We introduce a mathematically rigorous analysis of a generalized spinboson system for the treatme... more We introduce a mathematically rigorous analysis of a generalized spinboson system for the treatment of a donor-acceptor (reactant-product) quantum system coupled to a thermal quantum noise. The donor/acceptor probability dynamics describes transport reactions in chemical processes in presence of a noisy environment-such as the electron transfer in a photosynthetic reaction center. Besides being rigorous, our analysis has the advantages over previous ones that (1) we include a general, non energyconserving system-environment interaction, and that (2) we allow for the donor or acceptor to consist of multiple energy levels lying closely together. We establish explicit expressions for the rates and the efficiency (final donor-acceptor population difference) of the reaction. In particular, we show that the rate increases for a multi-level acceptor, but the efficiency does not.
There is growing evidence that eukaryotic microalgae can become a more sustainable and profitable... more There is growing evidence that eukaryotic microalgae can become a more sustainable and profitable alternative than terrestrial crops to produce feed, fuels, and valuable coproducts. The major factor driving progress in algal biomass production is the potential of microalgae to produce substantially greater biomass per unit land area than terrestrial crops. To be financially feasible, however, current algal biomass yields must be increased. Given the fact that algal biomass production is in its infancy there exist multiple opportunities to improve biomass yields. For example, recent bioprospecting efforts have led to the identification of new microalgal strains having biomass yields that compete economically with plant biomass. Substantial increases in biomass yields have also been achieved using advanced genetic engineering approaches. Targeted improvements in photosynthetic efficiency have led to three-fold increases in algal biomass yields. One genetic tool that has seen limited a...
Algal Research-Biomass Biofuels and Bioproducts, 2017
Energy-efficient and scalable harvesting and lipid extraction processes must be developed in orde... more Energy-efficient and scalable harvesting and lipid extraction processes must be developed in order for the algal biofuels and bioproducts industry to thrive. The major challenge for harvesting is the handling of large volumes of cultivation water to concentrate low amounts of biomass. For lipid extraction, the major energy and cost drivers are associated with disrupting the algae cell wall and drying the biomass before solvent extraction of the lipids. Here we review the research and development conducted by the Harvesting and Extraction Team during the 3-year National Alliance for Advanced Biofuels and Bioproducts (NAABB) algal consortium project. The harvesting and extraction team investigated five harvesting and three wet extraction technologies at lab bench scale for effectiveness, and conducted a techoeconomic study to evaluate their costs and energy efficiency compared to available baseline technologies. Based on this study, three harvesting technologies were selected for further study at larger scale. The selected harvesting technologies: electrocoagulation, membrane filtration, and ultrasonic harvesting, were evaluated in a field study at minimum scale of 100 L/h. None of the extraction technologies were determined to be ready for scale-up; therefore, an emerging extraction technology (wet solvent extraction) was selected from industry to provide scale-up data and capabilities to produce lipid and lipidextracted materials for the NAABB program. One specialized extraction/adsorption technology was developed that showed promise for recovering high value co-products from lipid extracts. Overall, the NAABB Harvesting and Extraction Team improved the readiness level of several innovative, energy efficient technologies to integrate with algae production processes and captured valuable lessons learned about scale-up challenges. 1. Introduction 1.1. Preface Harvesting algae and extracting the lipids are significant cost drivers in the biofuel production process [1-6]. Therefore, the goal of the Harvesting and Extraction task within the National Alliance for Biofuels and Bioproducts (NAABB) was to develop low-energy, low-cost harvesting and extraction technologies that could feed lipids into highly efficient fuel-conversion processes [7]. At the outset of the project, the NAABB leadership team determined that the harvesting and extraction technologies developed in this task must have: • Low capital expense (CAPEX) and operating expense (OPEX) • Ease of operation and low maintenance requirements
and 2007, its production and acreage tripled across africa and quadrupled in nigeria, the contine... more and 2007, its production and acreage tripled across africa and quadrupled in nigeria, the continent’s and the world’s largest producer (fao, 00 ). SubSaharan africa produced over million tons of fresh roots of cassava in 00 , of which no less than 95% was consumed as food; the starch provides > 5% of dietary energy for an estimated 00 million africans (Dorosh, 00 ). frequent consumers of cassava are at greater risk for malnutrition—especially deficiencies in vitamin-a, iron, and zinc—than consumers of other diets, particularly those that are cereal-based (Gegios et al., 0 0). a nutrition survey in cassava-consuming areas of nigeria and Kenya revealed inadequate intake of vitamin a in 3% and % and inadequate iron intake in 3% and % of pre-school-aged children, respectively (Gegios et al., 0 0). vitamin-a deficiency causes a loss of 9 ,000 disability-adjusted life years (DaLYs1) in nigeria and ,000 DaLYs in Kenya annually; iron deficiency causes loss of 59 ,000 DaLYs in nigeria and...
