Cyanotoxin Detection in Dairy Waste Water Treatment

We have developed molecular techniques to identify classes of cyanobacteria for early toxin threat detection and control before the growth becomes toxic. This screening is part of a larger sustainability project focused on converting dairy cow manure effluent into a fast-growing, algae-based, safe animal feed crop. The algae are produced in paddle-wheeled model ponds on the dairy farm which could become contaminated by toxic cyanobacteria. To ensure the integrity of our analysis, multiple species of pure cyanobacteria cultures were grown and their DNA purified. The 16S rRNA and rpoC1 gene sequences were targeted for their universal detection of cyanobacteria using PCR. To distinguish cyanobacteria species that have the potential to produce toxins, four gene clusters in the toxin synthesis pathways were targeted: mcy (microcystin), nda (nodularin), cyr (cylindrospermopsin), and ana (anatoxin-a). PCR-amplified fragments were separated using agarose gel electrophoresis and sequenced. 16S rRNA/rpoC1 PCR assays allowed for positive identification of quality control strains (QC) and exhibited the sensitivity and specificity to discriminate against multiple genera of non-cyanobacteria strains. Within our QC strains we have identified strains that indicate potential to produce microcystins, and anatoxin-a. Toxin production was quantitated using Abraxis/Beacon ELISA kits. Our future goal is quantitation of the toxin genes with qPCR. This protocol will routinely detect and identify the presence of cyanobacteria species that could produce toxins in algae production ponds and allow for their control in algae-based livestock feed operations.