A Simple and Non-destructive Method for Chlorophyll Quantification of Chlamydomonas Cultures Using Digital Image Analysis

Nicola J Wood; Alison Baker; Rupert J Quinnell; Miller Alonso Camargo-Valero

doi:10.3389/fbioe.2020.00746

A Simple and Non-destructive Method for Chlorophyll Quantification of Chlamydomonas Cultures Using Digital Image Analysis

Front Bioeng Biotechnol. 2020 Jul 21:8:746. doi: 10.3389/fbioe.2020.00746. eCollection 2020.

Authors

Nicola J Wood^{1

2}, Alison Baker³, Rupert J Quinnell⁴, Miller Alonso Camargo-Valero^{2

5}

Affiliations

¹ Centre for Doctoral Training in Bioenergy, School of Chemical and Process Engineering, University of Leeds, Leeds, United Kingdom.
² BioResource Systems Research Group, School of Civil Engineering, University of Leeds, Leeds, United Kingdom.
³ Centre for Plant Sciences and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
⁴ School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
⁵ Departamento de Ingeniería Química, Universidad Nacional de Colombia, Manizales, Colombia.

Abstract

Growing interest in the use of microalgae as a sustainable feedstock to support a green, circular, bio-economy has led to intensive research and development initiatives aimed at increasing algal biomass production covering a wide range of scales. At the heart of this lies a common need for rapid and accurate methods to measure algal biomass concentrations. Surrogate analytical techniques based on chlorophyll content use solvent extraction methods for chlorophyll quantification, but these methods are destructive, time consuming and require careful disposal of the resultant solvent waste. Alternative non-destructive methods based on chlorophyll fluorescence require expensive equipment and are less suitable for multiple sampling of small cultures which need to be maintained under axenic growth conditions. A simple, inexpensive and non-destructive method to estimate chlorophyll concentration of microalgal cultures in situ from digital photographs using the RGB color model is presented. Green pixel intensity and chlorophyll a, b and total chlorophyll concentration, measured by conventional means, follow a strong linear relationship (R ² = 0.985-0.988). In addition, the resulting standard curve was robust enough to accurately estimate chlorophyll concentration despite changes in sample volume, pH and low concentrations of bacterial contamination. In contrast, use of the same standard curve during nitrogen deprivation (causing the accumulation of neutral lipids) or in the presence of high quantities of bacterial contamination led to significant errors in chlorophyll estimation. The low requirement for equipment (i.e., a simple digital camera, available on smartphones) and widely available standard software for measuring pixel intensity make this method suitable for both laboratory and field-based work, particularly in situations where sample, qualified personnel and/or equipment is limited. By following the methods described here it should be possible to produce a standard curve for chlorophyll analysis in a wide range of testing conditions including different microalga cultures, culture vessel and photographic set up in any particular laboratory.

Keywords: C. reinhardtii; RGB color model; chlorophyll; digital image analysis; microalgae.