EPR spectra attributed to the redox active tyrosine residues on the oxidizing side of photosystem... more EPR spectra attributed to the redox active tyrosine residues on the oxidizing side of photosystem II (TyrZ and TyrD) have almost identical line shapes, although the tyrosyl radicals differ in stability and redox characteristics. Strongly modified spectra of oxidized TyrD in site-directed mutants in a histidine residue, H189 on the D2 reaction center protein in the cyanobacterium Synechocystis 6803, support a structural model where H189 interacts closely, probably via a hydrogen bond, to TyrD (Tommos, C., Davidsson, L., Svensson, B., Madsen, C., Vermass, W., and Styring, S. (1993) Biochemistry 32, 5436-5441). To determine whether TyrZ and the corresponding histidine on the D1 protein (D1-H190) interacts similarly, we have generated His-Phe (H190F) and His-Tyr (H190Y) mutations in the C2 symmetry related H190 residue on the D1 reaction center protein by site-directed mutagenesis in Chlamydomonas reinhardtii. The H190F and H190Y mutants assemble photosystem II reaction centers capable of primary photochemistry but unable to oxidize water. We have obtained kinetic spectra of a flash-induced transient EPR signal that we assign to oxidized TyrZ in the D1-H190 mutants. The spectra are identical in line width (18-20 G) and hyperfine structure to the wild-type spectrum from oxidized TyrZ and exhibit decay kinetics (t 1/2 approximately 500 ms) typical for the TyrZ radical in managenese-depleted photosystem II membranes. However, both TyrZ and TyrD were oxidized with reduced (10-15%) quantum yield in these mutants, indicating that the kinetics of electron donation to P+680 were significantly modified as a result of the mutation. Thus, the altered kinetics of TyrZ in the mutants suggest that there is an interaction between TyrZ and His-190 on the D1 protein. However, unlike the situation on the D2 side, the presence of a hydrogen bond between TyrZ and H190 on the D1 protein is improbable.
An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DN... more An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.
One of the major constraints limiting biomass production in autotrophs is the low thermodynamic e... more One of the major constraints limiting biomass production in autotrophs is the low thermodynamic efficiency of photosynthesis, ranging from 1 to 4%. Given the absorption spectrum of photosynthetic pigments and the spectral distribution of sunlight, photosynthetic efficiencies as high as 11% are possible. It is well-recognized that the greatest thermodynamic inefficiencies in photosynthesis are associated with light absorption and conversion of excited states into chemical energy. This is due to the fact that photosynthesis light saturates at one quarter full sunlight intensity in plants resulting in the dissipation of excess energy as heat, fluorescence and through the production of damaging reactive oxygen species. Recently, it has been demonstrated that it is possible to adjust the size of the light harvesting antenna over a broad range of optical cross sections through targeted reductions in chlorophyll b content, selectively resulting in reductions of the peripheral light harvesting antenna size, especially in the content of Lhcb3 and Lhcb6. We have examined the impact of alterations in light harvesting antenna size on the amplitude of photoprotective activity and the evolutionary fitness or seed production in Camelina grown at super-saturating and sub-saturating light intensities to gain an understanding of the driving forces that lead to the selection for light harvesting antenna sizes best fit for a range of light intensities. We demonstrate that plants having light harvesting antenna sizes engineered for the greatest photosynthetic efficiency also have the greatest capacity to mitigate high light stress through nonphotochemical quenching and reduction of reactive oxygen associated damage. Under sub-saturating growth light intensities, we demonstrate that the optimal light harvesting antenna size for photosynthesis and seed production is larger than that for plants grown at super-saturating light intensities and is more similar to the antenna size of wild-type plants. These results suggest that the light harvesting antenna size of plants is designed to maximize fitness under low light conditions such as occurs in shaded environments and in light competition with other plants.
We examine the dynamics of entanglement entropy of all parts in an open system consisting of a tw... more We examine the dynamics of entanglement entropy of all parts in an open system consisting of a two-level dimer interacting with an environment of oscillators. The dimer-environment interaction is almost energy conserving. We find the precise link between decoherence and production of entanglement entropy. We show that not all environment oscillators carry significant entanglement entropy and we identify the oscillator frequency regions which contribute to the production of entanglement entropy. For energy conserving dimer-environment interactions the models are explicitly solvable and our results hold for all dimer-environment coupling strengths. We carry out a mathematically rigorous perturbation theory around the energy conserving situation in the presence of small non-energy conserving interactions.
In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium bega... more In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy's Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played by metabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels.
We model and simulate the performance of charge-transfer in nonphotochemical quenching (NPQ) in t... more We model and simulate the performance of charge-transfer in nonphotochemical quenching (NPQ) in the CP29 lightharvesting antenna-complex associated with photosystem II (PSII). The model consists of five discrete excitonic energy states and two sinks, responsible for the potentially damaging processes and charge-transfer channels, respectively. We demonstrate that by varying (i) the parameters of the chlorophyll-based dimer, (ii) the resonant properties of the protein-solvent environment interaction, and (iii) the energy transfer rates to the sinks, one can significantly improve the performance of the NPQ. Our analysis suggests strategies for improving the performance of the NPQ in response to environmental changes, and may stimulate experimental verification.
