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A new species of marine algae Picochlorum celeri was obtained by enriching for fast reproductive rates under constant high irradiance in semi-continuous culture. Several rapidly-growing cell lines were isolated from the enrichment culture by bringing up cultures from single cells in very high light. Therefore, individual cells are very resilient to inhibition by light. This is a prominent characteristic of this P. celeri in all cell lines. Measured oxygen evolution rates were unusually high. One of these lines is able to double in as short as 2.0 h. Doubling times of other cell lines were between 2.4 and 3.5 h. This species is characterized by high pigment content, high absorption cross section, and an antenna size that is very responsive to irradiance becoming very small at high irradiance. more...

As a result of a secondary endosymbiosis of a red alga, stramenopiles retained β-1,3 glucans as the primary soluble storage carbohydrate, in contrast to the starch and glycogen of green plants and animals. This storage carbohydrate has been identified and characterized in representative diatoms, brown algae, and oomycetes, but has not been biochemically characterized in species of Nannochloropsis, an industrially relevant genus of algae favored for its lipid content and productivity. In this study, the soluble storage carbohydrate of Nannochloropsis is characterized by 1H NMR, linkage analysis, and size exclusion chromatography. The putative genes encoding enzymes required for synthesis of the carbohydrate oligosaccharide from glucose 6-phosphate are identified. A glycogenin-like glycosyltransferase family 8 (GT8) protein was also identified to be conserved among all species of Nannochloropsis, despite the lack of glycogen in the genus. Homologs were identified in available genomes of brown algae, diatoms, and oomycetes, all documented to utilize β-1,3 glucans for carbon storage. Finally, a set of three likely laminarinases are highlighted from the glycosyl hydrolases by phylogenetic analyses. more...

Picochlorum celeri has among the fastest photoautotrophic growth rates (~2 h doubling time in optimal conditions) reported to date for a marine alga. This study comprehensively analyzes the levels of Picochlorum celeri photosynthetic pigments, which can reach up to ~13% of the total particulate organic carbon (POC) under light-limiting conditions. The main Picochlorum celeri pigments identified include: chlorophyll a, chlorophyll b, lutein, β-carotene, canthaxanthin, violaxanthin, neoxanthin, zeaxanthin and antheraxanthin. The ketocarotenoid canthaxanthin can accumulate up to 120 mg L−1 in Picochlorum celeri liquid culture; ~12% of it was found in an extracellular polysaccharide matrix. Using a solar-simulating automated photobioreactor, we monitor the photoacclimation of cultures maintained in unshaded conditions ( more...

The model euryhaline cyanobacterium Synechococcus sp. PCC 7002 has potential as a platform for biotechnological applications because of its genetic amenability and fast growth rates. The purpose of this work was to measure the outdoor productivity metrics of Synechococcus sp. PCC 7002, which we tested by cultivation in 110 L flat-panel photobioreactors and 1025 L open raceway ponds at the Algae Testbed Public Private Partnership (ATP3) algae culturing testbed site at the Arizona Center for Algae Technology and Innovation (AzCATI). This work was carried out during the summer growing season when ambient temperatures and light availability are highest to support maximum photoautotrophic biomass yields. Modifications to the traditional A+ laboratory growth media for Synechococcus sp. PCC 7002 included the use of urea as a nitrogen source, and CO2 sparging for automated pH control in open ponds. Synechococcus showed robust growth in flat-panel photobioreactors, which are suited for the containment of modified organisms; however, cultivation in the open-raceway pond system was challenging due to pond crashes. We investigated the microbiota associated with Synechococcus pond cultures using Illumina amplicon sequencing of the 16S rRNA genes of bacteria and archaea and the 18S rRNA genes of microbial eukaryotes, to identify potential competitor and predator genera. These results pointed to likely predation of Synechococcus by a ciliated protozoan of the genus Euplotes. Development of crop protection strategies will be crucial for enabling the mass cultivation of Synechococcus in open pond systems. more...

Nannochloropsis species have garnered significant interest for biofuel production due to their ability to accumulate high levels of triacylglycerols (TAGs), especially following nitrogen-starvation. However, the first response to nutrient starvation is the synthesis of chrysolaminarin, a soluble β-1,3-glucan with β-1,6-branching. We employ CRISPR/Cas9 to knock out two key enzymes responsible for the synthesis of this oligosaccharide: a beta-glucan synthase (BGS) gene putatively responsible for the glucose β-1,3-linkages, and a transglycosylase (TGS) which putatively catalyzes β-1,6-branching. Analysis of the biomass from the generated mutants confirmed an ~5-fold decrease in the accumulation of soluble carbohydrate following nitrogen starvation, without an observed growth defect in a diel light-cycling regime compared to CRISPR-expressing controls. more...

