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Received Date : 12-Feb-2014 Revised Date : 22-Apr-2014 Accepted Date : 14-May-2014 Article type
: Research Letter
EFFECT OF PRESERVATION METHOD ON THE ASSESSMENT OF BACTERIAL COMMUNITY STRUCTURE IN SOIL AND WATER SAMPLES
Authors: Valeria Tatangelo1, Andrea Franzetti1, Isabella Gandolfi1, Giuseppina Bestetti1, Roberto Ambrosini2
Affiliations: 1
Dept. of Earth and Environmental Sciences – University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milan, Italy 2
Dept. of Biotechnology and Biosciences – University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy
Corresponding author: Andrea Franzetti, Dept. of Earth and Environmental Sciences – University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milan, Italy – email: [email protected], phone: +39 02 64482927
Abstract The methods used in sample preservation may affect the description of the microbial community structure by DNA-based techniques. This study aims at evaluating the effect of different storage conditions, including freezing, adding two liquid-based preservatives or simply storing samples with no preservative, on the structure of the microbial communities in aliquots of organic-rich soil and water samples as revealed by a T-FRLP. The results showed that the number of Terminal Restriction Fragments (TRF) detected in soil aliquots stored with LifeGuardTM solution was significantly lower
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12475 This article is protected by copyright. All rights reserved.
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than that of samples analysed immediately after sampling. Moreover, cluster and PCA analyses showed that soil aliquots stored using LifeGuardTM clustered separately from those stored with the other methods. Conversely, soil and water aliquots stored with DMSO-EDTA-salt solution (DESS) did not show either significant reduction in the number of TRFs or any change in the structure of the microbial community. Finally, the number of TRFs and the structure of microbial communities from soil aliquots stored with no preservative did not differ from those of aliquots analysed immediately after sampling. Preservation methods should therefore be accurately evaluated before collecting samples that have to be stored for long time before DNA extraction.
Keywords: microbial community storage conditions, T-RFLP, DNA extraction
1.
INTRODUCTION
An accurate description of the microbial community structure by DNA-based technologies relies on the amount and quality of nucleic acids recovered from environmental samples, which, in turn, are mostly affected by the methods used in sample collection and preservation, and in the extraction of the nucleic acids (Osborn & Smith 2005). Different protocols for sample collection and DNA extraction have been developed and proposed in the last 20 years, enabling microbiologists to achieve high yields and highly pure DNA/RNA extracts even from challenging materials (e.g. humic acid-rich soils) (Elsas et al. 2000; Carrigg et al. 2007; Leff et al. 1995; Sessitsch et al. 2002). Conversely, few investigations have addressed so far the effect of different sample preservation methods on the accuracy of the description of bacterial community structure that can be obtained by following DNAbased analyses (Lauber et al. 2010; Rissanen et al. 2010; Gray et al. 2013; Sessitsch et al. 2002; Elsas et al. 2000; Clark & Hirsch 2008). Indeed, improper sample preservation may determine DNA degradation and therefore bias the structure of the bacterial community that can be observed by any following DNA-based analysis. For soil samples in particular, contrasting evidences exist in the literature. Dolfing and colleagues (2004) demonstrated that it is possible to assess soil microbial community composition in samples that have been dried and stored for as long as 50 years, and other studies have showed that air-drying and sample storage at room temperature did not bias the assessment of the whole community composition (Klammer et al. 2005; Lauber et al. 2010; Rubin et al. 2013). Conversely, Tzeneva et al. (2009) found that sample drying and storage at room temperature significantly altered the composition of the observed microbial community. Moreover, most of the studies addressing the effect of storage conditions on the outcome of the analyses of soil microbial community have investigated only the effects of preservation time and temperature and physical treatments such as lyophilisation (Sessitsch et al. 2002; Lauber et al. 2010). However, in many cases, such as during sampling campaigns in remote areas of the world, low-temperature storage and lyophilisation are not feasible. In those cases the use of non-hazardous storage solutions may be a valuable alternative, provided that bacterial community structure can be reliably assessed when back in the lab. To the best of our knowledge, only two previous studies have tested the effectiveness of liquid-based preservatives for preserving bacterial communities in samples intended to be investigated by DNA-based analyses. (Gray et al. 2013; Rissanen et al. 2010). Indeed, DMSOEDTA-salt solution (DESS), which represents a cheap alternative to expensive proprietary products, has been tested so far on liquid cultures only (Gray et al. 2013). Therefore, an assessment of the
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performance of different preservation methods on samples intended to DNA-based analyses is necessary, especially before any sampling in remote areas. In this study we evaluated the effect of two liquid-based preservatives (the proprietary LifeGuardTM (MO BIO, Carlsbad, CA, USA) and the non-proprietary DESS) on the structure of the microbial communities in organic-rich soil and water samples as revealed by DNA-based Terminal Restriction Fragment Length Polymorphisms (T-RFLP). The results have been compared with those obtained from samples analysed immediately after sample collection and used as a reference of ‘true’ bacterial communities as well as with those obtained from samples stored at different temperatures and with frozen samples, which should represent samples stored with an ‘optimal’ preservation method.
2. 2.1
MATERIALS AND METHODS Sampling methods and handling
Surface soil samples were collected in the garden of the University of Milano-Bicocca (Milano, Italy, approximate coordinates of sampling site: 45°30’49’’ N, 9°12’39’’ E) while water samples were collected from a stream in an urban park near the University campus (Parco Nord, Milano-Italy, 45° 31' 54’’ N, 9° 12' 27’’E). The organic matter of the soil sample was 56 mg g-1, as determined on dry soil sample (62 °C, 24 h) after overnight combustion (Howard 1965). Soil and water samples were brought to the lab within 20 minutes without adding any preservative. In the lab the soil was homogenized by hand-mixing and divided into 2 g aliquots while water samples were homogenized, divided into 100 mL aliquots and filtered on nitrocellulose filters (Ø = 0.45 µm). Hence, in this study each aliquot consists of 2 g of soil or one nitrocellulose filter. Two aliquots of soil and two of water were immediately analysed (aliquots “analyzed immediately after collection”, AIAC hereafter), while the other ones were stored under different conditions as detailed below. The proprietary preservative solution LifeGuardTM was used as purchased according to manufacturer’s instructions (i.e. 6 mL of solution for 2 g of soil) while the non-proprietary DESS was prepared as described in Yoder et al. (2006) and used in the same proportion as the LifeGuardTM. Aliquots of samples without any preservatives were stored at –20 °C, +4 °C and +30 °C while LifeGuardTM- and DESS-preserved samples were stored at +4 °C and +30 °C. Storage times for each combination of preservative and temperature were 15 or 30 days for soils and 15 and 46 days for water samples. For brevity, hereafter we will refer to ‘preservation method’ as to the combination of type of preservative (none, LifeGuardTM, DESS), storage time (15 or 30 days for soil, 15 or 46 days for water) and temperature (-20 °C , +4 °C or +30 °C ). Figure 1 summarises the different preservation methods.