We present a theoretical analysis of exciton transfer and decoherence effects in a photosynthetic... more We present a theoretical analysis of exciton transfer and decoherence effects in a photosynthetic dimer interacting with collective (correlated) and local (uncorrelated) protein-solvent environments. Our approach is based on the framework of the spin-boson model. We derive explicitly the thermal relaxation and decoherence rates of the exciton transfer process, valid for arbitrary temperatures and for arbitrary (in particular, large) interaction constants between the dimer and the environments. We establish a generalization of the Marcus formula, giving reaction rates for dimer levels possibly individually and asymmetrically coupled to environments. We identify rigorously parameter regimes for the validity of the generalized Marcus formula. The existence of long living quantum coherences at ambient temperatures emerges naturally from our approach.
Induction of oil accumulation in algae for biofuel production is often achieved by withholding ni... more Induction of oil accumulation in algae for biofuel production is often achieved by withholding nitrogen. However, withholding nitrogen often reduces total biomass yield. In this report, it is demonstrated that Chlorella sorokiniana will not only accumulate substantial quantities of neutral lipids when grown in the absence of nitrogen but will also exhibit unimpeded growth rates for up to 2 weeks. To determine the physiological basis for the observed increase in oil and biomass accumulation, we compared photosynthetic and respiration rates and chlorophyll, lipid, and total energy content under ammonia replete and deplete conditions. Under Ndepleted growth conditions, there was a 64 % increase in total energy density and a ∼20-fold increase in oil accumulation relative to N-replete growth leading to a 1.6-fold greater total energy yield in N-depleted than in N-replete cultures. We propose that the higher energy accumulation in N-depleted cultures is due to enhanced photosynthetic energy capture and conversion associated with reduced chlorophyll levels and reduced self-shading as well as a shift in metabolism leading to the accumulation of oils.
Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic pr... more Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic products, photosynthetic rates, enhancement under low oxygen concentration, and CO2 compensation points. In addition, pulse-chase labeling experiments were conducted using one of the four populations. Depending on the plant population, C4 acids ranged from 40% to 11% of the primary products under short-term exposure to 14CO2. These compounds were also metabolized during pulse-chase experiments. All four populations had significantly different photosynthetic rates and those rates were correlated with the amounts of labelled C4 acids produced and C4-acid turnover. Three populations of M. verticillata had similar compensation points (40 gl/1) and degrees of photosynthetic enhancement under low [O2] (20%), the fourth population was much lower in both characteristics (CO2 compensation, 25 lal/1; low-O2 enhancement, 12%). The results verify the intermediate nature of photosynthesis in this species, and illustrate populational differences in its photosynthetic and photorespiratory carbon metabolism.
Early transcriptional responses of a cell walldeficient mutant of the green alga Chlamydomonas re... more Early transcriptional responses of a cell walldeficient mutant of the green alga Chlamydomonas reinhardtii to heavy-metal stress have been investigated using the method of mRNA differential display. We have identified, sequenced, and quantified the induction of a number of transcripts that are up-regulated by a brief (2-h) exposure to 25 lm cadmium chloride, including one transcript which is also highly responsive to iron (Fe) deficiency. These transcripts represent both nuclear-and chloroplast-encoded genes, and include both novel genes and genes with known or suspected functions. Among these is a gene with significant homology to HCR1, a high-CO 2-and Fe-deficiencyinducible gene from Chlorococcum littorale. We further characterized the regulation of the HCR1-like gene (H43) and found that this transcript is also induced by Fe-depletion of the medium. Heterologous expression of H43 in the Fe-uptake mutant fet3fet4 of Saccharomyces cerevisiae resulted in partial suppression of the slowgrowth phenotype of this mutant in minimal medium, and resulted in a 2-fold increase in Fe accumulation per cell. Our results demonstrate the utility of Chlamydomonas cwstrains for functional genomics studies of metal stress. The magnitudes of induction and functional analyses suggest possible utility for these genes in the study of metal stress sensing in green plants and development of novel Fe acquisition and phytoremediation strategies.
Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. pr... more Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N-acyl homoserine lactone (AHL) bacterial quorum-sensing (QS) signals and alter QS-regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal-mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepRstimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal-mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHLproducing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and ⁄ or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.
As one of the most pervasive environmental problems, Hg pollution in sediment is particularly dif... more As one of the most pervasive environmental problems, Hg pollution in sediment is particularly difficult to remediate because it cannot be decomposed. The application of ultrasound combined with biomass (transgenic Chlamydomonas reinhardtii (C. reinhardtii), a green alga) for the removal of Hg from model and contaminated sediments (Al(2)O(3), α-HgS, and PACS-2 marine sediment) was investigated in this study. Ultrasound was found to enhance Hg release from Al(2)O(3), α-HgS, and PACS-2 marine sediment into the aqueous phase compared to mechanical shaking. A transgenic C. reinhardtii (2AMT-2) expressing a plasmamembrane-anchored metallothionein polymer effectively recovered Hg(II) released into the aqueous phase by sonication over a broad pH range from 2.0 to 9.0. The results showed that this combined technique of ultrasound and alga biomass (2AMT-2) engineered for enhanced metal recovery was effective to remove Hg from solids and sediments, especially from Al(2)O(3) and α-HgS with no natural organic matter. The results of this study are discussed with respect to the development of in situ remediation techniques for Hg-contaminated sediments.
