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Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples

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Abstract

The anaerobic digestion (AD) of organic waste for biogas production has received much attention in recent years due to the increasing need for renewable energy and environmentally friendly waste management systems. Identification of the microbial community involved in AD aids in better understanding and optimising of the process. The choice of DNA extraction method is an integral step in any molecular biodiversity study. In the present study, potential biases introduced by DNA extraction methods were examined by comparing quality, quantity and representability of DNA extracted from AD samples using various extraction methods. In spite of the non-kit based method (cetyltrimethylammonium bromide) yielding the largest quantity of DNA (approximately 44 µg DNA per gram dry weight), the extracted DNA contained PCR inhibitors. Furthermore, the quantity of extracted DNA was not proportional to species diversity. Diversity, determined using denaturing gradient gel electrophoresis (DGGE), was strongly linked to the type of extraction method used. The spin-column filter-based kit that incorporated mechanical and chemical lysis (Macherey-Nagel kit) gave the best results in terms of bacterial and archaeal diversity (Shannon–Wiener indices: average 2.5 and 2.6, respectively). Furthermore, this kit was the most effective at lysing hard-to-lyse bacterial and archaeal cells. The choice of DNA extraction method significantly influences the reliability and comparability of results obtained during AD microbial ecology investigations. Moreover, the careful selection of the DNA extraction method is of particular importance when analysing AD samples since these samples are rich in PCR inhibitors and hard-to-lyse cells such as archaea and gram-positive bacteria.

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References

  • Adamowicz MS, Stasulli DM, Sobestanovich EM, Bille TW (2014) Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis. PLoS ONE 9(12):e116351

    Article  Google Scholar 

  • Albers SV, Meyer BH (2011) The archaeal cell envelope. Nat Rev Microbiol 9(6):414–426

    Article  CAS  Google Scholar 

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1):143–169

    CAS  Google Scholar 

  • Ariefdjohan MW, Savaiano DA, Nakatsu CH (2010) Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. J Nutr 9(1):1

    Article  Google Scholar 

  • Ariesyady HD, Ito T, Okabe S (2007) Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester. Water Res 41(7):1554–1568

    Article  CAS  Google Scholar 

  • Bergmann I, Mundt K, Sontag M, Baumstark I, Nettmann E, Klocke M (2009) Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters. Syst Appl Microbiol 33:78–84

    Article  Google Scholar 

  • Brownlow RJ, Dagnall KE, Ames CE (2012) A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods. J Forensic Sci 57(3):713–717

    Article  CAS  Google Scholar 

  • Cabeen MT, Jacobs-Wagner C (2005) Bacterial cell shape. Nat Rev Microbiol 3(8):601–610

    Article  CAS  Google Scholar 

  • 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(4):955–964

    Article  CAS  Google Scholar 

  • Chaudhary PP, Sirohi SK, Kumar S (2011) Improved extraction of quality DNA from methanogenic archaea present in rumen liquor for PCR application. Asian J Anim Sci 5(3):166–174

    Article  CAS  Google Scholar 

  • Claassen S, du Toit E, Kaba M, Moodley C, Zar HJ, Nicol MP (2013) A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. J Microbiol Methods 94(2):103–110

    Article  CAS  Google Scholar 

  • Delmont TO, Robe P, Clark I, Simonet P, Vogel TM (2011) Metagenomic comparison of direct and indirect soil DNA extraction approaches. J Microbiol Methods 86(3):397–400

    Article  CAS  Google Scholar 

  • Endres HN, Johnson JA, Ross CA, Welp JK, Etzel MR (2003) Evaluation of an ion-exchange membrane for the purification of plasmid DNA. Biotechnol Appl Biochem 37(3):259–266

    Article  CAS  Google Scholar 

  • Fahle GA, Fischer SH (2000) Comparison of six commercial DNA extraction kits for recovery of cytomegalovirus DNA from spiked human specimens. J Clin Microbiol 38(10):3860–3863

    CAS  Google Scholar 

  • Fang X, Willis RC, Burrell A, Evans K, Hoang Q, Xu W, Bounpheng M (2007) Automation of nucleic acid isolation on KingFisher magnetic particle processors. J Lab Autom 12(4):195–201

