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

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).

本手稿基于AIMS@JCU博士生Danilo Malara的博士研究项目成果撰写。其博士论文标题为：用于虾苗孵化场致病性弧菌防控的光动力抗菌化学疗法（Photodynamic antimicrobial chemotherapy）。第4章：水产养殖鲜活微藻饵料对光动力抗菌化学疗法的敏感性。JCU指导教师为Kirsten Heimann与Michael Oelgemoeller。

研究概述与研究意义：光动力抗菌化学疗法是一类新兴的灭菌技术，其原理是利用光激活特定化学化合物。本研究针对该技术在水产养殖饵料微藻培养体系中清除污染细菌的适用性进行了验证。研究发现，微藻的细胞壁是决定该技术是否对微藻自身产生毒性的关键因素。本次测试的微藻物种中，仅眼状微拟球藻（Nannochloropsis oculata）适用于该灭菌工艺。反之，该技术具备杀灭水产养殖缸及养殖设施中有害微藻的潜力，但相关应用仍需开展进一步研究。

实验方案说明：本研究于JCU开展实验，以阳离子卟啉（TMPyP）作为光敏剂，测试的微藻培养物包括：伊索克里西斯藻（Tisochrysis lutea）、眼状微拟球藻（Nannochloropsis oculata）、四枝藻（Tetraselmis chui）、皮克绿藻（Picochlorum atomus）以及牟氏角毛藻（Chaetoceros muelleri）。针对每一种微藻物种，我们均测试了梯度浓度的卟啉（0~50微摩尔）与不同处理时长（1~6小时）的组合效应。实验结果显示，仅眼状微拟球藻对卟啉处理具有抗性。我们选取该抗性微藻开展灭菌实验，通过添加三种不同浓度（1×10³ CFU/mL、1×10⁵ CFU/mL、1×10⁷ CFU/mL）的天然发光细菌坎贝尔弧菌（Vibrio campbellii ISO7）来模拟细菌污染场景。每组实验均采用基于前期实验优化得到的灭菌方案：20微摩尔卟啉浓度与6小时处理时长。灭菌实验的操作环节，包括琼脂平板接种与最大可能数（most probable number, MPN）富集培养，均于JCU完成。封装完成的琼脂平板与煮沸后的细胞沉淀分别被送至AIMS，用于在凝胶成像系统下观察发光菌落，以及开展多重聚合酶链式反应（multiplex PCR）检测。

本数据集包含以下内容：
1. 用于测定微藻活性的流式细胞术（Flow cytometry）数据；
2. 用于生物量标准化的微藻培养物干重与细胞计数测定数据；
3. 用于测定纯细菌培养物浓度的酶标仪（Plate reader）发光检测数据；
4. 展示添加的模式发光细菌菌落的琼脂平板照片；
5. 用于定量添加模式细菌的MPN富集培养物多重PCR结果凝胶电泳照片。