Exposed portions of the 32 kcl chloroplast membrane quinone-binding and triazine herbicide-bindin... more Exposed portions of the 32 kcl chloroplast membrane quinone-binding and triazine herbicide-binding protein of photosystem II have been mapped to the lumenal or to the outer (stromal) surface of the thylakoid by following reactions of antibodies generated against synthetic peptides corresponding to predicted hydrophilic amino acid sequences with normally oriented or everted membrane vesicles. These data have led to the construction of a model with five membranespanning domains. The model has been verified, in part, by immunoblots of fragments of the protein produced by trypsin treatment of thylakoids with peptidespecific antibodies. Some of the hydrophilic loops appear to be in close contact with proteins of the oxygen evolving complex of photosystem II inasmuch as their removal increases the antibody reaction.
Various bacterial species produce and monitor low-molecular weight signaling molecules that regul... more Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wildtype Vibrio harVeyi but exhibited no response to culture filtrates from V. harVeyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent K d) 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed. † We thank NOAA/Ohio Sea Grant for the financial support.
Flash-induced chlorophyll fluorescence kinetics from photosystem II in thylakoids from the dark-g... more Flash-induced chlorophyll fluorescence kinetics from photosystem II in thylakoids from the dark-grown wild type and two site-directed mutants of the D1 protein His190 residue (D1-H190) in Chlamydomonas reinhardtii have been characterized. Induction of the chlorophyll fluorescence on the first flash, reflecting electron transport from YZ to P680(+), exhibited a strong pH dependence with a pK of 7.6 in the dark-grown wild type which lacks the Mn cluster. The chlorophyll fluorescence decay, measured in the presence of DCMU, which reflects recombination between QA- and YZox, was also pH-dependent with a similar pK of 7.5. These results indicate participation by the same base, which is suggested to be D1-H190, in oxidation and reduction of YZ in forward electron transfer and recombination pathways, respectively. This hypothesis was tested in the D1-H190 mutants. Induction of chlorophyll fluorescence in these H190 mutants has been observed to be inefficient due to slow electron transfer from YZ to P680(+) [Roffey, R. A., et al. (1994) Biochim. Biophys. Acta 1185, 257-270]. We show that this reaction is pH-dependent, with a pK of 8. 1, and at pH >/=9, the fluorescence induction is efficient in the H190 mutants, suggesting direct titration of YZ. The efficient oxidation of YZ ( approximately 70% at pH 9.0) at high pH was confirmed by kinetic EPR measurements. In contrast to the wild type, the H190 mutants show little or no observable fluorescence decay. Our data suggest that H190 is an essential component in the electron transfer reactions in photosystem II and acts as a proton acceptor upon YZ oxidation. In the H190 mutants, this reaction is inefficient and YZ oxidation only occurs at elevated pHs when YZ itself probably is deprotonated. We also propose that H190 is able to return a proton to YZox during electron recombination from QA- in a reaction which does not take place in the D1-H190 mutants.
One of the unique features of electron transfer processes in photosystem II (PSII) reaction cente... more One of the unique features of electron transfer processes in photosystem II (PSII) reaction centers (RC) is the exclusive transfer of electrons down only one of the two parallel cofactor branches. In contrast to the RC core polypeptides (psaA and psaB) of photosystem I (PSI), where electron transfer occurs down both parallel redox-active cofactor branches, there is greater protein-cofactor asymmetry between the PSII RC core polypeptides (D1 and D2). We have focused on the identification of protein-cofactor relationships that determine the branch along which primary charge separation occurs (P(680)(+)/pheophytin(-)(Pheo)). We have previously shown that mutagenesis of the strong hydrogen-bonding residue, D1-E130, to less polar residues (D1-E130Q,H,L) shifted the midpoint potential of the Pheo(D1)/Pheo(D1)(-) couple to more negative values, reducing the quantum yield of primary charge separation. We did not observe, however, electron transfer down the inactive branch in D1-E130 mutants. The protein residue corresponding to D1-E130 on the inactive branch is D2-Q129 which presumably has a reduced hydrogen-bonding interaction with Pheo(D2) relative to the D1-E130 residue with Pheo(D1). Analysis of the recent 2.9 Å cyanobacterial PSII crystal structure indicated, however, that the D2-Q129 residue was too distant from the Pheo(D2) headgroup to serve as a possible hydrogen bond donor and directly impact its midpoint potential as well as potentially determine the directionality of electron transfer. Our objective was to characterize the function of this highly conserved inactive branch residue by replacing it with a nonconservative leucine or a conservative histidine residue. Measurements of Chl fluorescence decay kinetics and thermoluminescence studies indicate that the mutagenesis of D2-Q129 decreases the redox gap between Q(A) and Q(B) due to a lowering of the redox potential of Q(B). The resulting increased yield of S(2)Q(B)(-) charge recombination in the D2-Q129 mutants leads to an increased susceptibility to photoinhibitory light presumably due to (3)P(680)-mediated oxidative damage. The results indicate that the D2-Q129 residue plays a critical role in stabilizing the charge-separated state in PSII and further documents the structural and functional asymmetry between the two cofactor branches in PSII.
