Skip to main content
Log in

Rapid extraction of total RNA from an anaerobic sludge biocoenosis

  • Published:
Folia Microbiologica Aims and scope Submit manuscript

Abstract

In order to assess the activity of metabolic pathways during anaerobic biogas production, it is necessary to isolate total RNA from the anaerobic sludge. mRNA activity profiling complements the quantification of excreted metabolites for a comprehensive anaerobic digestion model (ADM1). Four non-commercial total RNA extraction protocols were examined to extract total RNA from suspended solids of anaerobic sludge. The most suitable protocol was identified and optimised. In relation to total RNA extraction efficiency, total RNA purity and RNA integrity, the best homogenisation method was a combined method of nitrogen grinding and bead beating. When bead beating or nitrogen grinding was used alone for homogenisation, total RNA extraction efficiency was lower than when both homogenisation methods were applied. Depending on the homogenisation method, the whole RNA extraction procedure takes approximately 2 to 3 h, which is as fast as when using commercial available soil RNA extraction kits. The proposed method is rapid in extracting total RNA from a biocoenosis present in an anaerobic sludge environment. Furthermore, we could apply any of the extracted homogenization methods for reverse transcription and subsequent PCR amplification of the gene for the methyl coenzyme M reductase alpha subunit (mcrA/mrtA).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

References

  • Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi S, Pavlostathis SG, Rozzi A, Sanders WTM, Siegrist H, Vavilin VA (2002) Anaerobic Digestion Model No.1. IWA Task Group for Mathematical Modelling of Anaerobic Digestion Processes, London

  • Bodrossy L, Stralis-Pavese N, Konrad-Koeszler M, Weilharter A, Reichenauer TG, Schoefer D, Sessitsch A (2006) mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray. Appl Environ Microbiol 72(2):1672–1676. doi:10.1128/aem.72.2.1672-1676.2006

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bürgmann H, Widmer F, Sigler WV, Zeyer J (2003) mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil. Appl Environ Microbiol 69(4):1928–1935. doi:10.1128/AEM.69.4.1928-1935.2003

    Article  PubMed Central  PubMed  Google Scholar 

  • Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11(2):113–116. doi:10.1007/bf02670468

    Article  CAS  Google Scholar 

  • Culley DE, Kovacik WP, Brockman FJ, Zhang W (2006) Optimization of RNA isolation from the archaebacterium Methanosarcina barkeri and validation for oligonucleotide microarray analysis. J Microbiol Methods 67(1):36–43. doi:10.1016/j.mimet.2006.02.014

    Article  CAS  PubMed  Google Scholar 

  • Delebès C, Lecerc M, Zumstein E, Godon J-J, Moletta R (2001) A molecular method to study population and activity dynamics in anaerobic digestors. Water Sci Technol 43(1):51–57

    Google Scholar 

  • Ferry James G (2010) How to make a living by exhaling methane. Ann rev microbiol 64:453–473. doi:10.1146/annurev.micro.112408.134051

    Article  CAS  Google Scholar 

  • Friedrich MW (2005) Methyl coenzyme M reductase genes: unique functional markers for methanogenic and anaerobic methane oxidizing archaea. Nucleic Acid Techniques 397:428–442. doi10.1016/S0076-6879(05)97026-2

  • Hurt RA, Qiu X, Wu L, Roh Y, Palumbo AV, Tiedje JM, Zhou J (2001) Simultaneous recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol 67(10):4495–4503. doi:10.1128/AEM.67.10.4495-4503.2001

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ibrahim A, Ahring BK (1999) Extraction of intact ribosomal RNA from anaerobic bioreactor samples for molecular ecological studies. Biotechniques 27(6):1132–1138

    CAS  PubMed  Google Scholar 

  • Kirk JL, Beaudette LA, Hart M, Moutoglis P, Klironomos JN, Lee H, Trevors JT (2004) Methods for studying soil microbial diversity. J Microbiol Methods 58:169–188. doi10.1016/j.mimet.2004.04.006

    Google Scholar 

  • Liu Y, Whitman WB (2008) Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci 1125:171–189. doi: 10.1196/annals.1419.019

    Google Scholar 

  • Mata-Alvarez J, Mace S, Llabres P (2000) Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresource Technol 74 (1):3–16. doi10.1016/S0960-8524(00)00023-7

    Google Scholar 

  • Rittmann S, Herwig C (2012) A comprehensive and quantitative review of dark fermentative biohydrogen production. Microb Cell Fact 11:115. doi:10.1186/1475-2859-11-115

    Google Scholar 

  • Sessitsch A, Gyamfi S, Stralis PN, 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 (2):171–179. doi10.1016/S0167-7012(02)00065-9

    Google Scholar 

  • Tebbe CC, Vahjen W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol 59(8):2657–2665

    CAS  PubMed Central  PubMed  Google Scholar 

  • Thauer RK, Kaster A-K, Seedorf H, Buckel W, Hedderich R (2008) Methanogenic archaea: ecologically relevant differences in energy conservation. Nat Rev Microbiol 6(8):579–591. doi:10.1038/nrmicro1931

    Article  CAS  PubMed  Google Scholar 

  • Wang P, Qi M, Barboza P, Leigh MB, Ungerfeld E, Selinger LB, McAllister TA, Forster RJ (2011) Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases. Can J Microbiol 57(7):590–598. doi:10.1139/W11-048

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This project was founded by the European Commission under contract no. SES6-CT-2004-502824. We greatly appreciate financial support from Krajete GmbH.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Simon Rittmann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rittmann, S., Holubar, P. Rapid extraction of total RNA from an anaerobic sludge biocoenosis. Folia Microbiol 59, 127–132 (2014). https://doi.org/10.1007/s12223-013-0274-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12223-013-0274-2

Keywords

Navigation