Optimisation of nucleic acid extraction methods for a low pH soil, quantification of denitrification gene expression, and the analysis of gas kinetics from agricultural peat soils

Abstract

Denitrification is one of the main processes in the nitrogen cycle, and it is the reduction of nitrate to N2 through a series of intermediates, removing biologically available nitrate from the biosphere. NO and N2O are both gaseous intermediates of denitrification which influence atmospheric reactions due to the formation of reactive nitrate radicals in the atmosphere. In addition, N2O is a potent greenhouse gas that has been on the rise in the last few decades. Thus, it is essential to the agricultural industry to explore the genetic reasons behind high N2O emissions from cultivated soils. Previous studies from our laboratory group utilised quantitative polymerase chain reaction (qPCR) and advanced laboratory-based gas measurements in complement to characterise denitrification gene expression and gas production/utilisation profiles. In those studies, soil pH was discovered to be a very important variable controlling the final reduction step of N2O to N2 in denitrification. However, further molecular work on acidic soil samples was stalled by ineffective nucleic acid extraction and DNA-contaminated RNA samples. Even with current technological advancements, successful extraction of nucleic acids from inhibitor-rich peat soil samples has been recognised as a difficult task. Often, separate extraction reactions or even extraction methods have to be used in order to achieve nucleic acids which are usable for downstream applications. However, this has created a potential source of technical bias, since the DNA and RNA extracted may not be directly comparable due to the heterogeneity of soil environments. Thus, this study first aimed to identify a suitable nucleic acid extraction method for the above mentioned acidic peat soils. Currently available methods were assessed for their ability to co-extract DNA and RNA from acidic peat soils, but were unable to yield mRNA suitable for downstream application. A new method, NM-OSP, was then designed with the information gained from the three failed methods and with maximum flexibility and transparency in mind, unlike many commercially available products. The NM-OSP method was tested on high and low pH peat soils to test its robustness. Although the new method was unable to yield RNA samples that were free of genomic DNA from acidic soils, DNA isolated from both high and low pH soils were of amplifiable quality. Also, high quality mRNA was successfully extracted from high pH soils, reverse-transcribed and quantified using in a qPCR. Denitrification gene expression patterns of the high pH soil matched a previous study using the same soil, confirming that the new extraction method was comparable to more traditional extraction methods and was not likely to create any new method-based bias of the samples. Furthermore, the new method yielded higher DNA and mRNA yields than one of the most commonly used methods in environmental studies. Combining this new extraction method with the aforementioned laboratory-based robotised gas measuring incubation system, the denitrification potential of high and low pH peat soils was analysed. Nucleic acids (DNA and mRNA) were extracted from the soils at multiple time points during incubation. The transcripts of denitrification enzymes were quantified and the expression patterns were correlated with the gas production/utilisation rates. Similar to previous studies, complete denitrification to N2 without external alteration of soil pH was possible but retarded in acidic soils. Comparison of gas profiles from soils with different pH values show a strong pH effect on denitrification and the delayed N2O reduction in low pH soils may be indicative of dissimilar Denitrification Regulatory Phenotypes (DRP) in soils of different pH. In conclusion, although the underlying genetic mechanisms have yet to be revealed, complete denitrification to N2 in acidic soils is possible in closed systems. However, this does not occur in situ because of the delayed activation of the N2O reductase (N2OR). This delayed N2OR activation may be caused by two different DRP in high and low pH soils, hinting at the extent of DRP effects on NOx gas production. The discovery of DRP possibly playing a major role in N2 production has helped to reveal the potential of low pH soils in performing complete denitrification to N2.

The work in this thesis was conducted in the Environmental Microbiology group of the Department of Chemistry, Biotechnology and Food Science (IKBM) of the Norwegian University of Life Sciences (UMB) in Ås, Norway.