FISH Handbook for Biological Wastewater Treatment: Identification and Quantification of Microorganisms in Activated Sludge and Biofilms by FISH

137 Pages·2009·8.3 MB·English

by  Per Halkjaer Nielsen, Holger Daims, Hilde Lemmer

FISH Handbook for Biological
Wastewater Treatment

FISH Handbook for Biological
Wastewater Treatment
Identification and quantification of microorganisms in
activated sludge and biofilms by FISH
Edited by
Per Halkjær Nielsen, Holger Daims and Hilde Lemmer
Publishedby IWAPublishing
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ISBN10:1843392316
ISBN13:9781843392316
Table of contents
List of contributors ................................................................................................................... ix
Abbreviations ............................................................................................................................... xi
1 INTRODUCTION .................................................................................................................. 1
1.1 Identification of microorganisms in activated sludge and biofilms ................. 1
1.2 The microbiology of biological wastewater treatment ....................................... 2
1.3 Factors of importance for the growth of microorganisms ................................. 4
1.4 The use of this FISH handbook ............................................................................. 6
2 THE NITRIFYING MICROBES: AMMONIA OXIDIZERS, NITRITE OXIDIZERS,
AND ANAEROBIC AMMONIUM OXIDIZERS .................................................................... 9
2.1 Introduction ............................................................................................................... 9
2.2 Ammonia oxidizers .................................................................................................. 10
2.2.1 Probes for the detection of AOB ................................................................. 11
2.3 Nitrite oxidizers ........................................................................................................ 13
2.3.1 Probes for the detection of NOB ................................................................. 14
2.4 Anammox bacteria ................................................................................................... 14
2.4.1 Probes for the detection of anammox organisms ....................................... 17
3 IDENTIFICATION OF DENITRIFYING MICROORGANISMS IN ACTIVATED
SLUDGE BY FISH .............................................................................................................. 19
3.1 Introduction ............................................................................................................... 19
3.2 Identity of denitrifiers in wastewater treatment systems ................................... 20
3.3 Abundant denitrifiers in full-scale plants ............................................................. 20
3.4 Probes for detection of denitrifiers ....................................................................... 21
4 IDENTIFICATION OF POLYPHOSPHATE-ACCUMULATING AND
GLYCOGEN-ACCUMULATING ORGANISMS BY FISH .................................................. 25
4.1 Introduction ............................................................................................................... 25
4.2 Identity of PAOs ....................................................................................................... 26
4.2.1 Probes for detection of PAOs ...................................................................... 28
vi FISH Handbook for Biological Wastewater Treatment
4.3 Identity of GAOs ....................................................................................................... 28
4.3.1 Probes for detection of GAOs ..................................................................... 29
5 IDENTIFICATION OF FILAMENTOUS BACTERIA BY FISH .......................................... 33
5.1 Introduction ............................................................................................................... 33
5.2 FISH detection offilamentous bacteria ................................................................. 36
5.3 Filamentous bacteria detection and identification .............................................. 38
5.3.1 Beggiatoa morphotype ................................................................................. 41
5.3.2 Haliscomenobacter hydrossis morphotype ................................................. 41
5.3.3 Leucothrix mucor morphotype ..................................................................... 45
5.3.4 Microthrix parvicella morphotype ................................................................. 45
5.3.5 Nostocoida limicola morphotypes ................................................................ 48
5.3.6 Nocardioform actinomycetes/Mycolata morphotype ................................... 52
5.3.7 Sphaerotilus natans and Leptothrix discophora morphotype ..................... 56
5.3.8 Streptococcus morphotype .......................................................................... 57
5.3.9 Thiothrix and Type 021N morphotypes ....................................................... 57
5.3.10 0041/0675 morphotype ................................................................................ 59
5.3.11 0092 morphotype ......................................................................................... 61
5.3.12 1701 morphotype ......................................................................................... 63
5.3.13 1851 morphotype ......................................................................................... 65
5.3.14 1863 morphotype ......................................................................................... 65
5.3.15 0803, 0914 morphotypes and other still unidentified
filamentous species ..................................................................................... 67
6 IDENTIFICATION OF OTHER MICROORGANISMS IN ACTIVATED SLUDGE AND
BIOFILMS BY FISH ............................................................................................................ 69
6.1 Introduction ............................................................................................................... 69
6.2 Epiphytic bacteria involved in protein hydrolysis ............................................... 69
6.3 Sulfate-reducing bacteria ........................................................................................ 70
6.4 Fermenting bacteria ................................................................................................. 70
6.5 Escherichia coli as indicator organism for entero-pathogens .......................... 70
7 PROTOCOL FOR FLUORESCENCE IN SITU HYBRIDIZATION (FISH) WITH
rRNA-TARGETED OLIGONUCLEOTIDES ....................................................................... 73
7.1 Introduction ............................................................................................................... 73
7.2 FISH protocol ............................................................................................................ 75
7.2.1 Materials and solutions ................................................................................ 75
7.2.2 Equipment and supplies needed for FISH .................................................. 76
7.3 Protocol ..................................................................................................................... 76
7.3.1 Sample collection and fixation ..................................................................... 76
7.3.2 Sample preparation ...................................................................................... 77
7.3.3 Immobilization of the samples on glass slides ........................................... 77
7.3.4 Dehydration .................................................................................................. 78
7.3.5 Permeabilization by enzymatic or chemical treatment ............................... 78
7.3.6 Preparation and quality check of probes .................................................... 79
Table of contents vii
7.3.7 Hybridization ................................................................................................. 79
7.3.8 Counterstaining with DAPI ........................................................................... 81
7.4 Microscopy ............................................................................................................... 81
7.5 Recommendations and troubleshooting .............................................................. 81
8 QUANTITATIVE FISH FOR THE CULTIVATION-INDEPENDENT QUANTIFICATION
OF MICROBES IN WASTEWATER TREATMENT PLANTS ........................................... 85
8.1 Introduction ............................................................................................................... 85
8.2 Quantitative FISH: A brief overview ...................................................................... 86
8.3 A protocol for quantitative FISH and image analysis to measure
biovolume fractions ................................................................................................. 88
8.4 Concluding remarks ................................................................................................ 91
9 COLOR IMAGE SECTION ................................................................................................. 93
10 REFERENCE LIST ............................................................................................................ 109
Index .......................................................................................................................................... 121