We introduce a mathematically rigorous analysis of a generalized spinboson system for the treatme... more We introduce a mathematically rigorous analysis of a generalized spinboson system for the treatment of a donor-acceptor (reactant-product) quantum system coupled to a thermal quantum noise. The donor/acceptor probability dynamics describes transport reactions in chemical processes in presence of a noisy environment-such as the electron transfer in a photosynthetic reaction center. Besides being rigorous, our analysis has the advantages over previous ones that (1) we include a general, non energyconserving system-environment interaction, and that (2) we allow for the donor or acceptor to consist of multiple energy levels lying closely together. We establish explicit expressions for the rates and the efficiency (final donor-acceptor population difference) of the reaction. In particular, we show that the rate increases for a multi-level acceptor, but the efficiency does not.
There is growing evidence that eukaryotic microalgae can become a more sustainable and profitable... more There is growing evidence that eukaryotic microalgae can become a more sustainable and profitable alternative than terrestrial crops to produce feed, fuels, and valuable coproducts. The major factor driving progress in algal biomass production is the potential of microalgae to produce substantially greater biomass per unit land area than terrestrial crops. To be financially feasible, however, current algal biomass yields must be increased. Given the fact that algal biomass production is in its infancy there exist multiple opportunities to improve biomass yields. For example, recent bioprospecting efforts have led to the identification of new microalgal strains having biomass yields that compete economically with plant biomass. Substantial increases in biomass yields have also been achieved using advanced genetic engineering approaches. Targeted improvements in photosynthetic efficiency have led to three-fold increases in algal biomass yields. One genetic tool that has seen limited a...
Algal Research-Biomass Biofuels and Bioproducts, 2017
Energy-efficient and scalable harvesting and lipid extraction processes must be developed in orde... more Energy-efficient and scalable harvesting and lipid extraction processes must be developed in order for the algal biofuels and bioproducts industry to thrive. The major challenge for harvesting is the handling of large volumes of cultivation water to concentrate low amounts of biomass. For lipid extraction, the major energy and cost drivers are associated with disrupting the algae cell wall and drying the biomass before solvent extraction of the lipids. Here we review the research and development conducted by the Harvesting and Extraction Team during the 3-year National Alliance for Advanced Biofuels and Bioproducts (NAABB) algal consortium project. The harvesting and extraction team investigated five harvesting and three wet extraction technologies at lab bench scale for effectiveness, and conducted a techoeconomic study to evaluate their costs and energy efficiency compared to available baseline technologies. Based on this study, three harvesting technologies were selected for further study at larger scale. The selected harvesting technologies: electrocoagulation, membrane filtration, and ultrasonic harvesting, were evaluated in a field study at minimum scale of 100 L/h. None of the extraction technologies were determined to be ready for scale-up; therefore, an emerging extraction technology (wet solvent extraction) was selected from industry to provide scale-up data and capabilities to produce lipid and lipidextracted materials for the NAABB program. One specialized extraction/adsorption technology was developed that showed promise for recovering high value co-products from lipid extracts. Overall, the NAABB Harvesting and Extraction Team improved the readiness level of several innovative, energy efficient technologies to integrate with algae production processes and captured valuable lessons learned about scale-up challenges. 1. Introduction 1.1. Preface Harvesting algae and extracting the lipids are significant cost drivers in the biofuel production process [1-6]. Therefore, the goal of the Harvesting and Extraction task within the National Alliance for Biofuels and Bioproducts (NAABB) was to develop low-energy, low-cost harvesting and extraction technologies that could feed lipids into highly efficient fuel-conversion processes [7]. At the outset of the project, the NAABB leadership team determined that the harvesting and extraction technologies developed in this task must have: • Low capital expense (CAPEX) and operating expense (OPEX) • Ease of operation and low maintenance requirements
and 2007, its production and acreage tripled across africa and quadrupled in nigeria, the contine... more and 2007, its production and acreage tripled across africa and quadrupled in nigeria, the continent’s and the world’s largest producer (fao, 00 ). SubSaharan africa produced over million tons of fresh roots of cassava in 00 , of which no less than 95% was consumed as food; the starch provides > 5% of dietary energy for an estimated 00 million africans (Dorosh, 00 ). frequent consumers of cassava are at greater risk for malnutrition—especially deficiencies in vitamin-a, iron, and zinc—than consumers of other diets, particularly those that are cereal-based (Gegios et al., 0 0). a nutrition survey in cassava-consuming areas of nigeria and Kenya revealed inadequate intake of vitamin a in 3% and % and inadequate iron intake in 3% and % of pre-school-aged children, respectively (Gegios et al., 0 0). vitamin-a deficiency causes a loss of 9 ,000 disability-adjusted life years (DaLYs1) in nigeria and ,000 DaLYs in Kenya annually; iron deficiency causes loss of 59 ,000 DaLYs in nigeria and...
