Effects of temperature, soil ammonium concentration and fertilizer on activity and community structure of ammonia oxidizers [Elektronische Ressource] / by Sharon Avrahami

Sharon

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2003
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Effects of temperature, soil ammonium
concentration and fertilizer on activity and
community structure of ammonia oxidizers


Doctoral thesis for the fulfilment of the grade of Doctor (Dr. rer. nat)
of the Philipps University of Marburg



Submitted to the faculty of Biology
of the Philipps University of Marburg



by Sharon Avrahami
from Jerusalem, Israel





Marburg/Lahn 2002






















Dedicated to my family



The present work was carried out between January 2000 to December 2002 at the Max-
Planck-Institute for Terrestrial Microbiology, Marburg, Germany under the supervision of
Prof. Dr. Ralf Conrad.



















By the Biology department of the Philipps University, Marburg as doctoral thesis accepted
on:

First reviewer: Prof. Dr. Ralf Conrad
Second reviewer: Prof. Dr. Wolfgang Buckel

Date of oral examination: The following papers were published by the date of submission of the present thesis:

1. Avrahami, S., G. Braker and R. Conrad. 2002. Effect of soil ammonium concentration
and temperature on N O release and the community structure of ammonia oxidizers and 2
denitrifiers. Applied Environmental Microbiology. 68: 5685-5692

2. Avrahami, S., W. Liesack, and R. Conrad. Effects of temperature and fertilizer on
activity and community structure of soil ammonia oxidizers. submitted.

3. Avrahami, S. and R. Conrad. Long-term effect of temperature on community structure
of ammonia oxidizers in different meadow soils. In preparation.

















Table of contents
Abbreviations
I. Introduction 1-23
1. The autotrophic ammonia oxidizers 3-5
2. Temperature effect 5-6
3. Ammonia oxidizers under anaerobic conditions 7
4. Other nitrifying bacteria 7-8
5. Phylogenetic affiliation 9
6. Culture methods for studying the ammonia oxidizer community 9-10
in natural environments
7. The molecular approach and 16S rRNA gene analysis 10-12
8. The ammonia monooxygenase (AMO) as a molecular marker 12-14
9. Community structure of ammonia oxidizers in different natural 14-16
environments
10. Community structure of ammonia oxidizers in different soils 16-19
11. Nitrifiers as trace gas producers 19-21
Objectives of the study 22-23
II. Material and Methods
1. Material used in this study 24-25
2. Soil samples 25-26
3. Ammonium measurements 26-27
4. Measurement of pH, gravimetric water and water holding capacity 27
5. Experiment’s set up 28
5.1. Short-term incubations 28
5.2. Long- 28-31
6. Molecular analysis 31
6.1. DNA Extraction 31-32
6.2. Test of primers 32-33
6.3. PCR amplification of amoA 33-34
6.4. DGGE and cloning 34-36 6.5. Sequencing 36
6.6. Phylogenetic analysis 36
6.7. Nucleotide sequence accession numbers available 37
6.8. Correspondence analysis 37
III. Chapter 1 - 39-45
Optimizing the amoA PCR system to study the community structure of
ammonia oxidizers using denaturing gradient gel electrophoresis (DGGE)
Results 39-44
Discussion 45
IV. Chapter 2 - 46-63
Effects of soil ammonium concentration, temperature, and fertilizer on activity
and community structure of soil ammonia oxidizers
Results 47-59
1. Short-term effect of ammonium soil concentration on N O release 2 47
2. Effect of temperature soil on nitrification activity 48-50
3. Short-term effect of ammonium soil concentration on community 51-53
structure
4. Long-term effect of temperature on community structure of ammonia 53-59
oxidizers
Discussion 60-63
V. Chapter 3 - 64-84
Long-term effect of temperature on community structure of ammonia oxidizers
in different meadow soils
Results 65-79
1. Ammonium measurements 65
2. Potential nitrification activity 66
3. Molecular analysis of environmental samples 66-68
4. Molecular analysis of moist soil and slurry incubation 68-71
4.1. KMS soil 71-73
4.2. GMS soil 73-74
4.3. OMS soil 74-79
Discussion 80-84 VII. Outlook 85-86
VIII. Summary 87- 89
IX. Reference 90-110
Curriculum Vitae
Acknowledgements
Appendix























