Density separation of petrous bone powders for optimized ancient DNA yields.

Density separation is a process routinely used to segregate minerals, organic matter, and even microplastics, from soils and sediments. Here we apply density separation to archaeological bone powders before DNA extraction to increase endogenous DNA recovery relative to a standard control extraction of the same powders. Using nontoxic heavy liquid solutions, we separated powders from the petrous bones of 10 individuals of similar archaeological preservation into eight density intervals (2.15 to 2.45 g/cm3, in 0.05 increments). We found that the 2.30 to 2.35 g/cm3 and 2.35 to 2.40 g/cm3 intervals yielded up to 5.28-fold more endogenous unique DNA than the corresponding standard extraction (and up to 8.53-fold before duplicate read removal), while maintaining signals of ancient DNA authenticity and not reducing library complexity. Although small 0.05 g/cm3 intervals may maximally optimize yields, a single separation to remove materials with a density above 2.40 g/cm3 yielded up to 2.57-fold more endogenous DNA on average, which enables the simultaneous separation of samples that vary in preservation or in the type of material analyzed. While requiring no new ancient DNA laboratory equipment and fewer than 30 min of extra laboratory work, the implementation of density separation before DNA extraction can substantially boost endogenous DNA yields without decreasing library complexity. Although subsequent studies are required, we present theoretical and practical foundations that may prove useful when applied to other ancient DNA substrates such as teeth, other bones, and sediments.

Efficiency of pilot-scale horizontal subsurface flow constructed wetlands and microbial community composition operating under tropical conditions.

This study assesses the microbial diversity of Thalia geniculate (L.) and Cyperus articulates (L.) in the rhizosphere in planted and unplanted systems with respect to removal efficiency in an experimental horizontal sub-surface constructed wetland pilot plant. The pilot-scale units consisted of six (6) cells of concrete of 0.94 × 0.6 × 0.4 m arranged in a parallel configuration. 29 L d-1 were distributed to the cells by gravity. The hydraulic retention time was 3 days and influent and effluent measurements of COD and nutrients were monitored with standard methodology. Bacteria samples were isolated from the roots of plants and gravel in selective media and incubated at 37 °C. Isolates were biochemically characterized and genotyped with group-specific primers. Results showed that systems planted with T. geniculata removed greater proportions of COD (82%), NH4+-N (83%) and PO42-P (83%) than C. articulatus (85, 74 and 72%, respectively) and unplanted wetland systems (80, 72 and 66%, respectively). Bacterial typing revealed several phyla were most abundant, α-Proteobacteria followed by ß-Proteobacteria and there was a significant difference (p < 0.05) in CFU between planted and unplanted treatments. The bacterial community varied with respect to plant species or unplanted and demonstrated significant effects to contaminants removal efficiency.