Visualization of Microorganisms in Bioprocesses
Abstract
Fluorescence in situ hybridization (FISH) is currently a standard technique for detecting, identifying, and enumerating microorganisms. The method allows for visualization of cell morphology as well as in situ localization of microorganisms at single-cell resolution. Although FISH has been used for more than 20 years, many challenges remain with regard to improving the technique for understanding microbial ecology and physiology. This chapter describes the recent developments in this field, such as improved sensitivity by catalyzed reporter deposition (CARD)-FISH and in situ DNA-hybridization chain reaction (HCR). Highly sensitive methods for detecting mRNA and/or functional genes are also described. Moreover, methods combining isotope probing to reveal microbial metabolic activities are introduced.
Keywords
Microorganisms Ecology Fluorescence Metabolic activity PhylogenyReferences
- Amann RI, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6:339–348. https://doi.org/10.1038/nrmicro1888 CrossRefPubMedGoogle Scholar
- Amann RI, Ludwing W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedPubMedCentralGoogle Scholar
- Araki N, Yamaguchi T, Yamazaki S, Harada H (2004) Quantification of amoA gene abundance and their amoA mRNA levels in activated sludge by real-time PCR. Water Sci Technol 50(8):1–8PubMedGoogle Scholar
- Behrens S, Fuchs BM, Mueller F, Amann RI (2003) Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit? Appl Environ Microbiol 69:4935–4941. https://doi.org/10.1128/AEM.69.8.4935-4941.2003 CrossRefPubMedPubMedCentralGoogle Scholar
- Behrens S, Losekann T, Pett-Ridge J, Weber PK, Ng WO, Stevenson BS, Hutcheon ID, Relman DA, Spormann AM (2008) Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (ELFISH) and nanoSIMS. Appl Environ Microbiol 74:3143–3150. https://doi.org/10.1128/AEM.00191-08 CrossRefPubMedPubMedCentralGoogle Scholar
- Ben-Dov E, Brenner A, Kushmaro A (2007) Quantification of sulphate reducing bacteria in industrial wastewater by real-time polymerase chain reaction (PCR) using dsrA and apsA genes. Microb Ecol 54:439–451. https://doi.org/10.1007/s00248-007-9233-2 CrossRefPubMedGoogle Scholar
- Biswas K, Taylor NW, Jurner SJ (2014) dsrAB-based analysis of sulphate-reducing bacteria in moving bed biofilm reactor (MBBR) wastewater treatment plants. Environ Biotechnol 98:7211–7222. https://doi.org/10.1007/s00253-014-5769-5 CrossRefGoogle Scholar
- Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M (2001) In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl Environ Microbiol 67:5273–5284. https://doi.org/10.1128/AEM.67.11.5273-5284.2001 CrossRefPubMedPubMedCentralGoogle Scholar
- DeLong EF, Wickhan GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of signal cells. Science 243:1360–1363. https://doi.org/10.1126/science.2466341 CrossRefPubMedGoogle Scholar
- Dirks RM, Pierce NA (2004) Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci U S A 101:15275–15288. https://doi.org/10.1073/pnas.0407024101 CrossRefPubMedPubMedCentralGoogle Scholar
- Fernández N, Gomez R, Amils R, Sierra-Alvarez R, Field JA, Sanz JL (2006) Microbiological and structural aspects of granular sludge from autotrophic denitrifying reactors. Water Sci Technol 54:11–17. https://doi.org/10.2166/wst.2006.480 CrossRefPubMedGoogle Scholar