2.2
DNA extraction and amplification
The total genomic DNA was extracted from 0.25 g of soil or half filter from each of the two AIAC aliquots or from each aliquot stored with a different preservation method using ZR Soil Microbe DNA
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Miniprep (Zymo Research Corporation, Irvine, CA, USA) according to manufacturer’s instructions. Before the extraction, the samples preserved with LifeGuardTM and DESS were centrifuged at 2500 x g for 5 min to remove the preservative solution, according to MO BIO protocol. 16S rRNA gene fragments were PCR-amplified and purified as previously described (Rivelli et al. 2013). Endonucleolytic digestions were performed in a 20 µL reaction, containing 10 U of MspI (Promega Corporation, Madison, WI, USA) and 600 ng of amplified DNA for 1.5 h at 37 °C. T-RFLP profiles were obtained and analyzed as reported in Rivelli et al. (2013). T-RFLP profiles provide the relative abundance of different Terminal Restriction Fragments (TRFs hereafter) which, in turn, represent different microbial strains roughly corresponding to genus level.
2.3
Statistical methods
Singletons (i.e. TRFs found in one sample only) were removed from the dataset before all the analyses because they may inflate variance explained by models (Legendre and Legendre 1998). Multivariate analyses of the structure of bacterial communities were based on several techniques (Borchard et al., 2011), namely hierarchical cluster analysis with the complete linkage method, principal component analysis (PCA), multivariate regression trees (MRT), and redundancy analysis (RDA). All analyses were based on Hellinger distances between bacterial communities as assessed by the relative abundance of TRFs. This distance is a measure of the difference in the structure of bacterial communities between aliquots (Legendre and Legendre 1998, De Cáceres et al. 2010). In MRT analyses, the best tree was chosen as the smallest one whose cross-validated relative error (CVRE) was within 1 standard error of the CVRE of the best tree (Borchard et al. 2011). CVRE ranges between zero (perfect predictors) and close to one (poor set of predictors, Borchard et al., 2011). In RDA, preservation method and storage temperature and time were entered as predictors. Post-hoc tests in RDA were performed by comparing each level of the factor of interest with a reference level, which consisted, alternatively, in the structure of bacterial communities obtained from AIAC samples or from frozen samples. These post-hoc comparisons therefore follow the same logic as the Dunnet method of performing post-hoc tests in ANOVA models, i.e. they assessed significance of selected comparisons only. As a consequence, a significant main effect may not necessarily imply significant Dunnet post-hoc comparisons, because significant differences may occur in comparisons that are not of interest. Significance values of post-hoc tests were corrected for multiple statistical tests with the False Discovery Rate (FDR) method (Banjamini and Yekutieli 2001). In a following step of RDAs, we excluded AIAC aliquots and analyzed separately those stored for different times. The logic behind these analyses is that sometimes samples cannot be analyzed immediately after collection (e.g. when samples are collected in remote areas) and therefore must be stored in some way. The reference level for post-hoc tests in these analyses is therefore the bacterial community of frozen aliquots, which represent the ‘optimal’ (but often impractical) preservation method.
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If a given preservation method is not effective in avoiding degradation of bacterial DNA, we expect that the number of OTUs that could be retrieved from samples stored with that method are lower than those observed in AIAC samples. To test for this hypothesis, we compared the number of OTUs of samples stored with different preservatives and AIAC samples, or frozen samples stored for the same amount of time, by Poisson generalized linear models (GLMs) followed by Dunnet post-hoc tests. All the analyses were performed in R 2.15.2 (R Core Team 2012).
3. 3.1
RESULTS Multivariate analyses
We were unable to amplify two soil samples stored for 30 d at +30 °C, one with no preservative and one with LifeGuardTM, the two water samples stored for 15 d with no preservative at +30 °C , and the two water samples stored for 46 d with no preservative at +30 °C. These samples were excluded from the subsequent analyses. Overall, 45 TRFs were identified in soil samples and 22 in water samples. After singletons removal, 31 TRFs were retained in the analyses of soil samples, and 16 in those of water samples. Cluster analyses and PCA plots indicated that soil aliquots stored with LifeGuardTM were separated from those stored with other preservatives (Figure 2a and 2b), while separation of water aliquots stored with LifeGuardTM or other preservation methods was less clear (Figure 3a and 3b). Importantly, soil aliquots stored with LifeGuardTM formed a different cluster than all the other ones (Figure 2a) and in PCA plot they were separated from AIAC aliquots (Figure 2b), while those stored with other preservatives were not. Water aliquots stored with LifeGuardTM were also separated from the others, albeit not so clearly as for soil samples (Figure 3b). MRT analyses on both soil and water aliquots indicated a tree with two leaves as the best one, although performance of the models was rather low (CVRE ≥ 0.61). In both cases aliquots stored with LifeGuardTM were separated from both those stored with other methods (including freezing) and AIAC aliquots. RDA on soil and water aliquots indicated that the structure of bacterial communities significantly differed among aliquots stored with different preservatives but not for different times (Table 1). In addition, soil bacterial community did not differ between aliquots stored at different temperatures, while those of water samples did (Table 1). However, post-hoc tests disclosed no significant difference between soil or water aliquots stored with different preservatives and AIAC ones neither between water aliquots stored at different temperatures and AIAC ones (F1,8 ≤ 6.89, PFDR ≥ 0.08 in all cases). Analyses run separately on aliquots stored for different times and excluding AIAC samples showed significant differences among aliquots stored with different preservatives (Table 2). However, Dunnet-type post-hoc tests indicated that bacterial communities of frozen aliquots did not differ from those of aliquots stored with any preservative for the same amount of time (F1,4 ≤ 7.22; PFDR ≥
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0.12 in all cases). A significant effect of temperature only appeared in water samples stored for 15 days (Table 2), but also in this case post-hoc tests did not disclose any significant difference between samples stored at +4 or +30 °C and samples stored at -20 °C (F1,6 ≤ 1.82; PFDR ≥ 0.39 in all cases).