EPR spectra attributed to the redox active tyrosine residues on the oxidizing side of photosystem... more EPR spectra attributed to the redox active tyrosine residues on the oxidizing side of photosystem II (TyrZ and TyrD) have almost identical line shapes, although the tyrosyl radicals differ in stability and redox characteristics. Strongly modified spectra of oxidized TyrD in site-directed mutants in a histidine residue, H189 on the D2 reaction center protein in the cyanobacterium Synechocystis 6803, support a structural model where H189 interacts closely, probably via a hydrogen bond, to TyrD (Tommos, C., Davidsson, L., Svensson, B., Madsen, C., Vermass, W., and Styring, S. (1993) Biochemistry 32, 5436-5441). To determine whether TyrZ and the corresponding histidine on the D1 protein (D1-H190) interacts similarly, we have generated His-Phe (H190F) and His-Tyr (H190Y) mutations in the C2 symmetry related H190 residue on the D1 reaction center protein by site-directed mutagenesis in Chlamydomonas reinhardtii. The H190F and H190Y mutants assemble photosystem II reaction centers capable of primary photochemistry but unable to oxidize water. We have obtained kinetic spectra of a flash-induced transient EPR signal that we assign to oxidized TyrZ in the D1-H190 mutants. The spectra are identical in line width (18-20 G) and hyperfine structure to the wild-type spectrum from oxidized TyrZ and exhibit decay kinetics (t 1/2 approximately 500 ms) typical for the TyrZ radical in managenese-depleted photosystem II membranes. However, both TyrZ and TyrD were oxidized with reduced (10-15%) quantum yield in these mutants, indicating that the kinetics of electron donation to P+680 were significantly modified as a result of the mutation. Thus, the altered kinetics of TyrZ in the mutants suggest that there is an interaction between TyrZ and His-190 on the D1 protein. However, unlike the situation on the D2 side, the presence of a hydrogen bond between TyrZ and H190 on the D1 protein is improbable.
An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DN... more An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.
One of the major constraints limiting biomass production in autotrophs is the low thermodynamic e... more One of the major constraints limiting biomass production in autotrophs is the low thermodynamic efficiency of photosynthesis, ranging from 1 to 4%. Given the absorption spectrum of photosynthetic pigments and the spectral distribution of sunlight, photosynthetic efficiencies as high as 11% are possible. It is well-recognized that the greatest thermodynamic inefficiencies in photosynthesis are associated with light absorption and conversion of excited states into chemical energy. This is due to the fact that photosynthesis light saturates at one quarter full sunlight intensity in plants resulting in the dissipation of excess energy as heat, fluorescence and through the production of damaging reactive oxygen species. Recently, it has been demonstrated that it is possible to adjust the size of the light harvesting antenna over a broad range of optical cross sections through targeted reductions in chlorophyll b content, selectively resulting in reductions of the peripheral light harvesting antenna size, especially in the content of Lhcb3 and Lhcb6. We have examined the impact of alterations in light harvesting antenna size on the amplitude of photoprotective activity and the evolutionary fitness or seed production in Camelina grown at super-saturating and sub-saturating light intensities to gain an understanding of the driving forces that lead to the selection for light harvesting antenna sizes best fit for a range of light intensities. We demonstrate that plants having light harvesting antenna sizes engineered for the greatest photosynthetic efficiency also have the greatest capacity to mitigate high light stress through nonphotochemical quenching and reduction of reactive oxygen associated damage. Under sub-saturating growth light intensities, we demonstrate that the optimal light harvesting antenna size for photosynthesis and seed production is larger than that for plants grown at super-saturating light intensities and is more similar to the antenna size of wild-type plants. These results suggest that the light harvesting antenna size of plants is designed to maximize fitness under low light conditions such as occurs in shaded environments and in light competition with other plants.
We examine the dynamics of entanglement entropy of all parts in an open system consisting of a tw... more We examine the dynamics of entanglement entropy of all parts in an open system consisting of a two-level dimer interacting with an environment of oscillators. The dimer-environment interaction is almost energy conserving. We find the precise link between decoherence and production of entanglement entropy. We show that not all environment oscillators carry significant entanglement entropy and we identify the oscillator frequency regions which contribute to the production of entanglement entropy. For energy conserving dimer-environment interactions the models are explicitly solvable and our results hold for all dimer-environment coupling strengths. We carry out a mathematically rigorous perturbation theory around the energy conserving situation in the presence of small non-energy conserving interactions.
In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium bega... more In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy's Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played by metabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels.
We model and simulate the performance of charge-transfer in nonphotochemical quenching (NPQ) in t... more We model and simulate the performance of charge-transfer in nonphotochemical quenching (NPQ) in the CP29 lightharvesting antenna-complex associated with photosystem II (PSII). The model consists of five discrete excitonic energy states and two sinks, responsible for the potentially damaging processes and charge-transfer channels, respectively. We demonstrate that by varying (i) the parameters of the chlorophyll-based dimer, (ii) the resonant properties of the protein-solvent environment interaction, and (iii) the energy transfer rates to the sinks, one can significantly improve the performance of the NPQ. Our analysis suggests strategies for improving the performance of the NPQ in response to environmental changes, and may stimulate experimental verification.
