(582m) The Complimentary Use of Sophisticated (SIMS) and Crude (Nile red) Chemical Screening to Improve Algae As a Biofuels Production Platform

We present several lines of research focused on improving algae oil production.  Secondary Ion Mass Spectrometry (SIMS) is being used to better understand the extracellular matrix of the hydrocarbon-producing alga Botryococcus braunii race B which consists of triterpene oils as well as a surrounding polysaccharide network.  Nile red provides a crude means of correlating oil content for screening of different algae lines as well as different growth conditions for oil formation.  These two methods therefore represent the opposite ends of the spectrum in terms of in depth, data-intensive chemical profiling and rapid, crude production screening.  

SIMS CHEMICAL IMAGING:  Botryococcus braunii is a hydrocarbon-rich microalga that has a poorly understood and complex extracellular matrix within which the algae grow as a colony of cells. Additionally, a polysaccharide network surrounding B. braunii colonies increases the difficulty of studying and cultivating these valuable algae. SIMS provides an unprecedented depth of spatially-resolved chemical information in 2 to 3 dimensions of B. braunii.  In this manner, one can develop a chemical image of several chemical species within the algae.  This is particularly useful for biofilms, or colony forming algae such as B. braunii race B. Accordingly, high-resolution chemical imaging has been used to investigate the hydrocarbon storage and distribution of this strong candidate for alternative fuels.  Organelles within the hydrocarbon-rich extracellular matrix surrounding the algal cells have been chemically imaged utilizing a 250 nm diameter C60+ primary ion beam on the J105 3D Chemical Imager (Ionoptika, LTD). Organelles of great interest are approximately 1 -2 µm round vesicles within the extracellular matrix, which have been proposed to contain C23 – C37 liquid hydrocarbons, termed “botryococcenes,” unique to B. braunii race B.  They are revealed after depth profiling through the algal cell colonies, thus, enabling the acquisition of chemical information in three dimensions.  Unique chemical components of B. braunii over m/z 600 may be chemically mapped.  Chemical signatures for the polysaccharide network, algal cell wall, and the liquid hydrocarbon extracellular matrix have been identified.  The botryococcenes and hydrocarbon-derivative chemical species are located in specific locations dependent on their function within the cell and in the colony.  Peak identification has been aided by the use of tandem MS capabilities.  With the use of this unique chemical imaging technique, questions surrounding the purpose and function of the B. braunii cell’s behavior and association with hydrocarbons may be investigated.

ESTABLISHING AXENIC CULTURES:  An outcome of the SIMS work was a better understanding of the polysaccharide matrix which appears to function in part by holding the colonies together. Since it is desirable to have axenic cultures of B. braunii to carry out chemical feeding and genetic transformation studies, the presence of cellulose in the surrounding matrix suggested we might dissipate the algae colonies with cellulase treatment.  This has been substantiated by chemically mapping the presence of cellulose within B. braunii colonies. Accordingly, the use of hydrolytic enzymes with endoxylanase and cellulase activity to degrade the polysaccharide wall surrounding these algal colonies resulted in the isolation of single cells. These results indicate that an axenic culture may be obtained through dilution plating or laser capture microdissection of these single cells. The outcome of this work will enable the mixotrophic growth of B. braunii resulting in an efficient and expedient means to scaling up for large scale biofuels production.

NILE RED SCREENING:  Complementary to the data-intensive chemical profiling provided by SIMS, Nile red fluorescence staining provides a means of rapidly screening for the hydrocarbon content in microalgae.  This method has been applied for the determination of the neutral hydrocarbon content in microalgae, where fluorescence intensity is correlated to the quantity of oil present.  Conventional solvent extraction methods are time consuming and require large culture samples as compared to the staining of algae in a 96-well plate. With very little sample preparation and low sample volume, a  fluorometer using 530 nm excitation and 575 nm emission enables the quick and accurate analysis of oil content in a given algal culture. The ability to sample small amounts of culture volume under a variety of culturing conditions enables high-throughput screening for maximum oil productivity.