List of contributors
Editors:
Professor Per Halkjær Nielsen
Department of Biotechnology, Chemistry and Environmental Engineering,
Aalborg University, Sohngaardsholmsvej 49,
DK-9000 Aalborg, Denmark
Fax: [phone]
Phone: [phone]
Email: [email protected]
Dr. Hilde Lemmer
Bavarian Environment Agency, Kaulbachstr. 37,
D-80539 Munich, Germany
Phone: þ49 89 2180 2783
Fax: þ49 89 2800838
Email: [email protected]
Dr. Holger Daims
Department of Microbial Ecology
Vienna Ecology Centre
University of Vienna, Althanstrasse 14
A-1090 Vienna, Austria
Phone: þ43 1 4277 54392
Fax: þ43 1 4277 54389
Email: [email protected]
Other contributors:
Dr. Aviaja A. Hansen
Department of Biotechnology, Chemistry and Environmental Engineering,
Aalborg University, Sohngaardsholmsvej 49,
DK-9000 Aalborg, Denmark
Email: [email protected]
Dr. Caroline Kragelund
Department of Biotechnology, Chemistry and Environmental Engineering,
Aalborg University, Sohngaardsholmsvej 49,
DK-9000 Aalborg, Denmark
Email: [email protected]
#2009IWAPublishing.FISHHandbookforBiologicalWastewaterTreatment:Identificationandquantificationofmicroorganisms
inactivatedsludgeandbiofilmsbyFISH.EditedbyPerHalkjærNielsen,HolgerDaimsandHildeLemmer.ISBN:9781843392316.
PublishedbyIWAPublishing,London,UK.