Optimisation of phase-dependent microalgal organic matter isolation and extraction methodology

Algal blooms are a global problem that lead to the formation of excessive algal organic matter, resulting in the deterioration of water sources and the disruption of water treatment processes. Methods for the extraction of algal organic matter from algal cells are time-consuming and sometimes ineffective in achieving cell lysis. This study aimed to optimise the extraction and characterisation of extracellular organic matter and intracellular organic matter from microalgae, as well as flow cytometry for assessment of microalgae population numbers and viability. Flow cytometry was applied to quantify a Chlorella vulgaris population and its viability based on chlorophyll integrity by using an image-based cell counter with a fluorescent filter for chlorophyll detection. This technique saved considerable time compared to conventional microscope-based evaluation methods. In the optimised algal organic matter extraction method, the intracellular organic matter fraction was released by subjecting the cells to five freeze (-196 °C)/thaw (70 °C) cycles, which were found to achieve complete lysis of the cells as shown by flow cytometry analysis. This optimized microalgae extraction provides a rapid and efficient method for isolation of intracellular organic matter. The algal organic matter from C. vulgaris was then characterised using multiple techniques and was assessed at three different growth phases: early stationary phase, late stationary phase and declining phase. The dissolved organic carbon concentration of the culture indicated that there was a significant development of soluble, extracellular organic material created by the microalgae which increased as the culture aged. The aromaticity of the organic matter in the extracellular and intracellular fractions was proposed to be low because the specific ultraviolet absorbance SUVA 254 values of the fractions were < 1.6 L mg −1 C m −1 . SUVA 254 values were found to decrease with culture age in the extracellular and intracellular fractions, indicating that the proportion of aromatic organic matter decreased. Fluorescence excitation-emission matrix spectroscopy showed that the extracellular organic matter fractions contained humic acid-like, soluble microbial product (SMP)-like and fulvic-like substances, while the intracellular organic matter fractions contained predominantly protein-like substances, indicating that the extraction method effectively isolated inner cell algal organic matter. This is the first study to clearly demonstrate that the fluorescence intensity of peaks from C. vulgaris extracellular organic matter fractions increased during the growth phases, suggesting a significant dependence of fluorescence intensity on growth phase. The optimized extraction method and efficient cell counting technique presented in this study should contribute to advancements in management of microalgal blooms in the environment, as well as management of microalgal cultures in both laboratory and industrial settings.