Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

Before the mid-2000s, the term “environmental DNA” was generally used to refer to DNA obtained from water and soil microbes although some use for the detection of human and animal DNA for purposes of fecal source tracking or non-native species detection had begun^1,2,3. Presently, environmental DNA is generally used to describe DNA from cells sloughed from eukaryotic organisms, and the analysis of this DNA has become a widely used management tool. The detection of cryptic species including those that are invasive or species at risk is the goal, typically accomplished by testing water or sediment for the presence of the characteristic DNA signatures of the target species. Studies may focus on a single species of interest using a specific PCR assay (active sampling), or many species may be detected simultaneously using metabarcoding and massively parallel sequencing.

Many improvements in sample collection, sample processing, and data analysis associated with eDNA research have been made during the last two decades. Early studies employed alcohol precipitation to capture DNA from water samples³. While tried and true, the need to analyze large water volumes to increase assay sensitivity make this method unattractive due to the typical requirement for two volumes of ethanol for each volume of water sample.

Later studies have generally used membrane filtration, rationalizing that eDNA is present in a continuum of states from cellular-bound to freely dissolved, but that dissolved DNA is subject to rapid degradation and therefore that cellular-bound DNA represents the most significant fraction. Filter material and pore size optimization is widely discussed and debated as is the optimal method(s) for extraction of captured eDNA from the filter^4,5,6.

A third approach to eDNA capture is flocculation, a process that has been used for centuries to clarify liquids including, water, beer, and wine. Several studies have used flocculation to capture virus from natural or potable water sources^7,8,9,10,11. These studies have typically used pre-flocculated skim milk or iron chloride as capture agents. Iron chloride methods typically result in downstream analysis molecular problems due to PCR reaction inhibition^12,13. Methods using pre-flocculated skim milk require the advance preparation of the flocculant, pH control, and thus far have only been applied to the capture of virus^7,8,9.

Recently, flocculation capture of bacteria and virus based on the use of lanthanum (III) chloride has been studied^12,13,14,15. The authors demonstrated that viable pathogens could be collected by this method as the binding of lanthanum chloride was effective at low concentration (0.2 mM). Furthermore, lanthanum flocculation is reversible via chelation under mild conditions in contrast to that of iron chloride.

Thus, flocculation using lanthanum chloride to capture biological particulates is attractive given that only a small amount of added concentrated flocculating agent is required, no strict pH control is needed, and sample volumes are scalable from milliliters to gallons. As applied to the capture of eDNA, cellular, sub-cellular (mitochondria and chloroplasts) as well as dissolved DNA are readily recovered. Bacteria and virus are collected simultaneously allowing for pathogen testing and (or) additional ecological studies from a single water sample. A flocculation protocol using lanthanum chloride is presented that enables collection of dissolved and particulate-bound nucleic acids from water samples.