The high oxygen (O2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1− hydA2−) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H2 photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O2 inactivation of CaI led to more sustained H2 photoproduction when cultures were challenged with O2 or kept under prolonged illumination at solar intensities. These results provide new insights into the requisites for attaining photobiological H2 production from water splitting using a more O2-tolerant hydrogenase. more...

Originally developed in bacterial and fungal models, Adaptive Laboratory Evolution (ALE) experimentally selects for targeted phenotypes by allowing cells containing beneficial random genetic mutations to replicate more quickly in the presence of environmental stress. When applied to microalgae, this technique has been used to increase growth rate, stress tolerance, substrate utilization, and product yield. This review discusses ALE experimental design parameters, and summarizes key results and lessons learned from recent microalgal ALE studies. By documenting the current state of the field, we provide insight on the ways microalgal ALE might be used to develop commercially viable biotechnology strains. more...

Cas9-guide RNA ribonucleoprotein (RNP) complexes were used to efficiently generate insertions in both alleles of two targeted genes (nitrate reductase and carotenoid isomerase) in the biotechnologically relevant alga Picochlorum celeri. Diploid loss-of-function mutants were present in ~6% of transformants targeting nitrate reductase and in ~50% of transformants targeting carotenoid isomerase. The protocol described here successfully generates mutants within weeks and can be used to mechanistically probe the remarkably fast growth rates and extreme physiological tolerances of this alga. more...

Flocculation provides an attractive route for the primary dewatering of dilute suspensions of microalgae and cyanobacteria, however, economical harvesting and separations remain challenging. In this article, recyclable flocculants are demonstrated in a novel approach for the harvesting of fresh- and saltwater microalgae and cyanobacteria. Polyampholytes, based on model acrylamide polymers, provide reversible electrostatic interactions with negatively-charged cellular microorganisms through changes in pH. The behavior of the polyampholytic flocculants is characterized for the harvesting of Chlamydomonas reinhardtii (Chlamydomonas Genetics Center CC124), Synechococcus PCC 7002, Aulacoseira ambigua (Varsity Lake, CU Boulder), Nannochloropsis gaditana (CCMP526), and Chlorella vulgaris (UTEX 395). The polyampholytic flocculants, with reversible electrostatic interactions, achieve greater than 97% flocculation efficiencies, can be recovered at greater than 90% yields following flocculation, and when recycled retain flocculation efficiencies of at least 95%. Additionally, the recyclable polymer flocculants, in contrast to what is possible with single-use commercial flocculants, are demonstrated to be more robust for the dewatering process, regardless of culture salinity and other differences (e.g., structural, motility, or surface charge) exhibited by the microorganisms. A techno-economic analysis of the dewatering process is also performed, with recyclable polyampholytic flocculants providing an opportunity to reduce flocculant operating costs by ~ 85% at standard conditions. more...

A promising renewable energy scenario involves growing photosynthetic microalgae as a biofuel feedstock that can be converted into fungible, energy-dense fuels. Microalgae transform the energy in sunlight into a variety of reduced-carbon storage products, including triacylglycerols, which can be readily transformed into diesel fuel surrogates. To develop an economically viable algal biofuel industry, it is important to maximize the production and accumulation of these targeted bioenergy carriers in selected strains. In an effort to identify promising feedstock isolates we developed, evaluated and optimized contemporary high-throughput cell-sorting techniques to establish a collection of microalgae isolated from highly diverse ecosystems near geographic areas that are potential sites for large-scale algal cultivation in the Southwest United States. These efforts resulted in a culture collection containing 360 distinct microalgal strains. We report on the establishment of this collection and some preliminary qualitative screening studies to identify important biofuel phenotypes including neutral lipid accumulation and growth rates. As part of this undertaking we determined suitable cultivation media and evaluated cryopreservation techniques critical for the long-term storage of the microorganisms in this collection. This technique allows for the rapid isolation of extensive strain biodiversity that can be leveraged for the selection of promising bioenergy feedstock strains, as well as for providing fundamental advances in our understanding of fundamental algal biology. more...