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Making Biofuel from Microalgae

So much potential coexists with so many scientific, environmental and economic challenges

Philip T. Pienkos, Lieve Laurens, Andy Aden

A New Agriculture

In order to produce that much algal biomass, it will be necessary to develop a new type of agriculture, comparable in scale to the amount of U.S. farmland devoted to growing corn, but focused on a microscopic crop. That will require novel methods for cultivation, harvesting and processing. As anyone with a poorly maintained swimming pool can attest, algae can grow without much prompting. But this agriculture will require crops that grow at maximum rates and achieve the highest possible concentration of cells per liter of cultivation medium. Successful algae crops must be able to thrive in the presence of pests, predators and pathogens. These include “weed” algal strains that are more robust than production strains but that are of no use in biofuel production, grazers such as rotifers, and infectious agents such as bacteria, fungi and viruses. All this will require a carefully engineered cultivation process.

2011-11PienkosF4.jpgClick to Enlarge ImageMuch of our research focuses on the growth rate and lipid content of algae. At this stage, that includes bioprospecting—the search for natural strains with high growth rate and high lipid content, as well as robustness. We look for species that are resistant to pests, predators and pathogens and have the ability to thrive under environmental conditions—sunlight, temperature and water chemistry—expected at a cultivation site. In this search, we use technologies developed for the biotechnology and pharmaceutical industries, such as robotics, liquid handling devices and fluorescence-activated cell sorting, to speed up the isolation of single algal cells from environmental samples and to test them for growth rate and lipid content. Learning from colleagues affiliated with the Culture Collection of Algae at the University of Texas, we developed methods to preserve culture samples cryogenically and to revive them at will, eliminating the laborious and sometimes counterproductive practice of maintaining algal cultures on agar plates and slants, which requires regular transfers to fresh media.

Our work in bioprospecting and our involvement with the Sustainable Algal Biofuels Consortium, led by Arizona State University, have made it clear to us that there is a widespread need to accelerate the quantification of lipids and tailor the analysis to rapidly screen several hundreds or thousands of individual strains, which is not feasible with traditional gravimetric or chromatographic separation processes. At NREL, we have developed rapid, infrared-spectroscopic, high-throughput methods for the estimation of algal lipid content based on multivariate calibration models. We have demonstrated the applicability of such methods to the quantification of exogenous lipids in algae. And we have applied improved methods to hundreds of algal biomass samples from more than 80 species, with lipid content varying from 10 to more than 60 percent over the growth of the culture. This method can distinguish between neutral and polar lipids, which is difficult using standard techniques, and could facilitate high-throughput screening to detect promising algal strains in metabolic engineering or bioprospecting projects.

In addition to C. vulgaris, mentioned above, NREL researchers are also evaluating other eukaryotic algae, including Chlamydomonas reinhardtii and species of Scenedesmus and Nannochloropsis. All three are well-researched laboratory strains. C. reinhardtii does not make much oil but is probably the most common eukaryotic algal lab strain. Its genome has been fully sequenced and many tools are available for genetic manipulation. Many of the Scenedesmus and Nannochloropsis cultivars have been exploited for oil production because both strains grow well in large-scale open ponds and photobioreactors, and both can make a significant amount of oil. Although algal researchers around the world are nowhere near exhausting the algal diversity that nature provides, many of us are also working on projects to improve on natural algal strains by classical genetics (mutation, selections, screening and breeding) as well as by genetic engineering. We are working on a number of “omics,” or system-biology projects, using genomics, transcriptomics and proteomics to understand the molecular details of high-lipid productivity and to use that information to inform experiments in genetic engineering. Goals include turning up or down gene-expression levels so that higher lipid levels can be achieved under conditions that allow rapid growth and to generate strains that are more amenable to harvesting. One example of our genetic-engineering research involves the cyanobacterium Synechocystis PCC 6803. Although untransformed cyanobacteria do not produce TAGs, we are engineering a species that can do so by redirecting carbon metabolism from carbohydrate production to fatty-acid production and by inserting missing genes for TAG production.

Genetic engineering can be a controversial topic because of fears that large-scale cultivation could result in the release of engineered strains whose recombinant genes could infiltrate other species. Regardless of whether engineered strains are ever allowed outside laboratories (where strict guidelines are in place to prevent accidental release), genetic-engineering research is critical for understanding the potential of a single algal strain to have all the properties needed for large-scale oil production. This research must be done in parallel with studies using natural strains or strains derived from classical genetics and breeding to understand the risks and to put safeguards in place for large- scale cultivation.

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