed by a specific phospha- tase releases diacylglycerol (DAG). In the final step of TAG synthesis, a third fatty acid is transferred to the vacant position 3 of DAG, and this reaction is catalysed by DAG acyltransferase (DGAT). Each coloured box represents a different organelle: red for mitochondria, green for chloroplast, and orange for dual localization (mitochondria and chloroplast). The yellow oblong represents a lipid body. Other abbreviation: PFL, pyruvate-formate lyase; HYDA, hydrogenase; ACCase, acetyl‑CoA carboxylase; KAS, β-ketoacyl-acyl carrier protein synthase; SAD, stearoyl-acyl carrier protein desaturase.

the reduced pyridine nucleotide that is co-produced microalgae and bacteria. In Chlamydomonas, the for- with the ATP must be reoxidized to sustain the activ- mation of ethanol occurs during fermentation through ity of the fermentative pathways. This reoxidation is two different pathways which both involve pyruvate linked to both organic acid and hydrogen formation10. decarboxylation. In one of the pathways, pyruvate is The hydrogen level measured under those conditions used as a substrate by the pyruvate formate lyase (PFL) is, however, very low compared with photobiological enzyme, which produces formate and releases acetyl- hydrogen and therefore fermentation remains as the CoA. Acetyl-CoA is then reduced to ethanol via an secondary hydrogen-production route. alcohol/aldehyde dehydrogenase (ADH1), resulting in the oxidation of two molecules of NADH10. Pyru- Production of alcohol vate can also be converted into ethanol via pyruvate decarboxylase 3 (PDC3) and a second ADH pathway, Ethanol synthesis pathway resulting in the production of ethanol and the oxida- As mentioned above, Chlamydomonas can synthesize tion of one NADH. Chlamydomonas is rare among ethanol under specific conditions. Ethanol produc- eukaryotes in having both pathways for the production tion pathways are not unusual and are found in many of ethanol1. It is possible that orthologues of the pro-

teins required for the conversion of acetyl-CoA into Conclusions either acetate (which yields ATP) or ethanol (which reoxidizes NADH allowing additional cycles of C. reinhardtii has been a powerful model system to glycolysis) are present in both mitochondria and understand the metabolic pathways involved in the chloroplasts. Propan‑2‑ol, butanol and other longer- production of both biofuels and valuable co-products. chain alcohols have not been shown to be naturally Those studies have underscored the potential value produced in the algal kingdom and would require of algal biofuels. The concept of using algae as an metabolic engineering in the organism to synthesize alternative and renewable source of biomass feedstock these compounds12. for biofuels is now widely explored. Currently, most estimates of algal biofuel productivity are based on Lipid metabolism small-scale experimental data. Outdoor facilities are

being examined and currently algal-based companies Downloaded from http://portlandpress.com/biochemist/article-pdf/33/2/20/5135/bio033020020.pdf by guest on 02 October 2021 TAG synthesis pathway are analysing mainly biodiesel production efficiency. Algae are known to accumulate lipids, typically in However, more basic research needs to be done to the form of TAGs, which are essential energy-storage better understand the biology behind those meta- molecules. Algae also synthesize fatty acids for es- bolic pathways and hopefully gain enough under- terification into glycerol-based membrane lipids, both standing to obtain improved and optimized biofuels phospholipids and glycolipids, which typically consti- production. We anticipate that, in the near future, a tute 5–20% of their dry cell weight. Under specific scalable, sustainable and commercially viable system stress condition, some algal species have been re- will emerge. ■ ported to double their TAG content under laboratory conditions13. In Chlamydomonas, the major pathway I would like to acknowledge the photobiology group at for the formation of TAG involves de novo fatty acid NREL and particularly Dr Maria Ghirardi for her helpful synthesis (Kennedy pathway) in the stroma of plastids comments on the review. and subsequent incorporation of the fatty acid into the glycerol backbone, leading to TAG via three sequen- Alexandra Dubini is a scientist at the tial acyl transfers from acyl‑CoA in the endoplasmic National Renewable Energy Laboratory, reticulum (ER)14 (see Figure 3). TAG can then be in Golden, CO, USA. Her main research is converted into biodiesel by a transesterification step, focused on understanding the metabolic to be used as biofuel. Typically, TAG accumulation pathways involved in biofuel production occurs under stress conditions (nutrient deficiency or including hydrogen production and high light) that strongly impair the productivity of the TAG accumulation in the green alga C. reinhardtii. More system. This implies that a performing system should specifically, she is investigating (i) the rate‑limiting factors be producing a high yield of TAG as well as a high bio- involved in hydrogen production and the competitive pathways mass; however, our knowledge of carbon partitioning at the level of ferredoxin, and (ii) the lipid biosynthesis and biosynthesis of TAGs in algae is very limited and process to characterize new pathways for TAG synthesis. does not allow us to accomplish that yet12. email: [email protected]

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