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Fluorescent in situ hybridisation; Artificial consortium; Quantitative PCR
Abstract :
[en] The use of an artificial consortium composed of selected hydrogen-producing species,
instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In
order to monitor such a consortium composed of different Clostridium spp., new protocols
were tested for two different assays, FISH and qPCR. New species-specific FISH probes and
qPCR primer sets were developed and optimised for three strains: Clostridium butyricum,
Clostridium felsineum and Clostridium pasteurianum, that were used in a consortium. Application
of a fast two-step FISH protocol, with pre-treatment step at 90 C for 5 min and
a subsequent hybridisation step at higher temperature (55 C) for 20 min resulted in a much
shorter analytical time compared to the standard FISH procedure (46 C for 2e3 h) and gave
a high hybridisation performance. Moreover, to accurately quantify each microorganism by
qPCR assay, two innovations were tested: the direct use of cell lysates (omitting the DNA
extraction step) and the use of two alternative molecular markers, recA and gyrA. These
markers are present in single copies in the genome, whereas there are multiple copies of
the ribosomal operons. This resulted in the development of accurate, reliable and fast FISH
and qPCR assays for routine monitoring of the dynamics of artificial hydrogen-producing
microbial consortia. Moreover, both techniques can be easily adapted to new Clostridium
strains.
Research Center/Unit :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Microbiology
Author, co-author :
Savichtcheva, Olga
Joris, Bernard ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes
Wilmotte, Annick ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes
Calusinska, Magdalena ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Novel FISH and quantitative PCR protocols to monitor artificial consortia composed of different hydrogen-producing Clostridium spp.
Alternative titles :
[fr] Protocoles nouveaux de FISH et PCR quantitative pour suivre un consortium artificiel de clostridies productrices d'hydrogène
R. Nandi, and S. Sengupta Microbial production of hydrogen: an overview Crit Rev Microbiol 24 1998 61 84 (Pubitemid 28166603)
D. Boyles Bioenergy, technology, thermodynamics and costs 1984 John Wiley & Sons N.Y p. 41
G. Davila-Vazquez, S. Arriaga, F. Alatriste-Mondragon, A. de Leon-Rodriguez, L.M. Rosales-Colunga, and E. Razo-Flores Fermentative biohydrogen production: trends and perspectives Rev Environ Sci Biotechnol 7 2008 27 45
D. Karakashev, S.M. Kotay, E. Trably, and I. Angelidaki A strict anaerobic extreme thermophilic hydrogen producing culture enriched from digested household waste J Appl Microbiol 106 2009 1041 1049
K. Brenner, Y. Lingchong, and F.H. Arnold Engineering microbial consortia: a new frontier in synthetic biology Trends Biotechnol 29 2008 483 489
G.L. Tang, J. Huang, Z.J. Sun, Q.Q. Tang, C.H. Yan, and G.Q. Liu Biohydrogen production from cattle wastewater by enriched anaerobic mixed consortia: influence of fermentation temperature and pH J Biosci Bioeng 106 2008 80 87
M.S. Kim, and D.Y. Lee Fermentative hydrogen production from tofu-processing waste and anaerobic digester sludge using microbial consortium Bioresour Technol 101 2010 48 52
M.J. Lee, J.H. Song, and S.J. Hwang Effects of acid pre-treatment on bio-hydrogen production and microbial communities during dark fermentation Bioresour Technol 100 2009 1491 1493
S. Ozmihci, and F. Kargi Comparison of different mixed cultures for biohydrogen-production from ground wheat starch by combined dark and light fermentation J Ind Microbiol Biotechnol 37 2010 341 347
J.J. Chang, C.H. Chou, C.Y. Ho, W.E. Chen, J.J. Lay, and C.C. Huang Syntrophic co-culture of aerobic Bacillus and anaerobic Clostridium for bio-fuels and bio-hydrogen production Int J Hydrogen Energy 33 2008 5137 5146
