A modified method for colorimetric quantification of lipids from cyanobacteria

Highlights

• Development of a method for high throughput quantification of lipids in microplates

• Rapid and cheap quantification of lipids in small sample volumes

• Investigation of the influence of solvents on the reaction vessel

• Colorimetric quantification of extracellular lipids of terrestrial cyanobacteria

Abstract

A colorimetric sulpho-phospho-vanillin method was developed for the quantification of total lipids in small sample volumes. The developed method is easy to perform and can be used for high throughput analysis of lipid contents in 96-well microplates. In contrast to previous studies, solvents had a great influence on the surface of microplates. Different solvents were tested, but methanol was the only one, which did not show any impact on surface material. Therefore, it was used to dissolve the extracted lipids quantitatively, which were then transferred to the microplates for further processing. In this study, the method was used to quantify the total lipid amounts in extracts of extracellular polymeric substances (EPS) from terrestrial cyanobacteria. These extracellular lipids are of great interest, especially with regard to the production of antimicrobial substances. The two strains Trichocoleus sociatus and Nostoc muscorum were cultivated under heterotrophic conditions with glucose as additional carbon source. The lipid content of the two strains differed during the cultivation period. On average, the amount of lipids reached the highest value in the beginning of heterotrophic cultivation. Although the experiments were conducted under submerged conditions, cyanobacterial cells showed a surface-associated growth. The fluctuations in lipid content might express the different stages of biofilm formation.

Introduction

Cyanobacteria are an emerging source for valuable biotechnological products. These include for example polysaccharides, vitamins [1], lipids [2], amino acids, enzymes, fine chemicals and pharmaceuticals [3] which can be applied in food, cosmetics and drug industry or as renewable energy sources. Cyanobacteria are divided into aquatic and terrestrial species depending on their original habitat [4]. Terrestrial cyanobacteria show a surface-associated growth and are embedded in a matrix of extracellular polymeric substances. In general, EPS consist of numerous compounds, such as water, lipids, polysaccharides, nucleic acids, alginate, dextran and many others [5,6]. It enables the cells to retain moisture for growth [7] and protects the organisms against various environmental stresses such as dehydration [8]. There are different types of EPS: the CPS (capsular polysaccharides) are directly associated to the cells, whereas the RPS (released polysaccharides) are released into the surrounding area. For industrial use of EPS, it is important to get quantitative and qualitative information about its composition. The biggest part of EPS is represented by carbohydrates, but despite of these, the minor components like lipids, as well as glycolipids and lipopolysaccharides, which are also interesting for biotechnological products (e.g. antimicrobial substances, food supplements), should be analysed as well [9,10]. The most commonly used standard techniques for the screening and quantification of lipids, are for example gravimetric and fluorescence dye methods [11,12]. Due to the fact that the lipid content of EPS of the applied strains is very low, gravimetrical determination is not an option in the lab-scale. The staining of lipids with fluorescent dyes like Nile Red is only possible when the lipid content of biomass should be quantified. A rapid, simple and colorimetric quantification of lipids from algal cultures was described by Cheng & Zheng [13]. It is based on the sulpho-phospho-vanillin (SPV) method for quantification of the total lipid content by Chabrol et al. [14]. The SPV method was modified for diverse applications, even for a high throughput quantification in 96-well microplates [13]. This method by Cheng & Zheng et al. [13] could not be transferred properly to our experimental setup. In our study, we modified the method and applied this technique to quantify lipids in small sample volumes of EPS-extracts of terrestrial cyanobacteria. This modified colorimetric quantification has several advantages. It is fast, inexpensive, requires only small amounts of solvents and can be used in connection with a standard lipid extraction, for example according to Bligh and Dyer [11]. It is well known that some solvents, for example chloroform, which are used in standard lipid extraction protocols may have an impact on surface material made of plastics and thus influence the experimental results. To ensure the applicability of the colorimetric quantification of lipids in combination with standard lipid extraction techniques, different solvents were tested with regard to their impact on the surface of 96-well microplates.