Sensitivity of live microalgal aquaculture feed to singlet oxygen-based photodynamic therapy

Full description

The manuscript builds on work performed as part of the PhD project of AIMS@JCU PhD student Danilo Malara. Thesis title: Photodynamic antimicrobial chemotherapy for pathogenic Vibrio control in prawn hatcheries. Chapter 4: Sensitivity of live microalgal aquaculture feed to photo antimicrobial chemotherapy. JCU supervisors were Kirsten Heimann and Michael Oelgemoeller.


Summary and Importance:


Photodynamic antimicrobial chemotherapy is an emerging sterilization technique based on chemical compounds activated by light.
We tested the suitability of this technique for cleaning up bacteria growing in microalgae cultures used as feed in aquaculture.
We found that the cell wall of the microalgae determines if the technique is toxic to the microalgae itself.
The techniques is suitable for sterilising only one of the tested microalgae-species, Nannochloropsis oculata.
Instead, the technique has potential for killing unwanted microalgae in aquaria and aquaculture facilities, but this would require further studies.


Description of experiments:


An experiment was performed at JCU using a cationic porphyrin (TMPyP) as the photosensitizer and the following microalgae cultures: Tisochrysis lutea, Nannochloropsis oculata, Tetraselmis chui, Picochlorum atomus, Chaetoceros muelleri. A range of porphyrin concentrations (0 - 50 micromolar) and treatment times (1 - 6 hours) were tested for each microalgae species.


Only Nannochloropsis oculata was resistant to the porphyrin treatment. This species was used in a disinfection experiment, where bacterial contamination was simulated by adding a naturally luminescent bacterium, Vibrio campbellii ISO7, at three different concentrations (1E3 CFUs/ml, 1E5 CFUs/ml, 1E7 CFUs/ml). In each case, an optimised disinfection protocol (based on experiments described above) was used with 20 micromolar porphyrin and 6 hour treatment.


The disinfection experiment itself, including the inoculation of agar plates and most probable number (MPN) enrichment cultures, was done at JCU. Sealed agar plates and boiled cell pellets were brought to AIMS for visualisation of luminescent colonies on the gel doc system and for multiplex PCR, respectively.


The data set includes:


Flow cytometry data to determine microalgae viability.


Dry weight and cell count determination of microalgae culture for biomass standardisation.


Plate reader data (luminescence) to determine the concentration of the bacterial pure culture.


Photos of agar plates showing luminescent bacterial colonies (added model bacterium)


Gel photos showing results of multiplex PCRs of MPN-enrichment cultures (to quantify added model bacterium).

Lineage

Maintenance and Update Frequency: notPlanned

Statement: Microalgae cultures were sourced from the North Queensland Algae Identification Facility (JCU), with the exception of Chatoceros muelleri (CS176), which was sourced from the microalgae culture facility at AIMS . The bacterial isolate used as a model bacterium, Vibrio campbellii ISO7, was originally isolated from diseased larvae of the oyster Saccostrea glomerata in the Veterinary Science Laboratory at James Cook University in 2002. Its precise identification and test of virulence against prawns is described in Danilo's PhD thesis and in Malara et al (2017) (Malara D, Hoj L, Heimann K, Citarrella G, Oelgemoeller M 2017 Capacity of cationic and anionic porphyrins to inactivate the potential aquaculture pathogen Vibrio campbellii. Aquaculture 473: 228-236). Glycerol stocks of the strain are stored in Freezer 7 in the South Wing PC2 laboratory at AIMS. Identification and quantification of the added model bacterium in complex samples was achieved using a method previously developed at AIMS and described in detail in Cano-Gomez et al (2015) (Cano-Gomez A, Hoj L, Owns L, Baillie B, Andreakis N 2015 A multiplex PCR-based protocol for identification and quantification of Vibrio harveyi-related species. Aquaculture 437: 195-200). All other materials and the protocols used are described in detail in Danilo's PhD thesis and in the published manuscript.

Notes

Credit
Malara, D AIMS@JCU PhD student, Australia

Credit
Hoj, L. Australian Institute of Marine Science (AIMS), Australia

Credit
Heimann, K James Cook University (JCU), Australia (Principal Investigator)