- Fernández N, Sierra-Alvarez R, Field JA, Amils R, Sanz JL (2008) Microbial community dynamics in a chemolithotrophic denitrification reactor inoculated with methanogenic granular sludge. Chemosphere 70:462–474. https://doi.org/10.1016/j.chemosphere.2007.06.062 CrossRefPubMedGoogle Scholar
- Hatamoto M, Miyauchi T, Kindaichi T, Ozaki N, Ohashi A (2011) Dissolved methane oxidation and competition for oxygen in down-flow hanging sponge reactor for post-treatment of anaerobic wastewater treatment. Bioresour Technol 102:10299–10304. https://doi.org/10.1016/j.biortech.2011.08.099 CrossRefPubMedGoogle Scholar
- Herbert RB, Winbjork H, Hellman M, Hallin S (2014) Nitrogen removal and spatial distribution of denitrifier and anammox communities in a bioreactor for mine drainage treatment. Water Res 66:350–360. https://doi.org/10.1016/j.watres.2014.08.038 CrossRefGoogle Scholar
- Hoshino T, Schramm A (2010) Detection of denitrification genes by in situ rolling circle amplification-fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells. Environ Microbiol 12:2508–2517. https://doi.org/10.1111/j.1462-2920.2010.02224.x CrossRefPubMedGoogle Scholar
- Hoshino T, Tsuneda S, Hirata A, Inamori Y (2003) In situ PCR for visualizing distribution of a functional gene “amoA” in a biofilm regardless of activity. J Biotechnol 105:33–40. https://doi.org/10.1016/S0168-1656(03)00171-8 CrossRefPubMedGoogle Scholar
- Hoshino T, Yilmaz LS, Noguera DR, Daims H, Wagner M (2008) Quantification of target molecules needed to detect microorganisms by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition-FISH. Appl Environ Microbiol 74:5068–5077. https://doi.org/10.1128/AEM.00208-08 CrossRefPubMedPubMedCentralGoogle Scholar
- Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ, Castelle CJ, Butterfield CN, Hernsdorf AW, Amano Y, Ise K, Suzuki Y, Dudek N, Relman DA, Finstad KM, Amundson R, Thomas BC, Banfield JF (2016) A new view of the tree of life. Nat Microbiol 2016:16048. https://doi.org/10.1038/NMICROBIOL.2016.48 CrossRefGoogle Scholar
- Iguchi A, Terada T, Narihiro T, Yamaguchi T, Kamagata Y, Sekiguchi Y (2009) In situ detection and quantification of uncultured members of the phylum Nitrospirae abundant in methanogenic wastewater treatment systems. Microbes Environ 24:97–104. https://doi.org/10.1264/jsme2.ME08562 CrossRefPubMedGoogle Scholar
- Imachi H, Sekiguchi Y, Kamagata Y, Ohashi A, Harada H (2000) Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl Environ Microbiol 66:3608–3615. https://doi.org/10.1128/AEM.66.8.3608-3615.2000 CrossRefPubMedPubMedCentralGoogle Scholar
- Ishii K, Mußmann M, MacGregor BJ, Amann RI (2004) An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiol Ecol 50:203–212. https://doi.org/10.1016/j.femsec.2004.06.015 CrossRefPubMedGoogle Scholar
- Kawakami S, Kubota K, Imachi H, Yamaguchi T, Harada H, Ohashi A (2010) Detection of single copy genes by two-pass tyramide signal amplification fluorescence in situ hybridization (two-pass TSA-FISH) with single oligonucleotide probes. Microbes Environ 25:15–21. https://doi.org/10.1264/jsme2.ME09180 CrossRefPubMedGoogle Scholar
- Kawakami S, Hasegawa T, Imachi H, Yamaguchi T, Harada H, Ohashi A, Kubota K (2012) Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes. J Microbiol Methods 88:218–223. https://doi.org/10.1016/j.mimet.2011.11.014 CrossRefPubMedGoogle Scholar
- Kim YM, Cho HU, Lee DS, Park D, Park JM (2011) Influence of operational parameters on nitrogen removal efficiency and microbial communities in a full-scale activated sludge process. Water Res 45:5785–5795. https://doi.org/10.1016/j.watres.2011.08.063 CrossRefPubMedGoogle Scholar
