Evaluation of disruption/permeabilization methodologies for Microcystis aeruginosa as alternatives to obtain high yields of microcystin release

Abstract

The extraction of the wide range of useful bioactive compounds produced by cyanobacteria is still a major bottleneck at industrial scale. In addition to the high costs, extraction efficiencies are also commonly low, with low cell disruption efficiencies playing a particularly significant role in intracellular compounds' release. To increase the chances of an extended use of the cyanobacteria toxin microcystin in several biotechnological fields, we aimed to optimize five different disruption techniques: bead milling, microwave, freeze-thaw cycles, highspeed homogenization, and sonication. For each of the methods tested, the conditions that maximized the intracellular organic matter release were: i) 20% of beads and treatment time of 7 min (bead milling); ii) 800 W for 1.5 min (microwave); iii) three 12-h freeze-thaw cycles at −20°C; iv) 15,000 rpm for 7 min (high-speed homogenization); and v) 40 kHz for 10 min (sonication). Sonication and freeze-thaw cycles followed by sonication revealed to be the most effective methodologies to ensure a maximum intracellular organic matter release and, consequently, microcystin availability for being extracted. The decrease of cells' viability was however more evident in freeze-thaw cycles, freeze-thaw cycles followed by sonication, and microwave where only 0.3, 0.05 and 0.9% of the initial cells, respectively, maintained their viability after being treated. On the other hand, sonication and bead milling reduced the viability of the original culture to 5 and 15.5%, respectively, while high-speed homogenization did not show any significant differences compared to control. According to the results obtained in this study, the most suitable methodology to maximize the release of microcystin was therefore the use of sonication (40 kHz) during 10 min.

Screening of several microalgae revealed biopesticide properties of Chlorella sorokiniana against the strawberry pathogen Phytophthora cactorum

“…We can speculate that the bead-milling resulted in less e cient cell disruption in our setup. A study testing various cell disruption techniques on the cyanobacterium Microcystis aeruginosa demonstrated that freeze-thawing is a more e cient cell disruption method than bead-milling (Geada et al 2019). Another reason could be that the bioactive compound(s) are sensitive to shears or oxidation processes occurring during the bead-milling and indeed it was previously suggested that the activity of some bioactive compound(s) is reduced in bead-milled microalgae extracts (Stirk et al 2020).…”

Optimization and validation of a Protein Phosphatase inhibition assay for accessible microcystin detection

“…Some authors indicated that freeze-thawing at −20°C in 2 hours cycles results in inefficient cyanobacterial lysis (Pestana et al, 2014). Hence, freeze-thaw cycle treatment was performed in 3 cycles of overnight freezing, where aliquots were stored at −20°C for more than 12 hours and then thawed, as proceeded by previous authors and standard ELISA protocols (Geada et al, 2019). Sonication was done on an ice bath, for 2 minutes in cycles of 10/30 seconds (ON/OFF) at 30 kHz.…”

Optimization and validation of a protein phosphatase inhibition assay for accessible microcystin detection

“…After each treatment, samples were centrifuged at 13500 g for 10 minutes, and the supernatant was kept. As an index of cellular rupture, we measured the Internal Organic Matter Release (IOMR) (Geada et al, 2019) by UV absorbance at 254 nm in a UV-VIS spectrophotometer (Jasco TM , Japan). Toxin release into the solution was measured by PP1 inhibition assay.…”

“…Some authors indicated that freeze-thawing at -20°C in 2 hours cycles results in inefficient cyanobacterial lysis (Pestana et al, 2014). Hence, freeze-thaw cycle treatment was performed in 3 cycles of overnight freezing, where aliquots were stored at -20ºC for more than 12 hours and then thawed, as proceeded by previous authors and standard ELISA protocols (Geada et al, 2019).…”