Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: Photosynthesis for the generation of fuels and chemicals from cyanobacteria and microalgae offers the promise of a single host organism acting both as photocatalyst and processor, performing sunlight absorption and utilization, as well as CO2 assimilation and conversion into product. However, there is a need to develop methods for generating, sequestering and trapping such bio-products in an efficient and cost-effective manner that is suitable for industrial scale-up and exploitation. A sealed gaseous/aqueous two-phase photobioreactor was designed and applied for the photosynthetic generation of volatile isoprene (C5H8) hydrocarbons, which operates on the principle of spontaneous diffusion of CO2 from the gaseous headspace into the microalgal or cyanobacterial-containing aqueous phase, followed by photosynthetic CO2 assimilation and isoprene production by the transgenic microorganisms. Volatile isoprene hydrocarbons were emitted from the aqueous phase and were sequestered into the gaseous headspace. Periodic replacement (flushing) of the isoprene (C5H8) and oxygen (O2) content of the gaseous headspace with CO2 allowed for the simultaneous harvesting of the photoproducts and replenishment of the CO2 supply in the gaseous headspace. Reduction to practice of the gaseous/aqueous two-phase photobioreactor is offered in this work with a fed-batch and a semi-continuous culturing system using Synechocystis sp. PCC 6803 heterologously-expressing the Pueraria montana (kudzu) isoprene synthase (IspS) gene. Constitutive isoprene production was observed over 192 h of experimentation, coupled with cyanobacterial biomass accumulation. The diffusion-based process in gaseous/aqueous two-phase photobioreactors has the potential to be applied to other high-value photosynthetically derived volatile molecules, emanating from a variety of photosynthetic microorganisms. PARTICIPANTS: The following postdoctoral research scholars contributed to the effort: Fiona K. Bentley, Mautusi Mitra, Jose Garcia, Andreas Zurbriggen, David Dewez, Sung-Yong Hong. The following graduate students contributed to this effort: Henning Kirst, Thilo Ruehle. The following undergraduate students contributed to this effort: Sam Ng TARGET AUDIENCES: Renewable fuels and biofuels audiences, including companies that operate in the energy and bioenergy sector. Carbon dioxide mitigation specialists and environmentalists. PROJECT MODIFICATIONS: None

Impacts
There is a need to develop renewable fuels and chemicals that will help meet global demands for energy and synthetic chemistry feedstock, but without contributing to climate change or other environmental degradation. Work in this period contributed to meeting technical requirement in this direction by providing novel methods for the sequestration and quantification of isoprene (C5H8) hydrocarbons in photosynthetic microorganisms. Isoprene, derived entirely via photosynthesis, i.e., from sunlight, carbon dioxide (CO2) and water (H2O), could serve as a renewable biofuel or feedstock in the synthetic chemistry industry. Photosynthetic microorganisms, such as cyanobacteria or unicellular microalgae, can grow to high densities within fully enclosed photo-bioreactors. Such a system would enable oxygenic photosynthesis to convert solar energy and store it in the form of hydrocarbons, while permitting collection and sequestration of this volatile product. This method was applied to a semi-continuous culturing system in a fed-batch bioreactor, enabling the continuous accumulation and harvesting of isoprene, proving suitability of this approach in industrial scale-up for the production of renewable photosynthetically-generated isoprene hydrocarbons.