- Kindaichi T, Ito T, Okabe S (2004) Eco-physiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by MAR-FISH. Appl Environ Microbiol 70:1641–1650. https://doi.org/10.1128/AEM.70.3.1641-1650.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- Kubota K (2013) CARD-FISH for environmental microorganisms: technical advancement and future applications. Microbes Environ 28:3–12. https://doi.org/10.1264/jsme2.ME12107 CrossRefPubMedGoogle Scholar
- Kubota K, Ohashi A, Imachi H, Harada H (2006) Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH). J Microbiol Methods 66:521–528. https://doi.org/10.1016/j.mimet.2006.02.002 CrossRefPubMedGoogle 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 72:54–59. https://doi.org/10.1016/j.mimet.2007.10.006 CrossRefPubMedGoogle Scholar
- Kubota K, Morono Y, Ito M, Terada T, Itezono S, Harada H, Inagaki F (2014) Gold-ISH: a nano-size gold particle-based phylogenetic identification compatible with NanoSIMS. Syst Appl Microbiol 37:261–266. https://doi.org/10.1016/j.syapm.2014.02.003 CrossRefPubMedGoogle Scholar
- Limpiyakorn T, Sonthiphand P, Rongsayamanont C, Polprasert C (2010) Abundance of amoA genes of ammonia-oxidizing archaea and bacteria in activated sludge of full-scale wastewater treatment plants. Bioresour Technol 102:3694–3701. https://doi.org/10.1016/j.biortech.2010.11.085 CrossRefPubMedGoogle Scholar
- Loy A, Horn M, Wagner M (2003) probeBase: an online resource for rRNA targeted oligonucleotide probes. Nucleic Acids Res 31:514–516. https://doi.org/10.1093/nar/gkg016 CrossRefPubMedPubMedCentralGoogle Scholar
- Ludwig W, Strunk O, Westram R, Richter L, Meier H, Buchner YA, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371. https://doi.org/10.1093/nar/gkh293 CrossRefPubMedPubMedCentralGoogle Scholar
- Luesken FA, Zhu B, van Alen TA, Butler MK, Rodriguez DM, Song B, Op den Camp HJM, Jetten MSM, Ettwig KF (2011) pmoA primers for detection of anaerobic methanotrophs. Appl Environ Microbiol 77:3877–3880. https://doi.org/10.1128/AEM.02960-10 CrossRefPubMedPubMedCentralGoogle Scholar
- Luo JF, Lin WT, Guo Y (2011) Functional gene based analysis of sulphur oxidizing bacteria community in sulfide removing bioreactor. Appl Microbiol Biotechnol 90:769–778. https://doi.org/10.1007/s00253-010-3061-x CrossRefPubMedGoogle Scholar
- Moraru C, Lam P, Fuchs BM, Kuypers MMM, Amann RI (2010) GeneFISH-an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environ Microbiol 12:3057–3073. https://doi.org/10.1111/j.1462-2920.2010.02281.x CrossRefPubMedGoogle Scholar
- Mota CR, So MJ, de los Reyes FL (2012) Identification of nitrite-reducing bacteria using sequential mRNA fluorescence in situ hybridization and fluorescence-assisted cell sorting. Microb Ecol 64:256–267. https://doi.org/10.1007/s00248-012-0018-x CrossRefPubMedGoogle Scholar
- Musat N, Halma H, Winterhollerb B, Hoppe P, Peduzzi S, Hillion F, Horreard F, Amann RI, Jørgensen BB, Kuypers MM (2008) A single-cell view on the ecophysiology of anaerobic phototrophic bacteria. Proc Natl Acad Sci U S A 105:17861–17866. https://doi.org/10.1073/pnas.0809329105 CrossRefPubMedPubMedCentralGoogle Scholar
- Nguyen HTT, Le VQ, Hansen AA, Nielsen JL, Nielsen PH (2011) High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems. FEMS Microbiol Ecol 76:256–267. https://doi.org/10.1111/j.1574-6941.2011.01049.x CrossRefPubMedGoogle Scholar