Abbreviations

AMO Ammonia monooxygenase
amoA Alpha subunit of the ammonia monooxygenase
Anammox Anaerobic ammonium oxidation
APS Ammoniumperoxodisulfate
ATCC American Type Culture Collection
bp Basepairs
DGGE Denaturant Gradient Gel Electrophoresis
DNRA Dissimilate nitrate to ammonium
-e Electron
EDTA Ethylendiaminetetraacetic acid
FISH Fluorescence In Situ Hybridization
GC-clamp Guanosine-cytosine clamp
GC Gas chromatograph
gdw Gram dry weight
GeneBank Nucleotide sequence database - http://www.ncbi.nlm.nih.gov/Entrez/
HAO Hydroxylamine oxidoreductase
IPTG Isopropyl-ß-D-thiogalactoside
LB Luria Broth
MPN Most probable number
O.D. Optical Density
PCR Polymerase Chain Reaction
PVPP Polyvinylpolypyrrolidone
RFLP Restriction Fragment Length Polymorphism
rpm Rounds per minute
SDS Sodium Dodecyl Sulfate
sp. Species (single)
spp. Species (plural)
TAE Tris-acetate-EDTA
TE Tris-EDTA
TEMED N,N,N’,N’-Tetramethylethyldiamine
T-RFLP Terminal Restriction Fragment Length Polymorphism
Tris Tris (hydroxymethyl)-aminomethane
WHC Maximal water holding capacity
w/w Weight per weight
wt/vol Weight per volume
X-Gal 5-Bromo-4-Chloro-3-indolyl-ß-D- galactopyranoside
I. Introduction
Nitrification, the conversion of the most reduced form of nitrogen (NH ammonia), to its 3
- most oxidized form, (NO nitrate), plays an important role in the nitrogen cycle of various 3
ecosystems including soils (Prosser, 1989). Nitrification has a great impact on environmental
processes, such as acidification of soils (Prosser, 1989; Biederbeck et al., 1996) and
biodeterioration of building materials (Meincke et al., 1989). Loss of nitrogen from fertilized
agriculture soils could lead to leaching of nitrite and nitrate since, as negative ions, these are
more mobile then ammonium, and therefore contamination of aquifers, springs and other
drinking water sources is possible (Bauhus and Melwes, 1991). Nitrification is also known to
produce nitric oxide (NO) and nitrous oxide (N O) as by-products, which are well known 2
greenhouse gasses and ozone scavengers (Crutzen, 1970; Dickinson and Cicerone, 1986).
Concurrently, nitrification has important positive effects, since high concentrations of
ammonium are toxic for life (Arthur et al., 1987) and create a large oxygen demand.
Nitrification therefore prevents eutrophication of surface and ground water from high input of
fertilizer (Hall and Jeffries, 1986), and also prevents the growth of phototrophs and
heterotrophs, which could lead to a decrease of biodiversity and the creation of anoxic
conditions. Nitrification can also be useful against anthropogenic damage to the environment,
by reducing the ammonium content of wastewater in sewage treatment before discharge into
aquatic environments (Painter, 1986).

Nitrification is composed of two stages. Ammonia oxidizers are involved in the first step,
when ammonia is oxidized to nitrite, and nitrite oxidizers are involved in the second step,
when nitrite is oxidized to nitrate (Prosser, 1989) (Fig. 1). Ammonia oxidation is thought to
be the rate-limiting step for nitrification in most systems, as nitrite is rarely found to
accumulate in the environment (Prosser, 1989; De Boer et al., 1990; 1992).
1

Figure 1: The nitrogen cycle

Nitrification is followed by denitrification, which is the production of di-nitrogen (N )2 ,
nitric oxide (NO) and nitrous oxide (N O) under anaerobic conditions. Interactions between 2
nitrifiers and denitrifiers are often mediated across oxic/anoxic interfaces such as soil
aggregates, which involve the diffusion of substrates and products from oxic to anoxic niches
(Zausig et al., 1993). Another possible process is chemodentrification, but it is not clear to
what extent this process affects nitrogen losses in natural environments (Kowalchuk and
Stephen, 2001). Release of di-nitrogen allows the biological fixation of nitrogen to
+ammonium (NH ), which is the available form of nitrogen for all other microorganisms. 4
Another process, where ammonium is released to the environment, is ammonification (or
mineralization) of organic nitrogen compounds. The majority of ammonium uptake is due to
assimilatory processes by most microorganisms in the environment. Theses are the main
competitors for