Nycodenz_study_2010
Bias in Bacterial Diversity as a Result of Nycodenz Extraction from Bulk Soil
The aim was to use 16S rRNA amplicon pyrosequencing to examined what, if any, bias there is in extraction of bacterial cells from soil by Nycodenz density gradient centrifugation. The soil was collected from three replica test plots in the Danish CRUCIAL field trial [1] that had not been treated with any kind of fertilizer for the last decade. The plots were designated U-B5, U-C3 and U-D7 but are here referred to as 1, 2 and 3 respectively. Soil was collected by taking 24 soil cores to a depth of 20 cm per plot and mixing them together. The soil samples were sieved through a 5-mm sieve to remove stones and roots and provide homogenous samples, and stored field moist at 4°C until use.    Cells were extracted in two replicates from 1 g of soil from each field plot by density gradient centrifugation with Nycodenz. The Nycodenz protocol used is by Burmølle et al. [2].    DNA was extracted in two replicates from the soil from each field plot (samples D). DNA was also extracted from the cells extracted by Nycodenz (samples N), and from the soil pellet left after the Nycodenz extraction (samples P). Technical replicates are designated with an A or B. All DNA extractions were done using the FastDNA SPIN for Soil kit (MP Biomedicals, Solon, Ohio, USA) as described by the manufacturer.    1. Magid, J., et al., Establishment of a long-term field trial with urban fertilizers - is recycling of nutrients from urban areas to peri-urban organic farms feasible?, in Long-term field experiments in organic farming, J. Raupp, et al., Editors. 2006, International Society of Organic Agriculture Research: Berlin, Germany. p. 59-78.  2. Burmølle, M., et al., Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry. Microbial Ecology, 2003. 45(3): p. 226-236.
Nycodenz density centrifugation allows extraction of both culturable and unculturable bacterial cells from soil, which can be used for further analysis; however there is a lack of information concerning the efficiency of this method. The aim of this study was to investigate the efficiency of Nycodenz extraction from soil.  Bacterial cells were extracted from three soil plots from the Danish CRUCIAL field trial using a Nycodenz protocol. DNA was extracted from soil, from Nycodenz extracted cells, and from the soil pellet left after Nycodenz extraction. The 16S rRNA gene was amplified from the extracted DNA. The PCR amplicons were tagged sequenced using a GS FLX pyrosequencing system. Sequences were processed and analyzed by using the Ribosomal Database Project’s (RDP) Pyrosequencing Pipline tools.  In this study, we show that extraction of bacteria from soil using Nycodenz density centrifugation is biased and results in over- or underrepresentation of common bacterial phyla. Rarefaction curves, analysis of similarity, multidimensional scaling plots and analysis of variance showed that the diversity in the Nycodenz extracted sample was lower than in the original soil or soil pellet. In addition, RDP classifier results showed a clear over- or underrepresentation of certain phyla after Nycodenz extraction. A mathematical model was constructed to estimate how many sequences would be necessary to find 95% of all operational taxonomic units (OTUs) in the soil. The model shows that the soil contains approximately 29 400 OTUs and that just 351 500 sequences are needed to cover 95% of the bacterial biodiversity. The equivalent of one full standard GS FLX run.