A.A. Zeidan, P. Rdström, and E.W. van Niel Stable coexistence of two Caldicellulosiruptor species in a de novo constructed hydrogen-producing co-culture Microb Cell Fact 9 2010 102 115
R.I. Amann, W. Ludwig, and K.H. Schleifer Phylogenetic identification and in situ detection of individual microbial cells without cultivation Microbiol Rev 59 1995 143 169
Y. Huang, W. Zong, X. Yan, R. Wang, C.L. Hemme, and J. Zhou Succession of the bacterial community and dynamics of hydrogen producers in a hydrogen-producing bioreactor Appl Environ Microbiol 76 2010 3387 3390
M.I. Van Dyke, and A.J. McCarthy Molecular biological detection and characterization of Clostridium populations in municipal landfill sites Appl Environ Microbiol 68 2002 2049 2053 (Pubitemid 34280020)
M. Quéméneur, J. Hamelin, E. Latrille, J.P. Steyer, and E. Trably Development and application of a functional CE-SSCP fingerprinting method based on [Fe-Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures Int J Hydrogen Energy 35 2010 13158 13167
M.T. Suzuki, and S.J. Giovannoni Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR Appl Environ Microbiol 62 1996 625 630 (Pubitemid 26047427)
A. Jousset, E. Lara, M. Nikolausz, H. Harms, and A. Chatzinotas Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil Sci Total Environ 408 2010 1221 1225
P.C. Burrell, C. O'Sullivan, H. Song, W.P. Clarke, and L.L. Blackall Identification, detection and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor Appl Environ Microbiol 70 2004 2414 2419 (Pubitemid 38500546)
C.J. Smith, D.B. Nedwell, L.F. Dong, and A.M. Osborn Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples Environ Microbiol 8 2006 804 815
A.E. Rasolofo, D. St-Gelais, G. LaPointe, and D. Roy Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk Int J Food Microbiol 138 2010 108 118
K.E.S. Tolvanen, P.E.P. Koskinen, A.I. Ylikoski, P.K. Ollikka, I.A. Hemmila, and J.A. Puhakka Quantitative monitoring of hydrogen-producing Clostridium butyricum strain from a continuous-flow, mixed culture bioreactor employing real-time PCR Int J Hydrogen Energy 33 2008 542 549 (Pubitemid 351210664)
J. Masset, S. Hiligsmann, C. Hamilton, L. Beckers, F. Franck, and P. Thonart Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009 Int J Hydrogen Energy 35 2010 3371 3378
J.E. Hobbie, R.H. Daley, and S. Jasper Use of nucleopore filters for counting bacteria by fluorescence microscopy Appl Environ Microbiol 33 1977 1225 1228 (Pubitemid 8108362)
S.A. Bustin, V. Benes, J.A. Garson, J. Hellemans, J. Huggett, and M. Kubista The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments Clin Chem 55 2009 611 622
S. Rozen, and H.J. Skaletsky Primer3 on the WWW for general users and for biologist programmers S. Krawetz, S. Misener, Bioinformatics methods and protocols: methods in molecular biology 2000 Humana Press Totowa, NJ 365 386
R.I. Amann, B.J. Binder, R.J. Olson, S.W. Chrisholm, R. Devereux, and D.A. Stahl Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations Appl Environ Microbiol 56 1990 1919 1925 (Pubitemid 20202165)
W. Ludwig, O. Strunk, R. Westram, L. Richter, H. Meier, and A. Yadhukumar ARB: a software environment for sequence data Nucleic Acids Res 25 2004 1363 1371 (Pubitemid 38854737)
J.R. Cole, B. Chai, R.J. Farris, Q. Wang, S.A. Kulam, and D.M. McGarrell The ribosomal database project (RDP-II): sequences and tools for high-throughput rRNA analysis Nucleic Acids Res 1 2005 294 296
A. Loy, R. Arnold, P. Tischler, T. Rattei, M. Wagner, and M. Horn ProbeCheck - a central resource for evaluating oligonucleotide probe coverage and specificity Environ Microbiol 10 2008 2894 2896
C. Lay, M. Sutren, V. Rochet, K. Saunier, J. Dore, and L. Rigottier-Gois Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota Environ Microbiol 7 2005 933 946 (Pubitemid 40817151)
B.M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes Appl Environ Microbiol 64 1998 4973 4982 (Pubitemid 28557868)
