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bioRxiv · 10.1101/2024.11.07.622432

Axenisation of oleaginous microalgal cultures via anoxic photosensitisation.

Iyer, A.·Monissen, M.·Ma, Q.·Osborne, M.·Schaedig, E.·Modin, O.·Halim, R.
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Abstract

Growing interest in sustainable biofuel research has necessitated high quality axenic oleaginous microalgal strains. Unfortunately, most strains available in culture banks contain commensal microbes such as bacteria and the default decontamination method involves antibiotic treatment which has begun to exacerbate the emergence of antibiotic resistance. To overcome this problem, anoxic photosensitisation was investigated as an alternate approach. Four oleaginous microalgal species (Tetradesmus obliquus, Desmodesmus armatus, Chlorella vulgaris and Nannochloropsis limnetica) were incubated in varying concentrations of Rose Bengal (0 {micro}M, 1 {micro}M, 3 {micro}M and 9 {micro}M) either in normal (oxic) or anoxic conditions, for 72 h under light (8.85 {+/-} 0.40 W m-2) in a specially designed heterotrophic growth complex (HGC) medium, followed by 72 h in standard Bolds Basal Medium (BBM). Commonly used antibiotics-based protocol was used as the control method. Post treatment, cell numbers and percentage populations were counted with Flow cytometry, and viability was tested using standard plating methods using BBM and LB. Additionally, the contaminating microbes in the cultures were profiled using 16s rRNA sequencing. Anoxic conditions were able to significantly decrease bacterial content, albeit with an equally detrimental effect on the microalgal population. Although the responses differed between the microalgae, anoxic incubation along with Rose Bengal at 3 {micro}M was able to completely decontaminate N. limnetica and C. vulgaris, while D. armatus and T. obliquus could be decontaminated with an additional streak-plating step. None of the cultures could be decontaminated using antibiotics treatment owing to the presence of gram-negative, multi-drug resistant bacteria such as S. maltophilia, M. foliorum and S. chilensis. These results suggest that decontamination of xenic microalgal cultures was largely due to anoxy, that was synergistically enhanced by Rose Bengal at a concentration of [≥]3 {micro}M. HighlightsO_LIRose Bengal at 3 {micro}M in anoxic conditions can help achieve axenic microalgal cultures. C_LIO_LIStandard antibiotics were unable to decontaminate any of the cultures owing to the presence of multi-drug resistant bacteria. C_LIO_LIStandard antibiotics treatment was deleterious to D. armatus cultures. C_LIO_LIStreak plating may be required for D. armatus and T. obliquus after anoxic Rose Bengal treatment to obtain individual colonies for further inoculation. C_LI

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Iyer, A., Monissen, M., Ma, Q., Osborne, M., Schaedig, E., Modin, O., Halim, R.. 2024-11-07. Axenisation of oleaginous microalgal cultures via anoxic photosensitisation.. https://doi.org/10.1101/2024.11.07.622432

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