Publications

• Bentley FK, Melis A (2012) Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous-aqueous two-phase photobioreactors by photosynthetic microorganisms. Biotech Bioeng 109:100-109
• Mitra M, Ng S, Melis A (2012) The TLA1 protein family members contain a variant of the plain MOV34-MPN domain. Amer J Biochem Mol Biol. 2(1):1-18
• Melis A (2012) Photosynthesis-to-Fuels: From sunlight to hydrogen, isoprene, and botryococcene production. Energy Environ. Sci. 5(2): 5531-5539
• Kirst H, Garcia-Cerdan JG, Zurbriggen A, Melis A (2012) Assembly of the light-harvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii requires expression of the TLA2-CpFTSY gene. Plant Physiol 158: 930-945
• Mitra M, Dewez D, Garcia-Cerdan JG, Melis A (2012) Polyclonal antibodies against the TLA1 protein also recognize with high specificity the D2 reaction center protein of PSII in the green alga Chlamydomonas reinhardtii. Photosynth Res 112:39-47
• Melis A (2012) Short chain volatile hydrocarbon production using genetically engineered microalgae, cyanobacteria or bacteria. United States Patent 8,133,708 (cyanobacteria; issued 13-Mar-2012)
• Zurbriggen A, Kirst H, Melis A (2012) Isoprene production via the mevalonic acid pathway in Escherichia coli (Bacteria). BioEnergy Res 5(4): 814-828
• Hong S-Y, Zurbriggen A, Melis A (2012) Isoprene hydrocarbons production upon heterologous transformation of Saccharomyces cerevisiae. J Appl Microbiol 113: 52-65
• Xie D-Y, Melis A (2012) Special Issue on metabolic plant biology (Editorial). Planta 236:763-764
• Kirst H, Garcia-Cerdan JG, Zurbriggen A, Ruehle T, Melis A (2012) Truncated photosystem chlorophyll antenna size in the green microalga Chlamydomonas reinhardtii upon deletion of the TLA3-CpSRP43 gene. Plant Physiol. 160(4):2251-2260
• Mitra M, Kirst H, Dewez D, Melis A (2012) Modulation of the light-harvesting chlorophyll antenna size in Chlamydomonas reinhardtii by TLA1 gene over-expression and RNA interference. Phil. Trans. R. Soc. B 367:3430-3443

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Six different strains of the green microalgae Botryococcus belonging to the A-race or B-race, accumulating alkadiene or botryococcene hydrocarbons, respectively, were compared for biomass and hydrocarbon productivities. Biomass productivity was assessed gravimetrically upon strain growth in the laboratory under defined conditions. Hydrocarbon productivities were measured by three different and independent experimental approaches, including density equilibrium of the intact cells and micro-colonies, spectrophotometric analysis of hydrocarbon extracts, and gravimetric quantitation of eluted hydrocarbons. All three hydrocarbon-quantitation methods yielded similar results for each of the strains examined. The B-race microalgae Botryococcus braunii var. Showa and Kawaguchi-1 constitutively accumulated botryococcene hydrocarbons equivalent to 30% and 20%, respectively, of their overall biomass. The A-race microalgae Botryococcus braunii, varieties Yamanaka, UTEX 2441 and UTEX LB572 constitutively accumulated alkadiene hydrocarbons ranging from 14% to 13% and 10% of their overall biomass, respectively. Botryococcus sudeticus (UTEX 2629), a morphologically different green microalga, had the lowest hydrocarbon accumulation, equal to about 3% of its overall biomass. Results validate the density equilibrium and spectrophotometric analysis methods in the quantitation of botryococcene-type hydrocarbons. These analytical advances will serve in the screening and selection of B. braunii and of other microalgae in efforts to identify those having a high hydrocarbon content for use in commercial applications. PARTICIPANTS: EROGLU E: Received postdoctoral training; OKADA S: Japanese collaborator; MELIS A: PI ORT DR: Collaborator from the UIUC; ZHU X-G: Collaborator from China;, MELIS A: PI EROGLU E: Received postdoctoral training; MELIS A: PI BLANKENSHIP RE, TIEDE DM, BARBER J, BRUDVIG GW, FLEMING G, GHIRARDI ML, GUNNER MR, JUNGE W, KRAMER DM, MELIS A, MOORE TA, MOSER CC, NOCERA DG, NOZIK AJ, ORT DR, PARSON WW, PRINCE RC, SAYRE RT (Senior scientists, DOE workshop participants and co-authors) MELIS A: Inventor TARGET AUDIENCES: Renewable Biofuels and Bioenergy Sectors PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
There is an urgent need to develop renewable biofuels that will help meet global demands for energy and synthetic chemistry feedstock, but without contributing to climate change or other environmental degradation. Findings in this work contributed to meeting these requirement by providing novel methods for the generation and quantification of longer chain (30-C) hydrocarbons in photosynthetic microorganisms. Alkadienes and botryococcenes, derived entirely via photosynthesis, i.e., from sunlight, carbon dioxide (CO2) and water (H2O), could serve as a renewable biofuel or feedstock in the synthetic chemistry industry. Photosynthetic microorganisms, such as cyanobacteria or unicellular microalgae, can grow to high densities within fully enclosed photo-bioreactors. Such a system would enable oxygenic photosynthesis to convert solar energy and store it in the form of hydrocarbons, while permitting collection and sequestration of this volatile product.