L. Monteiro, D. Bonnemaison, A. Vekris, K.G. Petry, J. Bonnet, and R. Vidal Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model J Clin Microbiol 35 1997 995 998 (Pubitemid 27146555)
A.G. Darvill, M.A. Hall, J.P. Fish, and J.G. Morris The intracellular reserve polysaccharide of Clostridium pasteurianum Can J Microbiol 23 1977 947 953 (Pubitemid 8177426)
F. Ausubel, R. Brent, R. Kingston, J. Seidman, J. Smith, and K. Struhl Current protocols in molecular biology Current protocols vol. I 1994 John Wiley and Sons, Inc. Supplement 27, Chapter II, p. 2.4.1-2.4.2
A. Clerc, C. Manceau, and X. Nesme Comparison of randomly amplified polymorphic DNA with amplified fragment length polymorphism to assess genetic diversity and genetic relatedness within genospecies III of Pseudomonas syringae Appl Environ Microbiol 64 1998 1180 1187 (Pubitemid 28188369)
D. Gevers, G. Huys, and J. Swings Applicability of rep-PCR fingerprinting for identification of Lactobacillus species FEMS Microbiol Lett 205 2001 31 36 (Pubitemid 33135435)
S.A. Bustin, V. Benes, T. Nolan, and M.W. Pfaffl Quantitative real-time RT-PCR - a perspective J Mol Endocrinol 34 2005 597 601 (Pubitemid 40903586)
U. Böckelmann, W. Manz, T. Neu, and U. Szewzyk Characterization of the microbial community of lotic organic aggregates ("river snow") in the Elbe River of Germany by cultivation and molecular methods FEMS Microbiol Ecol 33 2006 157 170
B.M. Fuchs, F.O. Glockner, J. Wulf, and R. Amann Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes Appl Environ Microbiol 66 2000 3603 3607 (Pubitemid 30624603)
J.J. McDevitt, P.S. Lees, W.G. Merz, and K.J. Schwab Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples Mycopathologia 158 2004 325 335 (Pubitemid 40167442)
L. Masco, T. Vanhoutte, R. Temmerman, and J. Swings Huys G.Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products Int J Food Microbiol 113 2007 351 357 (Pubitemid 46123637)
C.H. Hung, C.H. Cheng, L.H. Chenga, C.M. Lianga, and C.Y. Linb Application of Clostridium-specific PCR primers on the analysis of dark fermentation hydrogen-producing bacterial community Int J Hydrogen Energy 33 2008 1586 1592
M. Calusinska, T. Happe, B. Joris, and A. Wilmotte The surprising diversity of clostridial hydrogenases: a comparative genomic perspective Microbiology-SGM 156 2010 1575 1588
D.M. Stevenson, R.E. Muck, K.J. Shinners, and P.J. Weimer Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover Appl Microbiol Biotechnol 71 2006 329 338 (Pubitemid 43962138)
P.E. Saikaly, and M.A. Barlaz de los Reyes FL. Development of quantitative real-time PCR assays for detection and quantification of surrogate biological warfare agents in building debris and leachate Appl Environ Microbiol 73 2007 6557 6565 (Pubitemid 47623715)
M. Waleron, K. Waleron, K. Geider, and E. Lojkowska Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan Eur J Plant Pathol 121 2008 161 172
E. Malinen, A. Kassinen, T. Rinttila, and A. Palva Comparison of real-time PCR with SYBR green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria Microbiology-SGM 149 2003 269 277 (Pubitemid 36175697)
Y.Z. Tang, K.Y.H. Gin, and T.H. Lim High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry Appl Environ Microbiol 71 2005 8157 8164 (Pubitemid 41803940)
M. Durm, F.M. Haar, M. Hausmann, H. Ludwig, and C. Cremer Optimization of fast-fluorescence in situ hybridization with repetitive alphasatellite probes Z Naturforsch C 51 1996 253 261
C.C. da Silva, and A.D. da Cruz An easy procedure for cytogenetic analysis of aged chromosome preparation using FISH-WCP probes Chromosome Res 10 2002 233 238 (Pubitemid 34553497)
A. Schippers, A.A. Nagy, D. Kock, F. Melcher, and E.D. Gock The use of FISH and real-time PCR to monitor the biooxidation and cyanidation for gold and silver recovery from a mine tailings concentrate (Ticapampa, Peru) Hydrometallurgy 94 2008 77 81