3.2
Number of TRFs
Poisson GLMs indicated that the number of TRFs differed between samples stored with different preservatives and this occurred for both soil and water samples (LRT: χ24 ≥ 9.80, P ≤ 0.04). Post-hoc tests on soil samples indicated that samples stored with LifeGuardTM included a significantly lower number of TRFs than AIAC samples (-9.00 ± 1.22 SE OTUs, z = -3.18, P < 0.01; z| ≤ 1.15, P ≥ 0.52 in all the other cases). Conversely, post-hoc tests on water samples did not reveal any significant difference (|z| ≤ 2.36, P ≥ 0.06 in all cases). Analyses restricted to soil samples stored for 15 or 30 days (thus excluding AIAC samples) confirmed significant differences in the number of TRFs that could be observed in samples stored with different preservatives (15 days: χ23 = 8.23, P = 0.04, 30 days: χ23 = 8.46, P = 0.04). Post-hoc tests showed that samples stored for 15 days with LifeGuardTM contained a significantly smaller number of TRFs than samples frozen for the same amount of time (-8.00 ± 1.24 SE TRFs, z = -2.43, P = 0.04; |z| ≤ 1.05, P ≥ 0.56 in all the other cases), while post-hoc tests on soil samples stored for 30 days did not disclose any significant difference (|z| ≤ 1.56, P ≥ 0.26 in all cases). The same analyses conducted on water samples did not find any difference in the number of TRFs among samples stored with different preservatives (15 days, LRT: χ23 = 6.77, P = 0.08; 46 days : χ23 = 5.57, P = 0.13).
4.
DISCUSSION
In this study we investigated how storage condition, including the preservative added to samples, can alter the structure of bacterial communities compared to the one that could be retrieved if samples were analysed immediately after they were collected. A limit of this study is that we investigated only one soil and water sample, so that our results can be representative only of the performance of preservation methods on samples with similar characteristics. However, we stress that we chose a soil with high organic carbon content whose analysis is particularly challenging due the high activity of nucleases and the possible co-extraction of inhibitors affecting downstream analyses. During all the phases of the process of DNA-based characterization of the microbial community of soil and water samples collected in the field, the quality and the quantity of DNA should be preserved, from the time of collection to that of DNA extraction. Very often, storage of samples is unavoidable due to the lack of facilities in the field. In this case, storage at -20 °C (freezing) is considered the best preservation method for samples that should be used for analyses of bacterial community structure. However, even freezing samples may not be an option, particularly when samples are collected in remote areas of the world, and therefore the ability of alternative preservation methods of preserving the original microbial community structure should be explored.
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Our results did not disclose any difference in the structure of bacterial communities retrieved from samples stored with no preservative at different temperatures, including frozen samples, and that from samples analysed immediately after collection, in agreement with previous reports (Rissanen et al. 2010; Lauber et al. 2010; Rubin et al. 2013). In particular, Lauber and colleagues have even suggested that the samples they collected could be stored and shipped at room temperature without having a significant impact on the assessment of the overall community composition or on the relative abundances of most major bacterial taxa (Lauber et al. 2010). Our results confirm their findings, as samples stored with no preservative did not differ in the number of TRFs or in the structure of bacterial communities from AIAC and frozen samples. The lack of influence of storage temperature on bacterial community composition that they observed may be due to the fact that storage and shipping temperature did not differ significantly from ambient temperature at the place where sample where collected. Conversely, the relative abundance of the microbial populations may largely change if samples are stored and transported at temperatures that are markedly different from that at the collection site, due e.g. to differential growth of psycrophilic or thermophilic populations. Our results seem to contradict this hypothesis, because community structure observed in aliquots stored with no preservative did not differ from those of AIAC or frozen aliquots, independently of temperature and time of preservation. However, DNA seem to degrade in a few days in water aliquots stored at high temperature, as indicated by the fact that we failed in amplifying water aliquots stored at +30 °C . We expected that the use of bacteriostatic or bactericide preservative agents might enhance bacterial preservation. Unexpectedly, our results showed that LifeGuardTM solution led to a significant reduction in the number of TRFs that could be detected in soil aliquots compared to AIAC ones. Moreover, cluster and PCA analyses showed that soil aliquots stored using LifeGuardTM clustered separately from the samples stored with other methods. Although RDA did not find a significant difference between aliquots stored with LifeGuard™ and AIAC ones, the overall results might indicate that the addition of LifeGuard™ was not a suitable preservation strategy for our soil samples. Results from multivariate analyses on water samples were similar to those of soil samples, with only three aliquots stored with LifeGuardTM that clustered with the other aliquots, while all the other ones stored with this preservative grouped in a separate cluster. Preservative solutions can have different performance for water and soil samples. For example, DNAgardTM and RNAlater® have good preservation performances on liquid cultures (Gray et al. 2013) while RNAlater® lead to a significant reduction in extraction yield of DNA and RNA and to changes in the relative representation of some major taxa in sediment samples (Rissanen et al. 2010). This behavior may be due to the precipitation and fixation of protein and organic matter onto nucleic acids as a consequence of the presence of ammonium sulphate, which is used for protein precipitation, in the RNAlater® formulation (Rissanen et al. 2010). This may not apply to LifeGuardTM because, according to the patent (US patent 6,458,546), it contains no compound used for protein precipitation. The reason of this difference in the performance of LifeGuardTM in preserving soil and water samples needs therefore to be further investigated. The performance of the non-proprietary liquid solution DESS and the phenol–chloroform–isoamyl alcohol in storing bacterial communities without altering their structure have been already tested. In
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particular, phenol–chloroform–isoamyl alcohol was used on sediment samples and was able to preserve both the DNA and RNA yields and the bacterial community structure of sediments owing to the inactivation of nucleases (Rissanen et al. 2010), and DESS was able to properly preserve liquid cultures of culturable bacteria (Gray et al. 2013). In our study DESS was successfully applied also to soil and water samples as it did not show any significant reduction in TRF abundance and did not modify the structure of the community as revealed by DNA-based T-RFLP. LifeGuardTM and DESS share the presence of the divalent metal chelator EDTA and high salt concentration which inhibit DNA polymerase and other cleavage enzymes and prevent DNA precipitation, respectively. However, DESS contains also DMSO which is an excellent bi-polar extraction solution for soil organic matter. DESS is removed by centrifugation before the DNA extraction, thus removing from the samples possible inhibitors of downstream analyses. These differences in the formulation of the two preservative solutions may explain their different performances in preserving soil and water samples. In conclusion, our and previous studies demonstrated that storage conditions have different effects on the outcomes of molecular microbiological studies. These effects are sample-specific since they depend on the type of sample (soil or water) and its physico-chemical properties (Rissanen et al. 2010). Our results suggest that in all the cases when freezing is not possible, storage of soil samples can be safely performed with no preservative, while storage of water samples with no preservative should be avoided when samples are stored at high (about +30 °C ) temperature. The non-proprietary DESS solution is a valid alternative for storage of soil and water samples.