Publications

• EROGLU E, OKADA S, MELIS A (2011) Hydrocarbon productivities in different Botryococcus strains: comparative methods in product quantification. Journal of Applied Phycology 23:763-775
• ORT DR, ZHU X-G, MELIS A (2011) Optimizing antenna size to maximize photosynthetic efficiency. Plant Physiology 155(1):79-85
• EROGLU E, MELIS A (2011) Photobiological hydrogen production: Recent advances and state of the art. Bioresource Technology 102:8403-8413
• BLANKENSHIP RE, TIEDE DM, BARBER J, BRUDVIG GW, FLEMING G, GHIRARDI ML, GUNNER MR, JUNGE W, KRAMER DM, MELIS A, MOORE TA, MOSER CC, NOCERA DG, NOZIK AJ, ORT DR, PARSON WW, PRINCE RC, SAYRE RT (2011) Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement. Science 332:805-809
• MELIS A (2011) Short chain volatile hydrocarbon production using genetically engineered microalgae, cyanobacteria or bacteria. United States Patent 7,947,478 (issued 24-May-2011)

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The concept of PHOTOSYNTHETIC BIOFUELS envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from carbon dioxide (CO2) and water (H2O). Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO2, without a prior requirement for the harvesting, dewatering and processing of the respective biomass. PARTICIPANTS: - LINDBERG, PIA: Received postdoctoral training - PARK, SUNGSOON: Received postdoctoral training - MELIS, ANASTASIOS: Principal Investigator TARGET AUDIENCES: Renewable Biofuels and Bioenergy Sectors PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
There is an urgent need to develop renewable biofuels that will help meet global demands for energy and synthetic chemistry feedstock, but without contributing to climate change or other environmental degradation. Findings in this work addressed this need by providing novel methods for the generation of volatile isoprene hydrocarbons in photosynthetic microorganisms. Isoprene, derived entirely via photosynthesis, i.e., from sunlight, carbon dioxide (CO2) and water (H2O), could serve as a renewable biofuel or feedstock in the synthetic chemistry industry. Photosynthetic microorganisms, such as cyanobacteria or unicellular microalgae, can grow to high densities within fully enclosed photo-bioreactors. Such a system would enable oxygenic photosynthesis to convert solar energy and store it in the form of isoprene, while permitting collection and sequestration of this volatile product.

Publications

• LINDBERG P, PARK S, MELIS A (2010) Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metabolic Engineering 12:70-79

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The function of the REP27 protein in the photosystem-II repair process was elucidated. REP27 is a nuclear-encoded and chloroplast-targeted protein containing two tetratricopeptide repeated (TPR) motifs, two putative transmembrane domains, and an extended C-terminal region. Cell fractionation and Western blot analysis localized the REP27 protein in the C. reinhardtii chloroplast thylakoids. A folding model for REP27 suggested chloroplast stroma localization for N- and C-termini regions as well as the two tetratricopeptide repeats. A REP27 gene knockout strain of C. reinhardtii, termed rep27 mutant, was employed for complementation studies. The rep27 mutant was aberrant in the PSII-repair process and had substantially lower than wild type levels of D1 protein. Truncated REP27 cDNA constructs were made for complementation of the rep27, whereby TPR1, TPR2, TPR1+TPR2, or the C-terminal domains were deleted. rep27-complemented strains minus the TPR motifs showed elevated levels of D1 in thylakoids, comparable to those in the wild-type, but the PSII photochemical efficiency of these strains was not restored, suggesting that the functionality of the PSII reaction center could not be recovered in the absence of the TPR motifs. It is suggested that TPR motifs play a role in the functional activation of the newly integrated D1 protein in the PSII reaction center. rep27-complemented strains missing the C-terminal domain showed low levels of D1 protein in thylakoids, as well as low PSII photochemical efficiency, comparable to those in the rep27 mutant. Therefore, the C-terminal domain is needed for a de novo biosynthesis and/or assembly of D1 in the photodamaged PSII template. We conclude that REP27 plays a critical role in the regulation of D1 protein turnover by facilitating co-translational biosynthesis-insertion (C-terminal domain) and activation (TPR motifs) of the nascent D1 during the PSII repair process. PARTICIPANTS: - DEWEZ, DAVID; - PARK, SUNGSOON; - GARCIA-CERDAN, JOSE GINES; - LINDBERG, PIA; - MELIS, ANASTASIOS ALL AT UC BERKELEY TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The research elucidated specific steps of the molecular mechanism of a photosystem-II (PSII) damage and repair cycle in chloroplasts. An irreversible photo-oxidative damage occurs frequently in PSII of all chloroplasts, limiting photosynthesis and causing losses in plant growth and productivity. The PSII repair process rectifies this adverse effect by selectively removing and replacing the photo-inactivated D1/32 kD reaction center protein (the chloroplast-encoded psbA gene product) from the massive (>1,000 kD) H2O-oxidizing and O2-evolving PSII holocomplex. This repair is unique in the annals of biology; nothing analogous in complexity and specificity has been reported in other biological systems. The research employed genetic, molecular and biochemical approaches by which to identify a gene and its encoded protein that facilitates a critical step in the PSII repair process. Elucidation of the PSII repair mechanism may reveal the occurrence of hitherto unknown regulatory and catalytic reactions for the selective in situ replacement of specific proteins from within multi-protein complexes. This may have important applications in photosynthesis, agriculture, medicine, and other fields.

