SUMMARYThe CO 2 concentrating mechanism (CCM) represents an effective strategy for carbon acquisition that enables microalgae to survive and proliferate when the CO 2 concentration limits photosynthesis. The CCM improves photosynthetic performance by raising the CO 2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), simultaneously enhancing carbon fixation and suppressing photorespiration. Active inorganic carbon (Ci) uptake, Rubisco sequestration and interconversion between different Ci species catalyzed by carbonic anhydrases (CAs) are key components in the CCM, and an array of molecular regulatory elements is present to facilitate the sensing of CO 2 availability, to regulate the expression of the CCM and to coordinate interplay between photosynthetic carbon metabolism and other metabolic processes in response to limiting CO 2 conditions. This review intends to integrate our current understanding of the eukaryotic algal CCM and its interaction with carbon assimilation, based largely on Chlamydomonas as a model, and to illustrate how Chlamydomonas acclimates to limiting CO 2 conditions and how its CCM is regulated.

Temperature is one of the most important environmental factors affecting the growth and survival of microorganisms and in light of current global patterns is of particular interest. Here, we highlight studies revealing how vitamin B 12 (cobalamin)-producing bacteria increase the fitness of the unicellular alga Chlamydomonas reinhardtii following an increase in environmental temperature. Heat stress represses C. reinhardtii cobalamin-independent methionine synthase (METE) gene expression coinciding with a reduction in METE-mediated methionine synthase activity, chlorosis and cell death during heat stress. However, in the presence of cobalamin-producing bacteria or exogenous cobalamin amendments C. reinhardtii cobalamin-dependent methionine synthase METHmediated methionine biosynthesis is functional at temperatures that result in C. reinhardtii death in the absence of cobalamin. Artificial microRNA silencing of C. reinhardtii METH expression leads to nearly complete loss of cobalamin-mediated enhancement of thermal tolerance. This suggests that methionine biosynthesis is an essential cellular mechanism for adaptation by C. reinhardtii to thermal stress. Increased fitness advantage of METH under environmentally stressful conditions could explain the selective pressure for retaining the METH gene in algae and the apparent independent loss of the METE gene in various algal species. Our results show that how an organism acclimates to a change in its abiotic environment depends critically on co-occurring species, the nature of that interaction, and how those species interactions evolve.

Efficient lysis of microalgae for lipid extraction is an important concern when processing biofuels. Historically, ultrasound frequencies in the range of 10-40 kHz have been utilized for this task. However, greater efficiencies might be achievable if higher frequencies could be used. In our study, we evaluated the potential of using 1.1 MHz ultrasound to lyse microalgae for biofuel production while using Chlamydomonas reinhardtii as a model organism. The ultrasound was generated using a spherically focused transducer with a focal length of 6.34 cm and an active diameter of 6.36 cm driven by 20 cycle sine-wave tone bursts at a pulse repetition frequency of 2 kHz (3.6% duty cycle). The time-average acoustic power output was 26.2 W while the spatial-peak-pulse-average intensity (ISPPA) for each tone burst was 41 kW/cm(2). The peak compressional and rarefactional pressures at the focus were 102 and 17 MPa, respectively. The exposure time was varied for the different cases in the experiments from 5s to 9 min and cell lysis was assessed by quantifying the percentage of protein and chlorophyll release into the supernate as well as the lipid extractability. Free radical generation and lipid oxidation for the different ultrasound exposures were also determined. We found that there was a statistically significant increase in lipid extractability for all of the exposures compared to the control. The longer exposures also completely fragmented the cells releasing almost all of the protein and chlorophyll into the supernate. The cavitation activity did not significantly increase lipid oxidation while there was a minor trend of increased free radical production with increased ultrasound exposure.

SummaryThe genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.

Green and white variegation in the Arabidopsis immutans (im) mutant is caused by a nuclear recessive gene. The green sectors contain cells with normal-appearing chloroplasts, while cells in the white sectors have photooxidized plastids lacking organized lamellae. In the present experiments, we found that the green im sectors have enhanced rates of carbon assimilation (monitored by (14)CO(2) uptake) and that there are corresponding increases in the activities of Rubisco and SPS, elevated starch and sucrose pool sizes, and an altered pattern of carbohydrate partitioning that favors sucrose over starch. We hypothesize that these increases are due, at least in part, to interactions with white sectors, perhaps to compensate for reductions in total source tissue. Consistent with this idea, the im white sectors accumulate low levels of sucrose and acid invertase activities are markedly increased in the white versus green cells. This suggests that there is a sucrose gradient between the green and white sectors, and that sucrose is transported from the green to white cells in response to sink demand. The expression of photosynthetic genes is not appreciably altered in the green im sectors versus wild type, but rather there is an up-regulation of genes involved in defense against oxidative stress and down-regulation of genes involved in cell wall biosynthesis. We postulate that changes in photosynthesis in the im green cells are driven by a need for photoprotection (especially early in chloroplast biogenesis) and due to source-sink interactions.