Elevated atmospheric CO 2 (eCO 2 ) is anticipated to have marked effects on soil microbial populations. While there is experimental evidence to support this view there are also studies where changes in microbial populations, such as the abundance of bacteria, are suggested but cannot be statistically established. We conducted this study to identify whether the sampling and sample treatment methods used could influence the results obtained using bacterial abundance as the variable of interest. We tested three different sampling methods and two different DNA extraction kits. The first because microbes are distributed heterogeneously in soil so the sampling procedure might be expected to influence the accuracy and precision of the population estimate and the second because the quantity and quality of DNA extracted influences the microbial analyses that can be performed and can introduce bias. Samples were taken from a long-running FACE experiment on grassland from under plants of Agrostis capillaris . We found tha t bacterial abundance was consistently lower under eCO 2 but we were only able to establish a statistical difference where a more intense sampling regime was used and bulking of the soil sample was avoided. A reduction in bacterial abundance is a consistent outcome in eCO 2 field experiments but the only other occasion where this reduction has been found to be significant was also where individual soil cores were analysed rather than the samples being bulked. We conclude that while there is extra work and cost attached to more detailed sampling this approach is highly desirable if we are to make robust conclusions about the impacts of eCO 2 on soil microbes.
There is strong experimental evidence that soil properties are altered by elevated concentrations of atmospheric CO2 (eCO2) [
Measuring microbial populations is difficult. Two factors that are particularly challenging are a) ensuring that the soil sampling methodology provides an accurate estimation of the population given that microbes are frequently distributed heterogeneously in soil [
In this paper we compare methods for quantifying bacterial populations in a pasture exposed to eCO2 in a long-running Free Air Carbon Dioxide Enrichment (FACE) experiment on grassland [
The NZFACE is situated on a mixed plant species sheep-grazed pasture near
Ecosystem and CO2 enrichment system | Sampling protocol | DNA extraction kit | Bacterial abundance | Reference | ||||
---|---|---|---|---|---|---|---|---|
Individual soil cores | Bulked soil | Bulked DNA | MoBio | Fast | Other method | |||
Planted grassland; Open top chambers | √ | √ | Reduced, NS | (Castro et al. 2010) | ||||
Aspen; FACE | √ | Reduced, NS | (Dunbar et al. 2014) | |||||
Heathland: FACE | √ | √ | Reduced, NS | (Haugwitz et al. 2014) | ||||
Native grassland; mini-FACE | √ | √ | Reduced, NS | (Hayden et al. 2012) | ||||
Mixed grassland dominated by Agrostis capillaris near to the CO2 vent; CO2 spring | √ | √ | Reduced, sig. | (Oppermann et al. 2010) | ||||
This paper | √ | √ | Reduced, sig. | |||||
This paper | √ | √ | Reduced, sig. | |||||
This paper | √ | √ | Reduced, NS | |||||
This paper | √ | √ | Increased, NS | |||||
This paper | √ | √ | Reduced, NS | |||||
This paper | √ | | Reduced, NS |
Bulls (40˚14'S, 173˚16'E) in the North Island of New Zealand. The average annual temperature at the site is 12.9˚C and average total annual rainfall 870 mm. The experiment consists of six rings of 12 m in diameter with three of these left at ambient CO2 (aCO2) and three enriched with CO2 during the photoperiod, with enrichment continuous throughout the year. From October 1997 until December 2011 the enrichment was to 475 ppm. The CO2 enrichment was suspended in December 2011 until July 2013 to conduct technical maintenance and then resumed with a target concentration of 500 ppm. From 1997 to 2011 the rings were grazed periodically by adult sheep under a protocol described in Newton et al (2014) [
The pasture is a permanent grassland―uncultivated for at least 50 years― containing a mixture of C3 and C4 grasses, forbs and legumes [
The soil at the site is a black loamy fine-sand (a Mollic Psammaquent). During the course of the experiment fertiliser was added―superphosphate, potassium and magnesium sulphate―to maintain soil concentrations at recommended levels [
In January 2014 soil samples were collected for this experiment. Patches of browntop were identified and soil was sampled from these sites. Soil cores were taken to a depth of 75 mm using a 25 mm diameter steel soil corer. There were five browntop patches in each ring and a core was taken from each patch. Three experimental protocols were then used: first (