5.
ACKNOWLEDGEMENTS
The authors are grateful to two anonymous referees whose comments improved the quality of the manuscript. This study was partially funded by 2010-2011 MIUR PRIN grant (Grant No. 2010AYKTAB_006) to RA and by 2013 UNIMIB FAR grant to AF.
6.
REFERENCES
Borchard D, Gillet F, Legendre F. (2011). Numerical Ecology with R. Springer: New York.
Carrigg C, Rice O, Kavanagh S, Collins G & O’Flaherty V (2007) DNA extraction method affects microbial community profiles from soils and sediment. Appl. Microbiol. Biotechnol. 77: 955–964.
Clark I. M., Hirsch P. R. (2008). Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment. Soil Biol. Biochem. 40: 1090–1102.
This article is protected by copyright. All rights reserved.
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De Caceres M, Legendre P, Moretti M. (2010). Improving indicator species analysis by combining groups of sites. Oikos 119: 1674-1684.
Dolfing J, Vos A, Bloem J, Ehlert P a. I, Naumova NB & Kuikman PJ (2004) Microbial Diversity in Archived Soils. Science 80: 306: 813.
Elsas JD van, Smalla K & Tebbe CC (2000) Extraction and analysis of microbial community nucleic acids from environmental matrices. J. Jansson, J.D. van Elsas and M.J. Bailey (Eds.), Tracking genetically engineered microorganisms: method development from microcosms to the field. R.G. Landes Co., Georgetown, Texas, USA, pp. 29-51.
Gray MA, Pratte ZA & Kellogg CA (2013) Comparison of DNA preservation methods for environmental bacterial community samples. FEMS Microbiol. Ecol. 83: 468–477.
Howard PJA (1965) The Carbon-Organic Matter Factor in Various Soil Types. Oikos 15: 229.
Klammer S, Mondini C & Insam H (2005) Microbial community fingerprints of composts stored under different conditions. Ann. Microbiol. 55: 299–305.
Lauber CL, Zhou N, Gordon JI, Knight R & Fierer N (2010) Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiol. Lett. 307: 80–86.
Leff LG, Dana JR, McArthur J V & Shimkets LJ (1995) Comparison of methods of DNA extraction from stream sediments. Appl. Environ. Microbiol. 61: 1141–1143.
Legendre P, Legendre L. (1998). Numerical Ecology, 2nd English edn. Elsevier: Amsterdam.
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Osborn AM & Smith CJ (2005) Molecular microbial ecology. Taylor & Francis, New York.
Rissanen AJ, Kurhela E, Aho T, Oittinen T & Tiirola M (2010) Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies. Appl. Microbiol. Biotechnol. 88: 977–984.
Rivelli V, Franzetti A, Gandolfi I, Cordoni S & Bestetti G (2013) Persistence and degrading activity of free and immobilised allochthonous bacteria during bioremediation of hydrocarbon-contaminated soils. Biodegradation 24: 1–11.
Rubin BER, Gibbons SM, Kennedy S, Hampton-Marcell J, Owens S & Gilbert JA (2013) Investigating the impact of storage conditions on microbial community composition in soil samples. PLoS One 8: e70460.
Sessitsch A, Gyamfi S, Stralis-Pavese N, Weilharter A & Pfeifer U (2002) RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols. J. Microbiol. Methods 51: 171–179.
Tzeneva VA, Salles JF, Naumova N, de Vos WM, Kuikman PJ, Dolfing J & Smidt H (2009) Effect of soil sample preservation, compared to the effect of other environmental variables, on bacterial and eukaryotic diversity. Res. Microbiol. 160: 89–98.
Yoder M, Tandingan De Ley I, King IW, Mundo-Ocampo M, Mann J, Blaxter M, Poiras L & De Ley P (2006) DESS: A versatile solution for preserving morphology and extractable DNA of nematodes. Nematology 8: 367–376.
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LEGEND TO FIGURES
Figure 1. Chart of preservation conditions. Storage times for each combination of preservative and temperature were 15 or 30 days for soil and 15 and 46 days for water. Sample size is reported in brackets.
Figure 2. Soil samples: a) hierarchical cluster analysis of samples stored with different preservation methods (no: no preservative, fr: frozen, lg: LifeGuardTM, ds = DESS). Storage temperature and time are shown in the name. AIAC: samples analyzed immediately after collection. b) Two-dimensional PCA plot of bacterial communities. Polygons include samples stored with different preservatives (green: no preservative, blue: frozen, violet: LifeGuardTM, orange = DESS). The red dots represent AIAC samples. Other samples are not shown individually for simplicity. The percentage of variance explained by each axis is shown.
Figure 3. Water samples: a) hierarchical cluster analysis of samples stored with different preservation methods (no: no preservative, fr: frozen, lg: LifeGuardTM, ds = DESS). Storage temperature and time are shown in the name. AIAC: samples analyzed immediately after collection. b) Two-dimensional PCA plot of bacterial communities. Polygons include samples stored with different preservatives (green: no preservative, blue: frozen, violet: LifeGuardTM, orange = DESS). The red dots represent AIAC samples. Other samples are not shown individually for simplicity. The percentage of variance explained by each axis is shown.
TABLES
Table 1 Redundancy analyses on soil and water aliquots stored with different preservation methods. Significance was assessed by 999 permutations.
Effect
df
Variance
F
P
Preservative type
4
0.080
8.098
< 0.001
Temperature
1
0.005
2.006
0.085
Storage time
1
0.004
1.632
0.158
21
0.052
Soil samples
Residual
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Water samples Preservative type
4
0.050
4.276
< 0.001
Temperature
1
0.009
3.220
0.031
Storage time
1
0.004
1.505
0.203
19
0.056
Residual
Table 2 Redundancy analyses on aliquots stored for either 15 or 30 (soil) or 46 (water) days and excluding AIAC samples. Significance was assessed by 999 permutations.
