Validation of the detection of Pseudo-nitzschia spp. using specific RNA probes tested in a microarray format: Calibration of signal based on variability of RNA content with environmental conditions.

Harmful algal blooms (HAB) occur worldwide and cause health problems and economic damage to fisheries and tourism. Monitoring for toxic algae is therefore essential but is based primarily on light microscopy, which is time consuming and can be limited by insufficient morphological characters such that more time is needed to examine critical features with electron microscopy. Monitoring with molecular tools is done in only a few places world-wide. EU FP7 MIDTAL (Microarray Detection of Toxic Algae) used SSU and LSU rRNA genes as targets on microarrays to identify toxic species. In order to comply with current monitoring requirements to report cell numbers as the relevant threshold measurement to trigger closure of fisheries, it was necessary to calibrate our microarray to convert the hybridisation signal obtained to cell numbers. Calibration curves for two species of Pseudo-nitzschia for use with the MIDTAL microarray are presented to obtain cell numbers following hybridisation. It complements work presented by Barra et al. (2012b. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-012-1330-1v) for two other Pseudo-nitzschia spp., Dittami and Edvardsen (2012a. J. Phycol. 48, 1050) for Pseudochatonella, Blanco et al. (2013. Harmful Algae 24, 80) for Heterosigma, McCoy et al. (2013. FEMS. doi: 10.1111/1574-6941.12277) for Prymnesium spp., Karlodinium veneficum, and cf. Chatonella spp. and Taylor et al. (2014. Harmful Algae, in press) for Alexandrium.

Optimization of a total acid hydrolysis based protocol for the quantification of carbohydrate in macroalgae

Accurate quantification of carbohydrate content of biomass is crucial for many bio-refining applications. The standardised NREL two stage complete acid hydrolysis protocol was evaluated for its suitability towards seaweeds, as the protocol was originally developed for lignocellulosic feedstocks. The compositional differences between the major polysaccharides in seaweeds and terrestrial plants, and seaweed’s less recalcitrant nature, could suggest the NREL based protocol may be too extreme. Underestimations of carbohydrate content through the degradation of liberated sugars into furan compounds may yield erroneous data. An optimised analysis method for carbohydrate quantification in the brown seaweed L. digitata was thus developed and evaluated. Results from this study revealed stage 1 of the assay was crucial for optimisation however stage 2 proved to be less crucial. The newly optimised protocol for L. digitata yielded 210 mg of carbohydrate per g of biomass compared to a yield of only 166 mg/g from the original NREL protocol. Use of the new protocol on two other species of seaweed also gave consistent results; higher carbohydrate and significantly lower sugar degradation products generation than the original protocol. This study demonstrated the importance of specific individual optimisations of the protocol for accurate sugar quantification, particularly for different species of seaweed