Microbial characterisation of polyhydroxyalkanoates storing populations selected under different operating conditions using a cell-sorting RT-PCR approach
Tóm tắt
The identity of polyhydroxyalkanoates (PHA) storing bacteria selected under aerobic dynamic feeding conditions, using propionate as carbon source (reactor P), was determined by applying reverse transcriptase–polymerase chain reaction (RT-PCR) on micromanipulated cells and confirmed by fluorescence in situ hybridisation (FISH). Four genera, Amaricoccus, Azoarcus, Thauera and Paraccoccus were detected, the latter only rarely present. All the biomass was involved in PHA storage as shown by Nile Blue staining. By quantitative FISH, their specific amount was determined in this and two other systems using acetate as the carbon substrate (sequencing batch reactor [SBR] A and A1). SBR A and reactor P had the same sludge retention time (SRT, 10 days), while reactor A1 was operated with the SRT of 1 day and the double organic loading rate (OLR). Systems fed with acetate (41.1 ± 2.2 and 49.4 ± 1.4% total Bacteria, for A and A1, respectively) became enriched in Thauera independently on the SRT and OLR, while it was only present in a minor amount when propionate was used as a substrate (1.9 ± 0.2% total Bacteria). Amaricoccus was present in both reactors operated at 10 days SRT, favoured in the one fed with propionate (61.4 ± 1.9% total bacteria), and almost completely removed at the SRT of 1 day. Azoarcus cells were found in all the analysed systems (3.9 ± 0.3, 23.3 ± 1.5 and 45.9 ± 1.5 for P, A and A1, respectively), while Paracoccus was scarcely present.
Tài liệu tham khảo
Amann RI (1995) In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes. In: Akkermans A, van Elsas J, de Bruijn F (eds) Molecular microbial ecology manual. Kluwer, London, pp MMEM–3.3.6/1–MMEM-3.3.6/15
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
APHA, AWWA, WPCF (1995) Standard methods for the examination of water and wastewater. Port City Press, Baltimore (edited by Eaton AD, Clesceri LS, Greenberg AE)
Aulenta F, Dionisi D, Majone M, Parisi A, Ramadori R, Tandoi V (2003) Effect of periodic feeding in SBR reactor on substrate uptake and storage rates by a pure culture of Amaricoccus kaplicensis. Water Res 37:2764–27
Beccari M, Majone M, Massanisso P, Ramadori R (1998) A bulking sludge with high storage response selected under intermittent feeding. Water Res 32:3403–3413
Bergey’s Manual of Systematic Bacteriology Volume Two: the Proteobacteria (Part C): the Alpha-, Beta-, Delta-, and Epsilonproteobacteria (2005) Garrity G, Brenner DJ, Krieg NR, Staley JT (eds) 2nd edn. Williams and Wilkins, Springer
Beun JJ, Dircks K, van Loosdrecht MCM, Heijnen JJ (2002) Poly-β-hydroxybutyrate metabolism in dynamically fed mixed cultures. Water Res 36:1167–1180
Bouchez T, Patureau D, Dabert P, Wagner M, Delgenes JP, Moletta R (2000) Successful and unsuccessful bioaugmentation experiments monitored by fluorescent in situ hybridization. Water Sci Technol 41:61–68
Cho J, Song KJ, Yun H, Ahn KH, Kim JY, Chung TH (2005) Quantitative analysis of biological effect on membrane fouling in submerged membrane bioreactor. Water Sci Technol 51(6–7):9–18
Crocetti GR, Banfield JF, Keller J, Bond PL, Blackall LL (2002) Glycogen-accumulating organisms in laboratory-scale and full-scale wastewater treatment processes. Microbiology 148:3353–3364
Daims H, Bruehl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444
Dionisi D, Majone M, Papa V, Beccari M (2004) Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding. Biotechnol Bioeng 85:569–579
Dionisi D, Beccari M, Di Gregorio S, Majone M, Petrangeli Papini M, Vallini G (2005) Storage of biodegradable polymers by an enriched microbial community in a sequencing batch reactor operated at high organic load rate. J Chem Technol Biotechnol 80:1306–1318
Dionisi D, Majone M, Vallini G, Di Gregorio S, Beccari M (2006) Effect of applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactor. Biotechnol Bioeng 93:76–88
Direction Générale des Ressources Naturelles et de l’Environnement du Ministère de la Région du Valoni (2002) Technologies propres, no. 2. Kluwer, Dordrecht
Falvo A, Levantesi C, Rossetti S, Seviour RJ, Tandoi V (2001) Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrads forming bacterium present in activated sludge. J Appl Microbiol 91:299–305
Frølund B, Palmgren R, Keiding K, Nielsen PH (1996) Extraction of exopolymers from activated sludge using a cation exchange resin. Water Res 30(8):1749–1758
Hess A, Zarda B, Hahn D, Haner A, Stax D, Hohener P, Zeyer J (1997) In situ analysis of denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column. Appl Environ Microbiol 63:2136–2141
Jenkins D, Richards MG, Daigger GT (1993) Manual on the causes and control of activated sludge bulking and foaming, 2nd edn. Lewis, London
Juretschko S, Loy A, Lehner A, Wagner M (2002) The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach. Syst Appl Microbiol 25:84–99
Lemos PC, Serafim LS, Reis MAM (2006) Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding. J Biotechnol 122:226–238
Levantesi C, Rossetti S, Beimfohr C, Thelen K, Krooneman J, van der Waarde J, Tandoi V (2006) Description of filamentous bacteria present in industrial activated sludge WWTPs by conventional and molecular methods. Water Sci Technol 54:129–137
Loy A, Horn M, Wagner M (2003) probeBase—an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 3:514–516
Loy A, Schulz C, Lücker S, Schöpfer-Wendels A, Stoecker K, Baranyi C, Lehner A, Wagner M (2005) 16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order Rhodocyclales. Appl Environ Microbiol 71:1373–1386
Majone M, Massanisso P, Carucci A, Lindrea K, Tandoi V (1996) Influence of storage on kinetic selection to control aerobic filamentous bulking. Water Sci Technol 34:223–232
Manz W, Amann R, Ludwig W, Wagner M, Schleifer K-H (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600
Marchessault RH (1996) Tender morsels for bacteria: recent developments in microbial polyesters. Trends Polym Sci 4:163–168
Maszenan A-M, Seviour RJ, Patel BKC, Wanner J (2000) A fluorescently-labelled r-RNA targeted oligonucleotide probe for the in situ detection of G-bacteria of the genus Amaricoccus in activated sludge. J Appl Microbiol 88:826–835
Neef A (1997) Anwendung der in situ Einzelzell-Identifizierung von Bakterien zur Populationsanalyse in komplexen mikrobiellen Biozönosen. Doctoral thesis, Technische Universität München, München
Neef A, Zaglauer A, Meier H, Amann R, Lemmer H, Schleifer K-H (1996) Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms. Appl Environ Microbiol 62:4329–4339
Ostle AG, Holt JG (1982) Nile Blue A as a fluorescent stain for poly-β-hybroxybutyrate. Appl Environ Microbiol 44:238–241
Serafim LS, Lemos PC, Oliveira RF, Reis MAM (2004) Optimisation of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions. Biotechnol Bioeng 87:145–160
Serafim LS, Lemos PC, Rossetti S, Levantesi C, Tandoi V, Reis MAM (2006) Microbial community analysis with a high PHA storage capacity. Water Sci Tech 54:183–188