Storage time Effect
30 (soil) or 4
15 days df
Variance
F
P
df
Variance
Preservative type
3
0.075
6.269
< 0.001
3
0.125
Temperature
1
0.005
1.306
0.246
1
0.012
Residual
9
0.035
7
0.047
Preservative type
3
0.041
4.235
0.030
3
0.090
Temperature
1
0.017
5.143
0.014
1
0.018
Residual
7
0.023
7
0.066
Soil samples
Water samples
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Received Date : 12-Feb-2014 Revised Date : 22-Apr-2014 Accepted Date : 14-May-2014 Article type
: Research Letter
EFFECT OF PRESERVATION METHOD ON THE ASSESSMENT OF BACTERIAL COMMUNITY STRUCTURE IN SOIL AND WATER SAMPLES
Authors: Valeria Tatangelo1, Andrea Franzetti1, Isabella Gandolfi1, Giuseppina Bestetti1, Roberto Ambrosini2
Affiliations: 1
Dept. of Earth and Environmental Sciences – University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milan, Italy 2
Dept. of Biotechnology and Biosciences – University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy
Corresponding author: Andrea Franzetti, Dept. of Earth and Environmental Sciences – University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milan, Italy – email: [email protected], phone: +39 02 64482927
Abstract The methods used in sample preservation may affect the description of the microbial community structure by DNA-based techniques. This study aims at evaluating the effect of different storage conditions, including freezing, adding two liquid-based preservatives or simply storing samples with no preservative, on the structure of the microbial communities in aliquots of organic-rich soil and water samples as revealed by a T-FRLP. The results showed that the number of Terminal Restriction Fragments (TRF) detected in soil aliquots stored with LifeGuardTM solution was significantly lower
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12475 This article is protected by copyright. All rights reserved.
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than that of samples analysed immediately after sampling. Moreover, cluster and PCA analyses showed that soil aliquots stored using LifeGuardTM clustered separately from those stored with the other methods. Conversely, soil and water aliquots stored with DMSO-EDTA-salt solution (DESS) did not show either significant reduction in the number of TRFs or any change in the structure of the microbial community. Finally, the number of TRFs and the structure of microbial communities from soil aliquots stored with no preservative did not differ from those of aliquots analysed immediately after sampling. Preservation methods should therefore be accurately evaluated before collecting samples that have to be stored for long time before DNA extraction.
Keywords: microbial community storage conditions, T-RFLP, DNA extraction
1.
INTRODUCTION
An accurate description of the microbial community structure by DNA-based technologies relies on the amount and quality of nucleic acids recovered from environmental samples, which, in turn, are mostly affected by the methods used in sample collection and preservation, and in the extraction of the nucleic acids (Osborn & Smith 2005). Different protocols for sample collection and DNA extraction have been developed and proposed in the last 20 years, enabling microbiologists to achieve high yields and highly pure DNA/RNA extracts even from challenging materials (e.g. humic acid-rich soils) (Elsas et al. 2000; Carrigg et al. 2007; Leff et al. 1995; Sessitsch et al. 2002). Conversely, few investigations have addressed so far the effect of different sample preservation methods on the accuracy of the description of bacterial community structure that can be obtained by following DNAbased analyses (Lauber et al. 2010; Rissanen et al. 2010; Gray et al. 2013; Sessitsch et al. 2002; Elsas et al. 2000; Clark & Hirsch 2008). Indeed, improper sample preservation may determine DNA degradation and therefore bias the structure of the bacterial community that can be observed by any following DNA-based analysis. For soil samples in particular, contrasting evidences exist in the literature. Dolfing and colleagues (2004) demonstrated that it is possible to assess soil microbial community composition in samples that have been dried and stored for as long as 50 years, and other studies have showed that air-drying and sample storage at room temperature did not bias the assessment of the whole community composition (Klammer et al. 2005; Lauber et al. 2010; Rubin et al. 2013). Conversely, Tzeneva et al. (2009) found that sample drying and storage at room temperature significantly altered the composition of the observed microbial community. Moreover, most of the studies addressing the effect of storage conditions on the outcome of the analyses of soil microbial community have investigated only the effects of preservation time and temperature and physical treatments such as lyophilisation (Sessitsch et al. 2002; Lauber et al. 2010). However, in many cases, such as during sampling campaigns in remote areas of the world, low-temperature storage and lyophilisation are not feasible. In those cases the use of non-hazardous storage solutions may be a valuable alternative, provided that bacterial community structure can be reliably assessed when back in the lab. To the best of our knowledge, only two previous studies have tested the effectiveness of liquid-based preservatives for preserving bacterial communities in samples intended to be investigated by DNA-based analyses. (Gray et al. 2013; Rissanen et al. 2010). Indeed, DMSOEDTA-salt solution (DESS), which represents a cheap alternative to expensive proprietary products, has been tested so far on liquid cultures only (Gray et al. 2013). Therefore, an assessment of the
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performance of different preservation methods on samples intended to DNA-based analyses is necessary, especially before any sampling in remote areas. In this study we evaluated the effect of two liquid-based preservatives (the proprietary LifeGuardTM (MO BIO, Carlsbad, CA, USA) and the non-proprietary DESS) on the structure of the microbial communities in organic-rich soil and water samples as revealed by DNA-based Terminal Restriction Fragment Length Polymorphisms (T-RFLP). The results have been compared with those obtained from samples analysed immediately after sample collection and used as a reference of ‘true’ bacterial communities as well as with those obtained from samples stored at different temperatures and with frozen samples, which should represent samples stored with an ‘optimal’ preservation method.
2. 2.1
MATERIALS AND METHODS Sampling methods and handling
Surface soil samples were collected in the garden of the University of Milano-Bicocca (Milano, Italy, approximate coordinates of sampling site: 45°30’49’’ N, 9°12’39’’ E) while water samples were collected from a stream in an urban park near the University campus (Parco Nord, Milano-Italy, 45° 31' 54’’ N, 9° 12' 27’’E). The organic matter of the soil sample was 56 mg g-1, as determined on dry soil sample (62 °C, 24 h) after overnight combustion (Howard 1965). Soil and water samples were brought to the lab within 20 minutes without adding any preservative. In the lab the soil was homogenized by hand-mixing and divided into 2 g aliquots while water samples were homogenized, divided into 100 mL aliquots and filtered on nitrocellulose filters (Ø = 0.45 µm). Hence, in this study each aliquot consists of 2 g of soil or one nitrocellulose filter. Two aliquots of soil and two of water were immediately analysed (aliquots “analyzed immediately after collection”, AIAC hereafter), while the other ones were stored under different conditions as detailed below. The proprietary preservative solution LifeGuardTM was used as purchased according to manufacturer’s instructions (i.e. 6 mL of solution for 2 g of soil) while the non-proprietary DESS was prepared as described in Yoder et al. (2006) and used in the same proportion as the LifeGuardTM. Aliquots of samples without any preservatives were stored at –20 °C, +4 °C and +30 °C while LifeGuardTM- and DESS-preserved samples were stored at +4 °C and +30 °C. Storage times for each combination of preservative and temperature were 15 or 30 days for soils and 15 and 46 days for water samples. For brevity, hereafter we will refer to ‘preservation method’ as to the combination of type of preservative (none, LifeGuardTM, DESS), storage time (15 or 30 days for soil, 15 or 46 days for water) and temperature (-20 °C , +4 °C or +30 °C ). Figure 1 summarises the different preservation methods.
