Investigation into cell disruption for the industrial processing of microalgae

© 2014 Dr. Erin SpidenThe development of economically viable processes for the production of biodiesel is one of the significant drivers for investigations leading to increased understanding of the behaviour of microalgae during industrial bioprocessing. The overall process is apparently simple, with lipids extracted from mass-cultured algae then purified and chemically converted into biodiesel that is compatible with existing transport infrastructure. However, microalgae represent a diverse range of microorganisms that have not traditionally been the subject of biochemical engineering studies. A detailed level of understanding of how microalgae behave is required to allow a high degree of process optimisation which is required for economic viability. Of particular importance is developing an understanding of how to efficiently isolate lipids from inside these unicellular organisms at an unprecedented scale. It is now becoming apparent that physically breaking the cells open prior to extracting lipids is a critical step, with cell rupture shown to greatly improve the efficacy and kinetics of subsequent lipid extraction. However, despite the apparent importance, very little is known about the rupture of microalgae, particularly in a large-scale industrial context. This work investigates techniques to monitor and quantify cell rupture, evaluates the rupture of industrially relevant species of microalgae, and establishes a possible means of improving the cell rupture that can integrate well with downstream processing steps. A series of protocols to monitor and quantify cell rupture by mechanical processing were developed and evaluated. Saccharomyces cerevisiae was chosen for this initial investigation as it is a well understood, commonly modelled and readily available microorganism. The selected method of cell rupture was high pressure homogenisation (HPH), a scalable and efficient mechanical technique. Samples were processed at pressures up to 1500 bar and up to 10 passes through a bench top HPH. The quantification techniques compared include cell counting, measuring particle size distributions, monitoring protein release, and measuring the physical properties of turbidity, viscosity and residual dry weights. The quantification techniques were evaluated in terms of reproducibility, accuracy and technical simplicity. It was found that all quantification techniques gave somewhat similar measures of rupture but direct measures (cell counting, turbidity) generally gave more conservative estimates compared to indirect measures (UV absorbance, protein release) of disruption. Measuring the turbidity of a sample and the UV absorbance of the corresponding supernatant in combination gave a simple, descriptive indication of cell rupture whilst requiring minimal processing time. The susceptibility to rupture of the industrially interesting microalgae Nannochloropsis sp., Chlorella sp., Navicula sp. and Tetraselmis suecica by HPH were determined and evaluated. Different methods were used to quantify cell rupture, and their utility assessed in this context, including cell counting, turbidity, metabolite release and particle sizing. Cell counting was the only reliable method for quantitative comparisons of all microalgae, with turbidity measurements complicated by agglomeration of cell debris for T. suecica, and measurement of metabolite release affected by debris micronization and degradation of cell components occurring for all microalgae at high levels of rupture. The rupture of all microalgae followed exponential decay as a function of HPH passes. The pressure required to achieve rupture of 50% of the cells per pass was determined to be 170, 390, 840, 1380, and ca 2000 bar for Tetraselmis sp, Navicula sp., Chlorella sp., S. cerevisiae, and Nannochloropsis sp. respectively. These results help extend the criteria for the selection of microalgae for industrial applications beyond consideration of growth and compositional attributes. Combinations of chemical and physical treatments, which have previously been shown to improve cell rupture and metabolite extraction from yeasts and bacteria, were applied to Chlorella sp. and Navicula sp. in advance of mechanical rupture. The chemical and physical pre-treatments chosen to weaken the cell walls included the use of acid (HCl, to pH 1.5), detergent (Triton X-100, up to 2%) and exposure to heat (up to 123°C for 10 minutes) and rupture was evaluated quantitatively using cell counting and turbidity readings and qualitatively using SEM imaging. As observed via cell counting and normalised turbidity readings the pre-treatments generally appeared to hinder rupture with subsequent HPH; whereas SEM images showed that thermal and extreme pH treatments damaged the cell walls and solidified the cellular protoplast. The solidified protoplast remained relatively stable with subsequent HPH even as the cell wall fragmented whereas the non-pre-treated protoplasts lost structural integrity with the destruction of the outer cell wall. The solidified protoplasts had a similar appearance to whole cells, which resulted in the apparent reduction in rupture with HPH and extreme pre-treatments as measured by cell counting and turbidity. The thermal pretreatment of Chlorella sp. was further investigated; lipid extraction by monophasic chloroform/methanol/water extraction was marginally improved and the larger post-rupture cell fragments offered potential benefits for downstream processing. This body of work is the first to comprehensively investigate the rupture of microalgae with HPH and focus on the reliability of the indicators of cell rupture. It is also the first to compare the rupturability of a selection of industrially relevant microalgae and to investigate the potential use of combined disruption techniques to enhance the rupture process for microalgae. This research adds to our existing understanding of the industrial processing of microalgae, specifically in the previously largely overlooked area of cell rupture