Rapid Detection And Customized Controlling Of Microbial Contamination In Microalgal Cultivation

Industrial oil-rich microalgae can transform carbon dioxide and light energy into oil and fat on a large scale through photosynthesis.As a potential large-scale clean energy production and carbon dioxide high value scheme,it has been widely concerned domestic and international.The identification and contamination control are the key bottlenecks that restrict the operation of microalgae laboratory and the process of industrial cultivation.Conventional methods for the detection and identification of contamination pathogens mainly rely on specific microbiological biochemical components and genotype.The inherent disadvantage of these methods is the time-consuming process of culture enrichment,and this delayed diagnosis frequently leads to adverse effects ranging from mild symptoms to catastrophes and is jeopardizing food security.Moreover,certain plant pathogens do not readily grow in a laboratory environment.Conventional methods can do nothing for this situation.Therefore,rapid and sensitive detection techniques for pathogen identification and precise controlling are playing a significant role in microalgae cultivation.This research includes two parts.The first part is the rapid and sensitive identification of microbial contamination.For the species complexity and cognition limitation of contamination in microalgae cultivation,our research chose plant pathogen as a model,whose genotype and pathological phenotype are relatively clear.A rapid and efficient detecting method in-situ was developed by Raman spectroscopy.The application of this approach to plant pathogens with deep phylogenetic diversity,broad host ranges,and an array of pathogenic variants revealed that Raman spectroscopy was able to discriminate phytopathogens at genus,species,subspecies,and even pathovar level.This approach can readily and specifically detect plant pathogens Burkholderia gladioli pv.alliicola and Erwinia chrysanthemi.By detecting Raman spectral,it was able to discriminate them from a reference Raman spectral library including multiple similar disease symptom pathogens with broad host ranges and an array of pathogenic variants.Furthermore,using PCA,Erwinia stewartii and E.chrysanthemi from the same genus were detected;Clavibacter michiganensis subsp.sepedonicus and Clavibacter michiganensis subsp.insidiosus from the same species were detected;and Pseudomonas syringae pv.pisi and Pseudomonas syringae pv.tomato at pathovar level were detected;all the identification ratios are higher than 80%.To show the usefulness of this assay,B.gladioli pv.alliicola and E.chrysanthemi are detected in situ at single-bacterium level in plant tissue lesions without pre-enrichment;the recognition ratios of infected separately and infected simultaneously are 15.6%、51.3%,53.4%;the results are confirmed by molecular approach,which display comparable recognition ratios to the Raman spectroscopy-based bioassay.A micro-Raman spectroscopy-based bioassay for culture-free pathogen at the single cell level within 40 min is presented.This method has obvious advantages in the rapid detection of microorganisms contaminated,without isolating and culturing,real-time,in-situ and efficient at the single cell level.The second part is the development of contamination control in algal culture.In order to achieve the precise control of the microorganism contamination,we deeply seek genetic differences between target algae and contaminants.Our research chose the"drugs"acting on the specific metabolic pathway from known target"drug bank",and developped customized therapies for microbial contamination through precise shotting.Sterol biosynthetic pathway is the targets for commercial fungicides.Comparing mevalonic acid（MVA）pathway and methylerythritol 4-phosphate（MEP）pathway of fungi and microalgae,several chemical inhibitors were targeted.Sterol 14 alpha-demethylase（CYP51）gene sequence comparison of fungi and microalgae inspired us to find insights into the possible distinctions of key enzymes and genetic structural of sterol biosynthetic pathway,several inhibitors were chosen,such as mevinolin（MEV）,clomazone（CLO）,terbinafine（TBF）,amphotericin（APT）,triticonazole（TTA）,and tebuconazole（TBA）.With a concentration of 2.5μg m L^-1,MEV,CLO,TBF,APT and TBA all influenced N.oceanica relative growth rate and maximum quantum yield of photosystem II;sterol profiling changed a lot with these inhibitors at the concentration of IC50;only TTA stimulated the growth of N.oceanica.Treated with different concentration of TTA,the growth of Saccharomyces cerevisiae,Phaeodactylum tricornutum and Thalassiosira sp.are inhibited obviously,and chemical inhibitors targeting sterol biosynthetic enzymes were further characterized.With the concentration as low as 1μg m L^-1,TTA was capable of effectively eliminating contaminated fungi and invasive algal strains,and able to stimulate the growth of desired microalga.The species-specific contamination control method tailored to N.oceanica has been developed.These findings suggest that crossing kingdom genome comparison helps to identify genetic distinctions which can be utilized to select appropriate and customized microbial contamination controlling approach for culturing individual microalgal species with desired production traits.This genome-oriented approach is relatively precise and straight to discover drugs controlling contaminants in microalgal cultivation.It provides unique insight into strategies to advance microalgal technology by the expanding genome information.