Publications

• DEWEZ D, PARK S, GARCIA-CERDAN JG, LINDBERG P, MELIS A (2009) Mechanism of the REP27 protein action in the D1 protein turnover and photosystem-II repair from photodamage. Plant Physiol. 151:88-99

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The Chlamydomonas reinhardtii tla1 (truncated light-harvesting chlorophyll antenna size) mutant was generated upon DNA insertional mutagenesis and shown to specifically possess a smaller than wild type (WT) chlorophyll antenna size in both photosystems. Molecular and genetic analysis revealed that the exogenous plasmid DNA was inserted at the end of the 5 (prime) UTR and just prior to the ATG start codon of a hitherto unknown nuclear gene (termed Tla1), which encodes a protein of 213 amino acids. The Tla1 gene in the mutant is transcribed with a new 5 (prime) sequence, derived from the 3 (prime) end of the transforming plasmid. This replacement of the native 5 (prime) UTR and promoter regions resulted in enhanced transcription of the tla1 gene in the mutant but inhibition in the translation of the respective tla1 mRNA. Transformation of the tla1 mutant with WT Tla1 genomic DNA successfully rescued the mutant. These results are evidence that polymorphism in the 5 (prime) UTR of the Tla1 transcripts resulted in the tla1 phenotype and that expression of the Tla1 gene is a prerequisite for the development/assembly of the Chl antenna in C. reinhardtii. A blast search with the Tla1 deduced amino acid sequence revealed that this protein is highly conserved in many eukaryotes. It showed homology to a protein of unknown function in Arabidopsis thaliana (73%), Oryza sativa (76%), Drosophila melanogaster (71%) and Homo sapiens (67%). The Tla1 gene apparently regulates genes that deWne the Chl antenna size in the photosynthetic apparatus of C. reinhardtii. Potential applications of the Tla1 gene in photosynthesis and biotechnology were discussed. PARTICIPANTS: MELIS Anastasios: Professor and Project Director; TETALI Sarada : Postdoctoral Scholar; MITRA Mautusi: Postdoctoral Scholar; SEIBERT Michael: Principal Scientist, NREL; GHIRARDI Maria L: Biochemist, NREL. TARGET AUDIENCES: The Academic Community; Research Institutions; Renewable Energy Companies; Venture Capital Firms PROJECT MODIFICATIONS: None

Impacts
The goal of the research is to improve the performance of photosynthesis under bright-sunlight and mass-culture conditions. A truncated Chl antenna size will permit sunlight to penetrate deeper into a high-density microalgal culture, thus enabling many more cells to contribute to useful photosynthesis and biofuels production. It has been shown that a truncated Chl antenna size will enable about 3-4 times greater solar energy conversion efficiency and photosynthetic productivity than could be achieved with fully pigmented cells. Accordingly, research is directed toward identification of those genes that confer a truncated chlorophyll antenna size in photosynthetic organisms. Functional properties of these genes and the corresponding regulatory and metabolic processes are being investigated. The research is both of fundamental and practical importance, as it may lead to the development of microalgal strains that can achieve superior performance in photosynthesis.

