Determining the biomass composition of a sponge holobiont for flux analysis.

The first step on the path of flux analysis of a new organism with little available literature is the determination of the biomass composition. Once the content of the macromolecular components (protein, RNA, DNA, carbohydrates, lipids) and their composition is known, this composition can be converted into a biomass equation. The biomass equation is an important part of metabolic flux analysis. This equation provides the information about the precursor and energy needs for growth. In many experiments the determination of the growth rate is the simplest flux to be determined, yet this rate determines the net fluxes of a whole range of anabolic pathways in the system and often is used as the objective function in FBA analysis. The challenge for the scientist is to create a biomass equation that represents the organisms of choice under the conditions studied. This chapter outlines basic protocols that can be applied to the quantification of the macromolecular components, using the marine demosponge Amphimedon queenslandica as a case study. As is true for all other sponges and indeed marine animals, A. queenslandica is a holobiont, comprising an animal host plus symbiotic and other associated microbial cells. We show how this complexity can be overcome by developing a fast, yet robust, method for biomass quantification of sponges using the displacement volume. The analytical protocols we describe herein are widely applicable not only to other organisms sampled from complex environments but also to cell cultures. The second part of the chapter highlights the procedures needed to convert a macromolecular composition into a biomass equation.