We present a theoretical analysis of exciton transfer and decoherence effects in a photosynthetic... more We present a theoretical analysis of exciton transfer and decoherence effects in a photosynthetic dimer interacting with collective (correlated) and local (uncorrelated) protein-solvent environments. Our approach is based on the framework of the spin-boson model. We derive explicitly the thermal relaxation and decoherence rates of the exciton transfer process, valid for arbitrary temperatures and for arbitrary (in particular, large) interaction constants between the dimer and the environments. We establish a generalization of the Marcus formula, giving reaction rates for dimer levels possibly individually and asymmetrically coupled to environments. We identify rigorously parameter regimes for the validity of the generalized Marcus formula. The existence of long living quantum coherences at ambient temperatures emerges naturally from our approach.
Induction of oil accumulation in algae for biofuel production is often achieved by withholding ni... more Induction of oil accumulation in algae for biofuel production is often achieved by withholding nitrogen. However, withholding nitrogen often reduces total biomass yield. In this report, it is demonstrated that Chlorella sorokiniana will not only accumulate substantial quantities of neutral lipids when grown in the absence of nitrogen but will also exhibit unimpeded growth rates for up to 2 weeks. To determine the physiological basis for the observed increase in oil and biomass accumulation, we compared photosynthetic and respiration rates and chlorophyll, lipid, and total energy content under ammonia replete and deplete conditions. Under Ndepleted growth conditions, there was a 64 % increase in total energy density and a ∼20-fold increase in oil accumulation relative to N-replete growth leading to a 1.6-fold greater total energy yield in N-depleted than in N-replete cultures. We propose that the higher energy accumulation in N-depleted cultures is due to enhanced photosynthetic energy capture and conversion associated with reduced chlorophyll levels and reduced self-shading as well as a shift in metabolism leading to the accumulation of oils.
Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic pr... more Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic products, photosynthetic rates, enhancement under low oxygen concentration, and CO2 compensation points. In addition, pulse-chase labeling experiments were conducted using one of the four populations. Depending on the plant population, C4 acids ranged from 40% to 11% of the primary products under short-term exposure to 14CO2. These compounds were also metabolized during pulse-chase experiments. All four populations had significantly different photosynthetic rates and those rates were correlated with the amounts of labelled C4 acids produced and C4-acid turnover. Three populations of M. verticillata had similar compensation points (40 gl/1) and degrees of photosynthetic enhancement under low [O2] (20%), the fourth population was much lower in both characteristics (CO2 compensation, 25 lal/1; low-O2 enhancement, 12%). The results verify the intermediate nature of photosynthesis in this species, and illustrate populational differences in its photosynthetic and photorespiratory carbon metabolism.
Early transcriptional responses of a cell walldeficient mutant of the green alga Chlamydomonas re... more Early transcriptional responses of a cell walldeficient mutant of the green alga Chlamydomonas reinhardtii to heavy-metal stress have been investigated using the method of mRNA differential display. We have identified, sequenced, and quantified the induction of a number of transcripts that are up-regulated by a brief (2-h) exposure to 25 lm cadmium chloride, including one transcript which is also highly responsive to iron (Fe) deficiency. These transcripts represent both nuclear-and chloroplast-encoded genes, and include both novel genes and genes with known or suspected functions. Among these is a gene with significant homology to HCR1, a high-CO 2-and Fe-deficiencyinducible gene from Chlorococcum littorale. We further characterized the regulation of the HCR1-like gene (H43) and found that this transcript is also induced by Fe-depletion of the medium. Heterologous expression of H43 in the Fe-uptake mutant fet3fet4 of Saccharomyces cerevisiae resulted in partial suppression of the slowgrowth phenotype of this mutant in minimal medium, and resulted in a 2-fold increase in Fe accumulation per cell. Our results demonstrate the utility of Chlamydomonas cwstrains for functional genomics studies of metal stress. The magnitudes of induction and functional analyses suggest possible utility for these genes in the study of metal stress sensing in green plants and development of novel Fe acquisition and phytoremediation strategies.
Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. pr... more Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N-acyl homoserine lactone (AHL) bacterial quorum-sensing (QS) signals and alter QS-regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal-mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepRstimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal-mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHLproducing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and ⁄ or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.
As one of the most pervasive environmental problems, Hg pollution in sediment is particularly dif... more As one of the most pervasive environmental problems, Hg pollution in sediment is particularly difficult to remediate because it cannot be decomposed. The application of ultrasound combined with biomass (transgenic Chlamydomonas reinhardtii (C. reinhardtii), a green alga) for the removal of Hg from model and contaminated sediments (Al(2)O(3), α-HgS, and PACS-2 marine sediment) was investigated in this study. Ultrasound was found to enhance Hg release from Al(2)O(3), α-HgS, and PACS-2 marine sediment into the aqueous phase compared to mechanical shaking. A transgenic C. reinhardtii (2AMT-2) expressing a plasmamembrane-anchored metallothionein polymer effectively recovered Hg(II) released into the aqueous phase by sonication over a broad pH range from 2.0 to 9.0. The results showed that this combined technique of ultrasound and alga biomass (2AMT-2) engineered for enhanced metal recovery was effective to remove Hg from solids and sediments, especially from Al(2)O(3) and α-HgS with no natural organic matter. The results of this study are discussed with respect to the development of in situ remediation techniques for Hg-contaminated sediments.