Publications

• TETALI SD, MITRA M, MELIS A (2007) Development of the light-harvesting chlorophyll antenna in the green alga CHLAMYDOMONAS REINHARDTII is regulated by the novel Tla1 gene. PLANTA 225: 813-829
• MELIS A, SEIBERT M, GHIRARDI ML (2007) Hydrogen fuel production by transgenic microalgae. In: Leon R, Gavan A, Fernandez E (eds) Transgenic Microalgae as Green Cell Factories. Landes Bioscience, Austin, Texas. Chapter 10, pp. 110-121
• MELIS A (2007) Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green algae). PLANTA 226: 1075-1086

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Biological systems offer a variety of ways by which to generate renewable energy. Among them, unicellular green algae have the ability of capturing the visible portion of sunlight and storing the energy as hydrogen. They hold promise in generating a renewable fuel from nature's most plentiful resources, sunlight and water. Anoxygenic photosynthetic bacteria have the ability of capturing the near infrared emission of sunlight to produce hydrogen while consuming small organic acids. Dark anaerobic fermentative bacteria consume carbohydrates, thus generating hydrogen and small organic acids. Whereas efforts are under way to develop each of these individual systems, little effort has been undertaken to combine and integrate these various processes for increased efficiency and greater yields. This work addresses the development of an integrated biological hydrogen production process based on unicellular green algae, which are driven by the visible portion of the solar spectrum, coupled with purple photosynthetic bacteria, which are driven by the near infrared portion of the spectrum, to generate hydrogen. Specific methods have been tested for the co-cultivation and production of hydrogen by the two different biological systems. Thus, a two-dimensional integration of photobiological hydrogen production has been achieved resulting in better solar irradiance utilization (visible and infrared) and integration of nutrient utilization for the cost-effective production of substantial amounts of hydrogen gas. Approaches are discussed for the co-cultivation and co-production of hydrogen in green algae and purple photosynthetic bacteria entailing broad utilization of the solar spectrum. The possibility to improve efficiency even further is discussed, with dark anaerobic fermentations of the photosynthetic biomass, enhancing the hydrogen production process and providing a recursive link in the system to regenerate some of the original nutrients. PARTICIPANTS: MELIS Anastasios: Professor and Project Director; ZHANG Liping: Postdoctoral Scholar; BENEMANN John R: Postdoctoral Scholar; FORESTIER Marc: Postdoctoral Scholar; GHIRARDI Maria L: Research Biochemist; SEIBERT Michael: Senior Research Biochemist; PARK Seunghye: Ph.D. student; POLLE Juergen: Postdoctoral Scholar; LEE Taek Kyun: Ph.D. student; JIN EonSeon: Assistant Professor; MELNICKI Matthew: Ph.D. student. TARGET AUDIENCES: The Academic Community; Research Institutions; Renewable Energy Companies; Venture Capital Firms. PROJECT MODIFICATIONS: None

Impacts
The goal of the research is to elucidate the molecular mechanism that defines the performance characteristics in photosynthetic organisms. Research is directed toward identification of those genes that confer metabolic intergartion and hydrogen production in green microalgae and purple photosynthetic bacteria. Functional properties of these genes and metablic processes are being investigated. The research is of both fundamental and practical importance. It may lead to the development of a renewable biofuel production via the natural process of photosynthesis.

Publications

• MELIS A, ZHANG L, BENEMANN JR, FORESTIER M, GHIRARDI ML and SEIBERT M (2006) Hydrogen production using hydrogenase-containing oxygenic photosynthetic organisms. United States Patent 6,989,252 B2 (issued 24-Jan-2006)
• PARK S, POLLE JE, MELIS A, LEE TK and JIN E (2006) Up-regulation of photoprotection and PSII-repair gene expression by irradiance in the unicellular green alga DUNALIELLA SALINA. Marine Biotechnoilogy 8: 120-128.
• MELIS A and MELNICKI M (2006) Integrated biological hydrogen production. International Journal of Hydrogen Energy 31: 1563-1573