    Article  CAS  Google Scholar 

  • Garcia-Peña EI, Parameswaran P, Kang DW, Canul-Chan M, Krajmalnik-Brown R (2011) Anaerobic digestion and co-digestion processes of vegetable and fruit residues: process and microbial ecology. Bioresour Technol 102(20):9447–9455

    Article  Google Scholar 

  • Guo F, Zhang T (2013) Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing. Appl Environ Microbiol 97(10):4607–4616

    CAS  Google Scholar 

  • Herrera A, Cockell CS (2007) Exploring microbial diversity in volcanic environments: a review of methods in DNA extraction. J Microbiol Methods 70(1):1–12

    Article  CAS  Google Scholar 

  • Ikenaga M, Asakawa S, Muraoka Y, Kimura M (2004) Methanogenic archaeal communities in rice roots grown in flooded soil pots: estimation by PCR-DGGE and sequence analyses. Soil Sci Plant Nutr 50(5):701–711

    Article  CAS  Google Scholar 

  • Jarrell KF, Faguy D, Hebert AM, Kalmokoff ML (1992) A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria). Can J Microbiol 38(1):65–68

    Article  CAS  Google Scholar 

  • Kampmann K, Ratering S, Kramer I, Schmidt M, Zerr W, Schnell S (2012) Unexpected stability of Bacteroidetes and Firmicutes communities in laboratory biogas reactors fed with different defined substrates. Appl Environ Microbiol 78(7):2106–2119

    Article  CAS  Google Scholar 

  • Kishore R, Reef Hardy W, Anderson VJ, Sanchez NA, Buoncristiani MR (2006) Optimization of DNA extraction from low-yield and degraded samples using the BioRobot® EZ1 and BioRobot® M48. J Forensic Sci 51(5):1055–1061

    Article  CAS  Google Scholar 

  • Kojima K, Ozawa S (2002) US Patent No 20,020,192,667. Washington, DC, US Patent and Trademark Office

  • König H (1988) Archaeobacterial cell envelopes. Can J Microbiol 34(4):395–406

    Article  Google Scholar 

  • Krsek M, Wellington EMH (1999) Comparison of different methods for the isolation and purification of total community DNA from soil. J Microbiol Methods 39(1):1–16

    Article  CAS  Google Scholar 

  • Kubota K, Imachi H, Kawakami S, Nakamura K, Harada H, Ohashi A (2008) Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens. J Microbiol Methods 72(1):54–59

    Article  CAS  Google Scholar 

  • Leff LG, Dana JR, McArthur JV, Shimkets LJ (1995) Comparison of methods of DNA extraction from stream sediments. Appl Environ Microbiol 61(3):1141–1143

    CAS  Google Scholar 

  • Liesack W, Weyland H, Stackebrandt E (1991) Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria. Microb Ecol 21(1):191–198

    Article  CAS  Google Scholar 

  • Magurran AE (1988) Ecological diversity and its measurement. Princeton University Press, Princeton

    Book  Google Scholar 

  • Mahalanabis M, Al-Muayad H, Kulinski MD, Altman D, Klapperich CM (2009) Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip. Lab Chip 9(19):2811–2817

    Article  CAS  Google Scholar 

  • Mertens B, Boon N, Verstraete W (2005) Stereospecific effect of hexachloro- cyclohexane on activity and structure of soil methanotrophic communities. Environ Microbiol 7:660–669

    Article  CAS  Google Scholar 

  • Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 65(11):4715–4724

    CAS  Google Scholar 

  • Minas K, McEwan NR, Newbold CJ, Scott KP (2011) Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures. FEMS Microbiol Lett 325(2):162–169

    Article  CAS  Google Scholar 

  • Montpetit SA, Fitch IT, O’Donnell PT (2005) A simple automated instrument for DNA extraction in forensic casework. J Forensic Sci 50(3):555–563

    Article  CAS  Google Scholar 

  • Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59(3):695–700

    CAS  Google Scholar 

  • Riviere D, Desvignes V, Pelletier E, Chaussonnerie S, Guermazi S, Weissenbach J, Sghir A (2009) Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge. ISME J 3(6):700–714

    Article  Google Scholar 

  • Roh C, Villatte F, Kim BG, Schmid RD (2006) Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples. Appl Biochem Biotechnol 134:97–112