from each of the five cores sampled per ring and qPCR completed on all the samples; second (
Two DNA extraction kits were evaluated in this study, the FastDNA Spin kit for soil (MP Biomedicals, Solon, OH, USA) (hereafter described as the Fast kit) and the MoBio PowerSoil DNA isolation kit (MO BIO, Carlsbad, CA, USA) (hereafter described as the MoBio kit). All operations were conducted according to the manufactures instructions. DNA was extracted from 0.5 g fresh soil (approximately 0.3 g dry soil) for the Fast kit and 0.25 g fresh soil (approximately 0.15 g dry soil) for the MoBio kit. The quantity and quality of extracted DNA was assessed on the basis of absorbance at 260 and 280 nm using the NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). An A260/A280 ratio of 1.8 - 2.0 is indicative of high purity [
The abundance of the 16S rRNA gene of bacteria was quantified in triplicate by real-time PCR using a LightCyclerTM 480П (Roche, Vienna, Austria). The real time PCR reaction mixture contained 10 µl 10 × SYBR Green iCycler iQ mixture (Roche Applied Science, Germany), 0.2 µl each of the primers Bacteria 968F; AACGCGAAGAACCTTAC and Bacteria 1378R; CGGTGTGTACAAGGCCCG GAACG [
The effects of atmospheric CO2, sampling protocol and DNA extraction kits were tested by a mixed effects model with heterogeneous variance structure using the packages “nlm” and “predictmeans” in R version 3.1 [
The DNA yield from the MoBio kit was significantly lower than that from the Fast kit (P < 0.001) (
Comparing the values between
pling method was evident when using the Fast kit (P < 0.001) (
The DNA extraction rates convert to about 7 µg DNA g dry soil-1 for the Fast kit and 2 µg DNA g dry soil-1 for the MoBio kit. These values are higher than those reported byJossi et al. (2006) [
We found that bacterial abundance measured as copy number of the 16S rRNA gene and showing whether the result was significant or non-significant was more impacted by the soil sampling approach than the DNA extraction kit (
Using the individual core method meant that in our case 30 samples were required to be analysed compared to 6 for the bulk sampled method. The approximate total cost for single gene abundance (DNA extraction and qPCR) for 30 samples was 5 fold higher than for 6 bulked samples but similar for the two kits. There was also 3 fold increase in technical time required for the individual soil core approach compared to the bulked soil sample approach. Although the cost of analysis of individual soil core approach can be reduced by 1.5 fold by extracting DNA from individual cores and then bulking the DNA for qPCR (method C) in
Three different sampling methods and two different DNA extraction kits tested. The first because microbes are distributed heterogeneously in soil so the sampling procedure might be expected to influence the accuracy and precision of the population estimate and the second because the quantity and quality of DNA extracted influences the microbial analyses that can be performed and can introduce bias. We found that bacterial abundance was consistently lower under eCO2 but we were only able to establish a statistical difference where a more intense sampling regime was used and bulking of the soil sample was avoided. A reduction in bacterial abundance is a consistent outcome in eCO2 field experiments but the only other occasion where this reduction has been found to be significant was also where individual soil cores were analysed rather than the samples being bulked. We conclude that while there is extra work and cost attached to more detailed sampling this approach is highly desirable if we are to make robust conclusions about the impacts of eCO2 on soil microbes.
Our study demonstrated that increasing number of replicates per CO2 treatment improves the precision of microbial population estimates and that the extra effort and cost is likely to be a justifiable investment. This aspect of experimentation has not received attention in CO2 experiments in the past and our results highlights that the choice of soil sampling protocol and DNA extraction method needs to be considered carefully if microbial responses to eCO2 are to be characterised accurately.
Research costs were provided by AgResearch. The China Scholarship Council provided a Research Fellowship to Fengxia Li. The authors are grateful to Phil Theobald and Shona Brock for technical assistance.
Li, F.X., Bowatte, S., Newton, P.C.D. and Luo, D.W. (2017) Impact of Different Methods of Soil Sampling and DNA Extraction on the Identification of Soil Bacterial Abundance under Elevated CO2. Open Access Library Journal, 4: e3527. https://doi.org/10.4236/oalib.1103527