Exposed portions of the 32 kcl chloroplast membrane quinone-binding and triazine herbicide-bindin... more Exposed portions of the 32 kcl chloroplast membrane quinone-binding and triazine herbicide-binding protein of photosystem II have been mapped to the lumenal or to the outer (stromal) surface of the thylakoid by following reactions of antibodies generated against synthetic peptides corresponding to predicted hydrophilic amino acid sequences with normally oriented or everted membrane vesicles. These data have led to the construction of a model with five membranespanning domains. The model has been verified, in part, by immunoblots of fragments of the protein produced by trypsin treatment of thylakoids with peptidespecific antibodies. Some of the hydrophilic loops appear to be in close contact with proteins of the oxygen evolving complex of photosystem II inasmuch as their removal increases the antibody reaction.
Various bacterial species produce and monitor low-molecular weight signaling molecules that regul... more Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wildtype Vibrio harVeyi but exhibited no response to culture filtrates from V. harVeyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent K d) 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed. † We thank NOAA/Ohio Sea Grant for the financial support.
Flash-induced chlorophyll fluorescence kinetics from photosystem II in thylakoids from the dark-g... more Flash-induced chlorophyll fluorescence kinetics from photosystem II in thylakoids from the dark-grown wild type and two site-directed mutants of the D1 protein His190 residue (D1-H190) in Chlamydomonas reinhardtii have been characterized. Induction of the chlorophyll fluorescence on the first flash, reflecting electron transport from YZ to P680(+), exhibited a strong pH dependence with a pK of 7.6 in the dark-grown wild type which lacks the Mn cluster. The chlorophyll fluorescence decay, measured in the presence of DCMU, which reflects recombination between QA- and YZox, was also pH-dependent with a similar pK of 7.5. These results indicate participation by the same base, which is suggested to be D1-H190, in oxidation and reduction of YZ in forward electron transfer and recombination pathways, respectively. This hypothesis was tested in the D1-H190 mutants. Induction of chlorophyll fluorescence in these H190 mutants has been observed to be inefficient due to slow electron transfer from YZ to P680(+) [Roffey, R. A., et al. (1994) Biochim. Biophys. Acta 1185, 257-270]. We show that this reaction is pH-dependent, with a pK of 8. 1, and at pH >/=9, the fluorescence induction is efficient in the H190 mutants, suggesting direct titration of YZ. The efficient oxidation of YZ ( approximately 70% at pH 9.0) at high pH was confirmed by kinetic EPR measurements. In contrast to the wild type, the H190 mutants show little or no observable fluorescence decay. Our data suggest that H190 is an essential component in the electron transfer reactions in photosystem II and acts as a proton acceptor upon YZ oxidation. In the H190 mutants, this reaction is inefficient and YZ oxidation only occurs at elevated pHs when YZ itself probably is deprotonated. We also propose that H190 is able to return a proton to YZox during electron recombination from QA- in a reaction which does not take place in the D1-H190 mutants.
One of the unique features of electron transfer processes in photosystem II (PSII) reaction cente... more One of the unique features of electron transfer processes in photosystem II (PSII) reaction centers (RC) is the exclusive transfer of electrons down only one of the two parallel cofactor branches. In contrast to the RC core polypeptides (psaA and psaB) of photosystem I (PSI), where electron transfer occurs down both parallel redox-active cofactor branches, there is greater protein-cofactor asymmetry between the PSII RC core polypeptides (D1 and D2). We have focused on the identification of protein-cofactor relationships that determine the branch along which primary charge separation occurs (P(680)(+)/pheophytin(-)(Pheo)). We have previously shown that mutagenesis of the strong hydrogen-bonding residue, D1-E130, to less polar residues (D1-E130Q,H,L) shifted the midpoint potential of the Pheo(D1)/Pheo(D1)(-) couple to more negative values, reducing the quantum yield of primary charge separation. We did not observe, however, electron transfer down the inactive branch in D1-E130 mutants. The protein residue corresponding to D1-E130 on the inactive branch is D2-Q129 which presumably has a reduced hydrogen-bonding interaction with Pheo(D2) relative to the D1-E130 residue with Pheo(D1). Analysis of the recent 2.9 Å cyanobacterial PSII crystal structure indicated, however, that the D2-Q129 residue was too distant from the Pheo(D2) headgroup to serve as a possible hydrogen bond donor and directly impact its midpoint potential as well as potentially determine the directionality of electron transfer. Our objective was to characterize the function of this highly conserved inactive branch residue by replacing it with a nonconservative leucine or a conservative histidine residue. Measurements of Chl fluorescence decay kinetics and thermoluminescence studies indicate that the mutagenesis of D2-Q129 decreases the redox gap between Q(A) and Q(B) due to a lowering of the redox potential of Q(B). The resulting increased yield of S(2)Q(B)(-) charge recombination in the D2-Q129 mutants leads to an increased susceptibility to photoinhibitory light presumably due to (3)P(680)-mediated oxidative damage. The results indicate that the D2-Q129 residue plays a critical role in stabilizing the charge-separated state in PSII and further documents the structural and functional asymmetry between the two cofactor branches in PSII.
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Papers by Richard Sayre