    Article  CAS  Google Scholar 

  • Roopnarain A, Adeleke R (2017) Current status, hurdles and future prospects of biogas digestion technology in Africa. Renew Sustain Energy Rev 67:1162–1179

    Article  CAS  Google Scholar 

  • Salonen A, Nikkilä J, Jalanka-Tuovinen J, Immonen O, Rajilić-Stojanović M, Kekkonen RA, de Vos WM (2010) Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods 81(2):127–134

    Article  CAS  Google Scholar 

  • Shaw KJ, Thain L, Docker PT, Dyer CE, Greenman J, Greenway GM, Haswell SJ (2009) The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths. Anal Chim Acta 652(1):231–233

    Article  CAS  Google Scholar 

  • Sironen A, Uimari P, Vilkki J (2008) Comparison of different DNA extraction methods from hair root follicles to genotype Finnish Landrace boars with the Illumina PorcineSNP60 BeadChip. Agric Food Sci 20(2):143–150

    Article  Google Scholar 

  • Slana I, Pribylova R, Kralova A, Pavlik I (2011) Persistence of Mycobacterium avium subsp. paratuberculosis at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle. Appl Environ Microbiol 77(9):3115–3119

    Article  CAS  Google Scholar 

  • Sundberg C, Al-Soud WA, Larsson M, Alm E, Yekta SS, Svensson BH, Karlsson A (2013) 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS Microbiol Ecol 85(3):612–626

    Article  CAS  Google Scholar 

  • Tan SC, Yiap BC (2009) DNA, RNA, and protein extraction: the past and the present. Biomed Res Int. doi:10.1155/2009/574398

    Google Scholar 

  • Theron J, Cloete TE (2000) Molecular techniques for determining microbial diversity and community structure in natural environments. Crit Rev Microbiol 26(1):37–57

    Article  CAS  Google Scholar 

  • Vanysacker L, Declerck SA, Hellemans B, De Meester L, Vankelecom I, Declerck P (2010) Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods. Appl Microbiol Biotechnol 88(1):299–307

    Article  CAS  Google Scholar 

  • Watanabe T, Asakawa S, Nakamura A, Nagaoka K, Kimura M (2004) DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil. FEMS Microbiol Lett 232(2):153–163

    Article  CAS  Google Scholar 

  • Weiss A, Jerome V, Freitag R (2007) Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants. J Chromatogr B 853:190–197

    Article  CAS  Google Scholar 

  • Wintzingerode FV, Göbel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21(3):213–229

    Article  Google Scholar 

  • Xu Z, Zhao M, Miao H, Huang Z, Gao S, Ruan W (2014) In situ volatile fatty acids influence biogas generation from kitchen wastes by anaerobic digestion. Bioresour Technol 163:186–192

    Article  CAS  Google Scholar 

  • Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online 1(1):40–47

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Biogas research at ARC-ISCW, South Africa is supported by the National Research Foundation (NRF; Grant Number 96735), the South Africa—Norway Research Co-operation (SANCOOP; Grant Number RCN 234203) and the Water Research Commission (WRC; Grant Number K5/2543). Opinions expressed and conclusions reached are those of the authors and not necessarily endorsed by the NRF, SANCOOP or WRC.

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Authors and Affiliations

  1. Microbiology and Environmental Biotechnology Research Group, Agricultural Research Council-Institute for Soil Climate and Water, Arcadia, Pretoria, 0001, South Africa

    Ashira Roopnarain, Mashudu Mukhuba & Rasheed Adeleke

  2. Department of Life and Consumer Sciences, College of Agriculture and Environmental Science, UNISA, P. O. Box 329, Pretoria, South Africa

    Mashudu Mukhuba

  3. Unit of Environmental Sciences and Management, North-West University, Potchefstroom Campus, Potchefstroom, 2520, South Africa

    Rasheed Adeleke

  4. Agricultural Research Council-Institute for Soil, Climate and Water, 600, Belvedere Street, Arcadia, Pretoria, 0083, South Africa

    Mokhele Moeletsi

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  1. Ashira Roopnarain
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Correspondence to Rasheed Adeleke.

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Roopnarain, A., Mukhuba, M., Adeleke, R. et al. Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples. 3 Biotech 7, 375 (2017). https://doi.org/10.1007/s13205-017-1009-x

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