- Nobu MK, Tamaki H, Kubota K, Liu WT (2014) Metagenomic characterization of ‘Candidatus Defluviicoccustetraformis strain TFO71’, a tetradforming organism, predominant in an anaerobic-aerobic membrane bioreactor with deteriorated biological phosphorus removal. Environ Microbiol 16:2739–2751. https://doi.org/10.1111/1462-2920.12383 CrossRefPubMedGoogle Scholar
- Nobu MK, Narihiro T, Rinke C, Kamagata Y, Tringe SG, Woyke T, Liu WT (2015) Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor. ISME J 9:1710–1722. https://doi.org/10.1038/ismej.2014.256 CrossRefPubMedPubMedCentralGoogle Scholar
- Okabe S, Kindaichi T, Ito T (2004) MAR–FISH—an ecophysiological approach to link phylogenetic affiliation and in situ metabolic activity of microorganisms at a single-cell resolution. Microbes Environ 19:83–98. https://doi.org/10.1264/jsme2.19.83 CrossRefGoogle Scholar
- Pavlekovic M, Schmid MC, Schmider-Poignee N, Spring S, Pilhofer M, Gaul T, Fiandaca M, Löffler FE, Jetten M, Schleifer KH, Lee NM (2009) Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment. J Microbiol Methods 78:119–126. https://doi.org/10.1016/j.mimet.2009.04.003 CrossRefPubMedGoogle Scholar
- Pernthaler A, Amann RI (2004) Simultaneous fluorescence in situ hybridization of mRNA and rRNAin environmental bacteria. Appl Environ Microbiol 70:5426–5433. https://doi.org/10.1128/AEM.70.9.5426-5433.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- Pratscher J, Stichternoth C, Fichtl K, Schleifer K-H, Braker G (2009) Application of recognition of individual genes-fluorescence in situ hybridization (RING-FISH) to detect nitrite reductase genes (nirK) of denitrifiers in pure cultures and environmental samples. Appl Environ Microbiol 75:802–810. https://doi.org/10.1128/AEM.01992-08 CrossRefPubMedGoogle Scholar
- Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ, Cheng J-F, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund BP, Tsiamis G, Sievert SM, Liu WT, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499:431–437. https://doi.org/10.1038/nature12352 CrossRefPubMedGoogle Scholar
- Saito Y, Aoki M, Hatamoto M, Yamaguchi T, Takai K, Imachi H (2015) Presence of a novel methanogenic archaeal lineage in anaerobic digesters inferred from mcrA and 16S rRNA gene phylogenetic analyses. J Water Environ Technol 13:279–289. https://doi.org/10.2965/jwet.2015.279 CrossRefGoogle Scholar
- Schönhuber W, Fuchs B, Juretschko S, Amann RI (1997) Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl Environ Microbiol 63:3268–3273PubMedPubMedCentralGoogle Scholar
- Sekiguchi Y (2006) Yet-to-be cultured microorganisms relevant to methane fermentation processes. Microbes Environ 21:1–15. https://doi.org/10.1264/jsme2.21.1 CrossRefGoogle Scholar
- Sekiguchi Y, Kamagata Y, Nakamura K, Ohashi A, Harada H (1999) Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules. Appl Environ Microbiol 65:1280–1288PubMedPubMedCentralGoogle Scholar
- Sekiguchi Y, Takahashi H, Kamagata Y, Ohashi A, Harada H (2001) In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green nonsulfur bacteria, subdivision I. Appl Environ Microbiol 67:5740–5749. https://doi.org/10.1128/AEM.67.12.5740-5749.2001 CrossRefPubMedPubMedCentralGoogle Scholar
- Steinberg LM, Regan JM (2008) Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge. Appl Environ Microbiol 74:6663–6671. https://doi.org/10.1128/AEM.00553-08 CrossRefPubMedPubMedCentralGoogle Scholar