2.2
DNA extraction and amplification
The total genomic DNA was extracted from 0.25 g of soil or half filter from each of the two AIAC aliquots or from each aliquot stored with a different preservation method using ZR Soil Microbe DNA
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Miniprep (Zymo Research Corporation, Irvine, CA, USA) according to manufacturer’s instructions. Before the extraction, the samples preserved with LifeGuardTM and DESS were centrifuged at 2500 x g for 5 min to remove the preservative solution, according to MO BIO protocol. 16S rRNA gene fragments were PCR-amplified and purified as previously described (Rivelli et al. 2013). Endonucleolytic digestions were performed in a 20 µL reaction, containing 10 U of MspI (Promega Corporation, Madison, WI, USA) and 600 ng of amplified DNA for 1.5 h at 37 °C. T-RFLP profiles were obtained and analyzed as reported in Rivelli et al. (2013). T-RFLP profiles provide the relative abundance of different Terminal Restriction Fragments (TRFs hereafter) which, in turn, represent different microbial strains roughly corresponding to genus level.
2.3
Statistical methods
Singletons (i.e. TRFs found in one sample only) were removed from the dataset before all the analyses because they may inflate variance explained by models (Legendre and Legendre 1998). Multivariate analyses of the structure of bacterial communities were based on several techniques (Borchard et al., 2011), namely hierarchical cluster analysis with the complete linkage method, principal component analysis (PCA), multivariate regression trees (MRT), and redundancy analysis (RDA). All analyses were based on Hellinger distances between bacterial communities as assessed by the relative abundance of TRFs. This distance is a measure of the difference in the structure of bacterial communities between aliquots (Legendre and Legendre 1998, De Cáceres et al. 2010). In MRT analyses, the best tree was chosen as the smallest one whose cross-validated relative error (CVRE) was within 1 standard error of the CVRE of the best tree (Borchard et al. 2011). CVRE ranges between zero (perfect predictors) and close to one (poor set of predictors, Borchard et al., 2011). In RDA, preservation method and storage temperature and time were entered as predictors. Post-hoc tests in RDA were performed by comparing each level of the factor of interest with a reference level, which consisted, alternatively, in the structure of bacterial communities obtained from AIAC samples or from frozen samples. These post-hoc comparisons therefore follow the same logic as the Dunnet method of performing post-hoc tests in ANOVA models, i.e. they assessed significance of selected comparisons only. As a consequence, a significant main effect may not necessarily imply significant Dunnet post-hoc comparisons, because significant differences may occur in comparisons that are not of interest. Significance values of post-hoc tests were corrected for multiple statistical tests with the False Discovery Rate (FDR) method (Banjamini and Yekutieli 2001). In a following step of RDAs, we excluded AIAC aliquots and analyzed separately those stored for different times. The logic behind these analyses is that sometimes samples cannot be analyzed immediately after collection (e.g. when samples are collected in remote areas) and therefore must be stored in some way. The reference level for post-hoc tests in these analyses is therefore the bacterial community of frozen aliquots, which represent the ‘optimal’ (but often impractical) preservation method.
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If a given preservation method is not effective in avoiding degradation of bacterial DNA, we expect that the number of OTUs that could be retrieved from samples stored with that method are lower than those observed in AIAC samples. To test for this hypothesis, we compared the number of OTUs of samples stored with different preservatives and AIAC samples, or frozen samples stored for the same amount of time, by Poisson generalized linear models (GLMs) followed by Dunnet post-hoc tests. All the analyses were performed in R 2.15.2 (R Core Team 2012).
3. 3.1
RESULTS Multivariate analyses
We were unable to amplify two soil samples stored for 30 d at +30 °C, one with no preservative and one with LifeGuardTM, the two water samples stored for 15 d with no preservative at +30 °C , and the two water samples stored for 46 d with no preservative at +30 °C. These samples were excluded from the subsequent analyses. Overall, 45 TRFs were identified in soil samples and 22 in water samples. After singletons removal, 31 TRFs were retained in the analyses of soil samples, and 16 in those of water samples. Cluster analyses and PCA plots indicated that soil aliquots stored with LifeGuardTM were separated from those stored with other preservatives (Figure 2a and 2b), while separation of water aliquots stored with LifeGuardTM or other preservation methods was less clear (Figure 3a and 3b). Importantly, soil aliquots stored with LifeGuardTM formed a different cluster than all the other ones (Figure 2a) and in PCA plot they were separated from AIAC aliquots (Figure 2b), while those stored with other preservatives were not. Water aliquots stored with LifeGuardTM were also separated from the others, albeit not so clearly as for soil samples (Figure 3b). MRT analyses on both soil and water aliquots indicated a tree with two leaves as the best one, although performance of the models was rather low (CVRE ≥ 0.61). In both cases aliquots stored with LifeGuardTM were separated from both those stored with other methods (including freezing) and AIAC aliquots. RDA on soil and water aliquots indicated that the structure of bacterial communities significantly differed among aliquots stored with different preservatives but not for different times (Table 1). In addition, soil bacterial community did not differ between aliquots stored at different temperatures, while those of water samples did (Table 1). However, post-hoc tests disclosed no significant difference between soil or water aliquots stored with different preservatives and AIAC ones neither between water aliquots stored at different temperatures and AIAC ones (F1,8 ≤ 6.89, PFDR ≥ 0.08 in all cases). Analyses run separately on aliquots stored for different times and excluding AIAC samples showed significant differences among aliquots stored with different preservatives (Table 2). However, Dunnet-type post-hoc tests indicated that bacterial communities of frozen aliquots did not differ from those of aliquots stored with any preservative for the same amount of time (F1,4 ≤ 7.22; PFDR ≥
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0.12 in all cases). A significant effect of temperature only appeared in water samples stored for 15 days (Table 2), but also in this case post-hoc tests did not disclose any significant difference between samples stored at +4 or +30 °C and samples stored at -20 °C (F1,6 ≤ 1.82; PFDR ≥ 0.39 in all cases).
