SAMPLING AND ISOLATION OF PURE CULTURES

Microalgae grow in most of the natural environments including water, rocks and soil, but interestingly also grow on and in other organisms. Their main habitats are freshwater, brackish and marine ecosystems. Microalgae can be found and collected not only in general aquatic ecosystems such as lakes, rivers and the oceans, but also in extreme environments such as vol­canic waters and salt waters. Local microalgae species should be collected because it can be expected that they have a competitive advantage under the local geographical, climatic and ecological conditions. Our experience has shown that water and sediment samples from aquatic environments that undergo fluctuating and/or occasional adverse conditions provide a higher chance of isolating high lipid accumulating microalgae. Most likely these conditions would favor robust and opportunistic (fast-growing) al­gae with superior survival skills (e. g., by accumulation of storage lipids). Examples of these environments are tidal rock pools, estuaries and rivers.

Isolation is a necessary process to obtain pure cultures and presents the first step towards the selection of microalgae strains with potential for biodiesel production. Traditional isolation techniques include the use of a micropipette for isolation under a microscope or cell dilution followed by cultivation in liquid media or agar plates. Single cell isolation, based on traditional methods from the original sample is time-consuming and requires sterilized cultivation media and equipment, but the result of this elaborate process is always a pure culture that is usually easily identifi­able. Another approach in the laboratory includes the enrichment of some microalgae strains by adding nutrients for algal growth. The most impor­tant nutrient sources for algal growth are nitrogen and phosphate. Some particular algae species may require trace minerals for their growth (e. g., silicon for diatoms). Soil water extract is an excellent source of nutrients for algae growth at this stage because this medium is easy to produce and satisfies nutrient intake of many algae strains. Although automatic isola­tion techniques have offered some advantages towards traditional methods (see below), single cell isolation by a micropipette (e. g., a glass capillary) is still a very effective method that can be used for a wide range of samples and is very cost-effective. An automated single cell isolation method that has been developed and widely used for cell sorting is flow cytometry [17]. This technique has been successfully used for microalgae cell sort­ing from water with many different algae strains [18], primarily based on properties of chlorophyll autofluorescence (CAF) and green autofluores­cence (GAF) to distinguish algae species such as diatoms, dinoflagellates or prokaryotic phytoplankton.

Unlike for many agricultural crops, a targeted selection and domes­tication of microalgae strains is still in its infancy, while technology to economically grow microalgae with high lipid content is still be­ing developed [4]. Each microalgae strain requires careful selection and optimization in order to increase lipid productivity with the aim to provide a cropping system with improved biofuel production and per­formance properties [19]. Each micro-environment may provide algae strains with very different properties. As opposed to only manipulating a few individual cultured algae strains in the laboratory for high lipid productivity, a more efficient and useful strategy to identify oleagi­nous microalgae would be an in-depth and systematic investigation of a whole taxonomic group of microalgae over a wide geographical and ecological distribution [20]. By correlating this with algal oil contents and optimal environmental growth conditions, a predictive tool for se­lecting optimal microalgae strains for biofuel production maybe devel­oped. A bioinformatics approach could assist with the discovery of new algae isolates capable of biodiesel production and their phylogenetic grouping may suggest that potentially many more species have this ability. Typically, the steps involved in obtaining data for phylogenetic analysis include primers design (Table 1), DNA and/or RNA extrac­tion, PCR amplification, denaturizing gradient gel electrophoresis and/ or sequencing.

In 2010, seven microalgae genomes had been completed [27] and cur­rent efforts to obtain many other microalgal genome sequences will en­hance gene-based biofuel feedstock optimization studies (e. g., by meta­bolic engineering). The accumulation of storage lipid precursors and the discovery of genes associated with their biosynthesis and metabolism is a promising topic for investigation. For example, genes encoding key en­zymes involved in biosynthesis and catabolism of fatty acids/TAG and their regulation are currently not well understood (for a review of the lipid biosynthesis pathway in microalgae see Schuhmann et al. [28]). By pro­viding insight into the mechanisms underpinning the relevant metabolic processes, efforts can be made to identify molecular markers for selection or to genetically manipulate microalgae strains to enhance the production of feedstock for commercial microalgal biofuels. To date, genetic engi­neering approaches have been successfully used to improve biofuel phe­notypes only in Chlamydomonas reinhardtii, Nannochloropsis gaditana and Phaeodactylum tricornutum [29].