- Sumino H, Takahashi M, Yamaguchi T, Abe K, Araki N, Yamazaki S, Shimozaki S, Nagano A, Nishio N (2007) Feasibility study of a pilot-scale sewage treatment system combining an up-flow anaerobic sludge blanket (UASB) and an aerated fixed bed (AFB) reactor at ambient temperature. Bioresour Technol 98:177–182. https://doi.org/10.1016/j.biortech.2005.10.020 CrossRefPubMedGoogle Scholar
- Thiele S, Fuchs BM, Amann RI (2011) Identification of microorganisms using the ribosomal RNA approach and fluorescence in situ hybridization. In: Wilderer P (ed) Treatise on water science. Elsevier, Oxford. ISBN: 0444531998, pp 171–189. https://doi.org/10.1016/B978-0-444-53199-5.00056-7 CrossRefGoogle Scholar
- Yamada T, Sekiguchi Y, Imachi H, Kamagata Y, Ohashi A, Harada H (2005) Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules. Appl Environ Microbiol 71:7493–7503. https://doi.org/10.1128/AEM.71.11.7493-7503.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- Yamada T, Yamauchi T, Shiraishi K, Hugenholtz P, Ohashi A, Harada H, Kamagata Y, Nakamura K, Sekiguchi Y (2007) Characterization of filamentous bacteria, belonging to candidate phylum KSB3, that are associated with bulking in methanogenic granular sludges. ISME J 1:246–264. https://doi.org/10.1038/ismej.2007.28 CrossRefPubMedGoogle Scholar
- Yamada T, Kikuchi K, Yamauchi T, Shiraishi K, Ito T, Okabe S, Hiraishi A, Ohashi A, Harada H, Kamagata Y, Nakamura K, Sekiguchi Y (2011) Ecophysiology of uncultured filamentous anaerobes belonging to the phylum KSB3 that cause bulking in methanogenic granular sludge. Appl Environ Microbiol 77:2081–2087. https://doi.org/10.1128/AEM.02475-10 CrossRefPubMedPubMedCentralGoogle Scholar
- Yamaguchi T, Fuchs BM, Amann RI, Kawakami S, Kubota K, Hatamoto M, Yamaguchi T (2015a) Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH). Syst Appl Microbiol 38:400–405. https://doi.org/10.1016/j.syapm.2015.06.007 CrossRefPubMedGoogle Scholar
- Yamaguchi T, Kawakami S, Hatamoto M, Imachi H, Takahashi M, Araki N, Yamaguchi T, Kubota K (2015b) In situ DNA-HCR: a facilitated in situ hybridization chain reaction system for the detection of environmental microorganisms. Environ Microbiol 17:2532–2541. https://doi.org/10.1111/1462-2920.12745 CrossRefPubMedGoogle Scholar
- Yilmaz LS, Noguera DR (2004) Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization. Appl Environ Microbiol 70:7126–7139. https://doi.org/10.1128/AEM.70.12.7126-7139.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- Yilmaz LS, Okten HE, Noguera DR (2006) Making all parts of the 16S rRNA of Escherichia coli accessible in situ to single DNA oligonucleotides. Appl Environ Microbiol 72:733–744. https://doi.org/10.1128/AEM.72.1.733-744.2006 CrossRefPubMedPubMedCentralGoogle Scholar
- Yilmaz LS, Parnerkar S, Noguera DR (2011) mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol 77:1118–1122. https://doi.org/10.1128/AEM.01733-10 CrossRefPubMedGoogle Scholar
- You SJ (2005) Identification of denitrifying bacteria diversity in an activated sludge system by using nitrite reductase genes. Biotechnol Lett 27:1477–1482. https://doi.org/10.1007/s10529-005-1314-z CrossRefPubMedGoogle Scholar
- Zwirglmaier K, Ludwig W, Schleifer KH (2004) Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization-RINGFISH. Mol Microbiol 51:89–96. https://doi.org/10.1046/j.1365-2958.2003.03834.x CrossRefPubMedGoogle Scholar