3.2
Number of TRFs
Poisson GLMs indicated that the number of TRFs differed between samples stored with different preservatives and this occurred for both soil and water samples (LRT: χ24 ≥ 9.80, P ≤ 0.04). Post-hoc tests on soil samples indicated that samples stored with LifeGuardTM included a significantly lower number of TRFs than AIAC samples (-9.00 ± 1.22 SE OTUs, z = -3.18, P < 0.01; z| ≤ 1.15, P ≥ 0.52 in all the other cases). Conversely, post-hoc tests on water samples did not reveal any significant difference (|z| ≤ 2.36, P ≥ 0.06 in all cases). Analyses restricted to soil samples stored for 15 or 30 days (thus excluding AIAC samples) confirmed significant differences in the number of TRFs that could be observed in samples stored with different preservatives (15 days: χ23 = 8.23, P = 0.04, 30 days: χ23 = 8.46, P = 0.04). Post-hoc tests showed that samples stored for 15 days with LifeGuardTM contained a significantly smaller number of TRFs than samples frozen for the same amount of time (-8.00 ± 1.24 SE TRFs, z = -2.43, P = 0.04; |z| ≤ 1.05, P ≥ 0.56 in all the other cases), while post-hoc tests on soil samples stored for 30 days did not disclose any significant difference (|z| ≤ 1.56, P ≥ 0.26 in all cases). The same analyses conducted on water samples did not find any difference in the number of TRFs among samples stored with different preservatives (15 days, LRT: χ23 = 6.77, P = 0.08; 46 days : χ23 = 5.57, P = 0.13).
4.
DISCUSSION
In this study we investigated how storage condition, including the preservative added to samples, can alter the structure of bacterial communities compared to the one that could be retrieved if samples were analysed immediately after they were collected. A limit of this study is that we investigated only one soil and water sample, so that our results can be representative only of the performance of preservation methods on samples with similar characteristics. However, we stress that we chose a soil with high organic carbon content whose analysis is particularly challenging due the high activity of nucleases and the possible co-extraction of inhibitors affecting downstream analyses. During all the phases of the process of DNA-based characterization of the microbial community of soil and water samples collected in the field, the quality and the quantity of DNA should be preserved, from the time of collection to that of DNA extraction. Very often, storage of samples is unavoidable due to the lack of facilities in the field. In this case, storage at -20 °C (freezing) is considered the best preservation method for samples that should be used for analyses of bacterial community structure. However, even freezing samples may not be an option, particularly when samples are collected in remote areas of the world, and therefore the ability of alternative preservation methods of preserving the original microbial community structure should be explored.
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Our results did not disclose any difference in the structure of bacterial communities retrieved from samples stored with no preservative at different temperatures, including frozen samples, and that from samples analysed immediately after collection, in agreement with previous reports (Rissanen et al. 2010; Lauber et al. 2010; Rubin et al. 2013). In particular, Lauber and colleagues have even suggested that the samples they collected could be stored and shipped at room temperature without having a significant impact on the assessment of the overall community composition or on the relative abundances of most major bacterial taxa (Lauber et al. 2010). Our results confirm their findings, as samples stored with no preservative did not differ in the number of TRFs or in the structure of bacterial communities from AIAC and frozen samples. The lack of influence of storage temperature on bacterial community composition that they observed may be due to the fact that storage and shipping temperature did not differ significantly from ambient temperature at the place where sample where collected. Conversely, the relative abundance of the microbial populations may largely change if samples are stored and transported at temperatures that are markedly different from that at the collection site, due e.g. to differential growth of psycrophilic or thermophilic populations. Our results seem to contradict this hypothesis, because community structure observed in aliquots stored with no preservative did not differ from those of AIAC or frozen aliquots, independently of temperature and time of preservation. However, DNA seem to degrade in a few days in water aliquots stored at high temperature, as indicated by the fact that we failed in amplifying water aliquots stored at +30 °C . We expected that the use of bacteriostatic or bactericide preservative agents might enhance bacterial preservation. Unexpectedly, our results showed that LifeGuardTM solution led to a significant reduction in the number of TRFs that could be detected in soil aliquots compared to AIAC ones. Moreover, cluster and PCA analyses showed that soil aliquots stored using LifeGuardTM clustered separately from the samples stored with other methods. Although RDA did not find a significant difference between aliquots stored with LifeGuard™ and AIAC ones, the overall results might indicate that the addition of LifeGuard™ was not a suitable preservation strategy for our soil samples. Results from multivariate analyses on water samples were similar to those of soil samples, with only three aliquots stored with LifeGuardTM that clustered with the other aliquots, while all the other ones stored with this preservative grouped in a separate cluster. Preservative solutions can have different performance for water and soil samples. For example, DNAgardTM and RNAlater® have good preservation performances on liquid cultures (Gray et al. 2013) while RNAlater® lead to a significant reduction in extraction yield of DNA and RNA and to changes in the relative representation of some major taxa in sediment samples (Rissanen et al. 2010). This behavior may be due to the precipitation and fixation of protein and organic matter onto nucleic acids as a consequence of the presence of ammonium sulphate, which is used for protein precipitation, in the RNAlater® formulation (Rissanen et al. 2010). This may not apply to LifeGuardTM because, according to the patent (US patent 6,458,546), it contains no compound used for protein precipitation. The reason of this difference in the performance of LifeGuardTM in preserving soil and water samples needs therefore to be further investigated. The performance of the non-proprietary liquid solution DESS and the phenol–chloroform–isoamyl alcohol in storing bacterial communities without altering their structure have been already tested. In
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particular, phenol–chloroform–isoamyl alcohol was used on sediment samples and was able to preserve both the DNA and RNA yields and the bacterial community structure of sediments owing to the inactivation of nucleases (Rissanen et al. 2010), and DESS was able to properly preserve liquid cultures of culturable bacteria (Gray et al. 2013). In our study DESS was successfully applied also to soil and water samples as it did not show any significant reduction in TRF abundance and did not modify the structure of the community as revealed by DNA-based T-RFLP. LifeGuardTM and DESS share the presence of the divalent metal chelator EDTA and high salt concentration which inhibit DNA polymerase and other cleavage enzymes and prevent DNA precipitation, respectively. However, DESS contains also DMSO which is an excellent bi-polar extraction solution for soil organic matter. DESS is removed by centrifugation before the DNA extraction, thus removing from the samples possible inhibitors of downstream analyses. These differences in the formulation of the two preservative solutions may explain their different performances in preserving soil and water samples. In conclusion, our and previous studies demonstrated that storage conditions have different effects on the outcomes of molecular microbiological studies. These effects are sample-specific since they depend on the type of sample (soil or water) and its physico-chemical properties (Rissanen et al. 2010). Our results suggest that in all the cases when freezing is not possible, storage of soil samples can be safely performed with no preservative, while storage of water samples with no preservative should be avoided when samples are stored at high (about +30 °C ) temperature. The non-proprietary DESS solution is a valid alternative for storage of soil and water samples.
5.
ACKNOWLEDGEMENTS
The authors are grateful to two anonymous referees whose comments improved the quality of the manuscript. This study was partially funded by 2010-2011 MIUR PRIN grant (Grant No. 2010AYKTAB_006) to RA and by 2013 UNIMIB FAR grant to AF.
6.
REFERENCES
Borchard D, Gillet F, Legendre F. (2011). Numerical Ecology with R. Springer: New York.
Carrigg C, Rice O, Kavanagh S, Collins G & O’Flaherty V (2007) DNA extraction method affects microbial community profiles from soils and sediment. Appl. Microbiol. Biotechnol. 77: 955–964.
Clark I. M., Hirsch P. R. (2008). Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment. Soil Biol. Biochem. 40: 1090–1102.
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De Caceres M, Legendre P, Moretti M. (2010). Improving indicator species analysis by combining groups of sites. Oikos 119: 1674-1684.
Dolfing J, Vos A, Bloem J, Ehlert P a. I, Naumova NB & Kuikman PJ (2004) Microbial Diversity in Archived Soils. Science 80: 306: 813.
Elsas JD van, Smalla K & Tebbe CC (2000) Extraction and analysis of microbial community nucleic acids from environmental matrices. J. Jansson, J.D. van Elsas and M.J. Bailey (Eds.), Tracking genetically engineered microorganisms: method development from microcosms to the field. R.G. Landes Co., Georgetown, Texas, USA, pp. 29-51.
Gray MA, Pratte ZA & Kellogg CA (2013) Comparison of DNA preservation methods for environmental bacterial community samples. FEMS Microbiol. Ecol. 83: 468–477.
Howard PJA (1965) The Carbon-Organic Matter Factor in Various Soil Types. Oikos 15: 229.
Klammer S, Mondini C & Insam H (2005) Microbial community fingerprints of composts stored under different conditions. Ann. Microbiol. 55: 299–305.
Lauber CL, Zhou N, Gordon JI, Knight R & Fierer N (2010) Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiol. Lett. 307: 80–86.
Leff LG, Dana JR, McArthur J V & Shimkets LJ (1995) Comparison of methods of DNA extraction from stream sediments. Appl. Environ. Microbiol. 61: 1141–1143.
Legendre P, Legendre L. (1998). Numerical Ecology, 2nd English edn. Elsevier: Amsterdam.
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Osborn AM & Smith CJ (2005) Molecular microbial ecology. Taylor & Francis, New York.
Rissanen AJ, Kurhela E, Aho T, Oittinen T & Tiirola M (2010) Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies. Appl. Microbiol. Biotechnol. 88: 977–984.
Rivelli V, Franzetti A, Gandolfi I, Cordoni S & Bestetti G (2013) Persistence and degrading activity of free and immobilised allochthonous bacteria during bioremediation of hydrocarbon-contaminated soils. Biodegradation 24: 1–11.
Rubin BER, Gibbons SM, Kennedy S, Hampton-Marcell J, Owens S & Gilbert JA (2013) Investigating the impact of storage conditions on microbial community composition in soil samples. PLoS One 8: e70460.
Sessitsch A, Gyamfi S, Stralis-Pavese N, Weilharter A & Pfeifer U (2002) RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols. J. Microbiol. Methods 51: 171–179.
Tzeneva VA, Salles JF, Naumova N, de Vos WM, Kuikman PJ, Dolfing J & Smidt H (2009) Effect of soil sample preservation, compared to the effect of other environmental variables, on bacterial and eukaryotic diversity. Res. Microbiol. 160: 89–98.
Yoder M, Tandingan De Ley I, King IW, Mundo-Ocampo M, Mann J, Blaxter M, Poiras L & De Ley P (2006) DESS: A versatile solution for preserving morphology and extractable DNA of nematodes. Nematology 8: 367–376.
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LEGEND TO FIGURES
Figure 1. Chart of preservation conditions. Storage times for each combination of preservative and temperature were 15 or 30 days for soil and 15 and 46 days for water. Sample size is reported in brackets.
Figure 2. Soil samples: a) hierarchical cluster analysis of samples stored with different preservation methods (no: no preservative, fr: frozen, lg: LifeGuardTM, ds = DESS). Storage temperature and time are shown in the name. AIAC: samples analyzed immediately after collection. b) Two-dimensional PCA plot of bacterial communities. Polygons include samples stored with different preservatives (green: no preservative, blue: frozen, violet: LifeGuardTM, orange = DESS). The red dots represent AIAC samples. Other samples are not shown individually for simplicity. The percentage of variance explained by each axis is shown.
Figure 3. Water samples: a) hierarchical cluster analysis of samples stored with different preservation methods (no: no preservative, fr: frozen, lg: LifeGuardTM, ds = DESS). Storage temperature and time are shown in the name. AIAC: samples analyzed immediately after collection. b) Two-dimensional PCA plot of bacterial communities. Polygons include samples stored with different preservatives (green: no preservative, blue: frozen, violet: LifeGuardTM, orange = DESS). The red dots represent AIAC samples. Other samples are not shown individually for simplicity. The percentage of variance explained by each axis is shown.
TABLES
Table 1 Redundancy analyses on soil and water aliquots stored with different preservation methods. Significance was assessed by 999 permutations.
Effect
df
Variance
F
P
Preservative type
4
0.080
8.098
< 0.001
Temperature
1
0.005
2.006
0.085
Storage time
1
0.004
1.632
0.158
21
0.052
Soil samples
Residual
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Water samples Preservative type
4
0.050
4.276
< 0.001
Temperature
1
0.009
3.220
0.031
Storage time
1
0.004
1.505
0.203
19
0.056
Residual
Table 2 Redundancy analyses on aliquots stored for either 15 or 30 (soil) or 46 (water) days and excluding AIAC samples. Significance was assessed by 999 permutations.
Storage time Effect
30 (soil) or 4
15 days df
Variance
F
P
df
Variance
Preservative type
3
0.075
6.269
< 0.001
3
0.125
Temperature
1
0.005
1.306
0.246
1
0.012
Residual
9
0.035
7
0.047
Preservative type
3
0.041
4.235
0.030
3
0.090
Temperature
1
0.017
5.143
0.014
1
0.018
Residual
7
0.023
7
0.066
Soil samples
Water samples
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