Enrichment and characterization of three ammonia-oxidizing

Enrichment and characterization of three ammonia-oxidizing Archaea from freshwater 1

environments. 2

3

Elizabeth French 1, Jessica A. Kozlowski 1,3, Maitreyee Mukherjee 2, George Bullerjahn 2, and 4

Annette Bollmann 1* 5

6

1 Miami University 7

Department of Microbiology 8

32 Pearson Hall, 700 East High Street 9

Oxford, OH 45056 10

USA 11

12

2 Bowling Green State University 13

Department of Biological Sciences 14

516 Life Sciences 15

Bowling Green, OH 43403 16

USA 17

18

3 present address: 19

University of Alberta 20

Department of Biological Sciences 21

CW405, Biological Sciences Building 22

Edmonton, Alberta T6G 2E9 23

Canada 24

25

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00432-12 AEM Accepts, published online ahead of print on 8 June 2012

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* Corresponding author: 26

email: [email protected] 27

phone: +1 513 529 0426 28

fax: +1 513 529 2431 29

30

31

Short title: Characterization of three AOA enrichments 32

33

Section: Microbial Ecology 34

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Abstract 36

Aerobic biological ammonia oxidation is carried out by two groups of microorganisms, 37

ammonia-oxidizing bacteria (AOB), and the recently discovered ammonia-oxidizing archaea 38

(AOA). Here we present a study using cultivation-based methods to investigate the differences in 39

growth of three AOA cultures and one AOB culture enriched from freshwater environments. The 40

enriched AOA belong to the Thaumarchaeal group I.1a, with one enrichment culture having the 41

highest identity with Candidatus Nitrosoarchaeum koreensis and the other two representing a 42

new genus of AOA. The AOB enrichment culture was also obtained from freshwater and had the 43

highest identity to AOB from the Nitrosomonas oligotropha group (Nitrosomonas cluster 6a). 44

We investigated the influence of ammonium, oxygen, pH, and light on the growth of AOA and 45

AOB. The growth rates of the AOB increased with increasing ammonium concentrations while 46

the growth rates of the AOA decreased slightly. Increasing oxygen concentrations led to a 47

increase in the growth rate of the AOB, while the growth rates of AOA were almost oxygen 48

insensitive. Light exposure (white and blue wavelengths) inhibited the growth of AOA 49

completely, and the AOA did not recover when transferred to the dark. AOB were also inhibited 50

by blue light, however, growth recovered immediately after transfer to the dark. Our results show 51

that the tested AOB has an competitive advantage over the tested AOA under most investigated 52

conditions. Further experiments will elucidate the niches of AOA and AOB in more detail. 53


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Introduction 54

Nitrification, the microbial oxidation of NH3 (ammonia) to NO3- (nitrate), is one of the key 55

processes of the global nitrogen cycle. The first and rate-limiting step of nitrification is the 56

oxidation of NH3 to NO2- (nitrite). Until recently, aerobic ammonia oxidation was attributed only 57

to a small subset of the Proteobacteria; most freshwater and terrestrial ammonia-oxidizing 58

bacteria (AOB) belong to a distinct group in the Betaproteobacteria, while a few marine AOB 59

species belong to the Gammaproteobacteria (30, 33, 34). The AOB have a 60

chemolithoautotrophic metabolism, oxidizing NH3 to NO2- via the intermediate NH2OH 61

(hydroxylamine), and fixing carbon from CO2 (carbon dioxide) via the Calvin cycle (1). 62

Recently, genes encoding ammonia monooxygenase (amoA), the first enzyme in the process of 63

ammonia oxidation, were discovered together with archaeal 16S rRNA genes in a metagenomic 64

study (62) and a soil fosmid library (59). At the same time, Nitrosopumilus maritimus, the first 65

archaeal ammonia oxidizer, was isolated into pure culture from a saltwater aquarium (31). 66

Ammonia-oxidizing archaea (AOA) in pure and enrichment cultures have essentially the same 67

metabolism as AOB; they oxidize NH3 stoichiometrically to NO2- and fix carbon from 68

bicarbonate (HCO3-) (15, 21, 31, 38, 45, 58). However, the genomes of N. maritimus and 69

Candidatus Nitrosoarchaeum limnia revealed differences between AOA and AOB, such as the 70

use of the 3-hydroxypropionate/4-hydroxybutyrate pathway for HCO3- fixation, the absence of 71

hydroxylamine oxidoreductase, and the presence of many copper-containing enzymes (5, 65). 72

AOA and AOB often co-occur in the same environment, but the contributions of AOA and AOB 73

to the total ammonia oxidation still need to be elucidated. Many previous studies focused on the 74

influence of environmental factors on niche differentiation between AOA and AOB using 75

cultivation-independent molecular methods. From those studies it can be concluded that AOA 76


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are frequently found in environments with lower substrate (NH4+ and O2) availability, and AOB 77

in environments with higher substrate availability (4, 13, 17, 25, 44, 57 among others). However, 78

most of these studies were conducted using methods that target the abundance and/or expression 79

of the archaeal and bacterial amoA genes. Unfortunately, it is not possible to draw direct 80

conclusions about the activity of the AOA and AOB based on abundance and expression of the 81

amoA gene, because amoA mRNA has been detected in AOB for weeks and 16S rRNA 82

(ribosomes) for up to a year after the onset of starvation (8, 26, 27). The response of AOA 83

towards starvation and resuscitation has not yet been investigated. In addition it has been shown 84

that not all amoA-encoding Thaumarchaeota are autotrophic ammonia oxidizers (41, 66). While 85

studies focusing on the analysis of abundance and activity of microbes using molecular methods 86

give very valuable insights, it is also necessary to investigate the response of microbes to 87

environmental factors using cultivation based approaches, because these experiments will 88

demonstrate changes in physiological activity more conclusively. 89

Here we present a study that used a cultivation-dependent approach to investigate the responses 90

of AOA and AOB to environmental factors. Three phylogenetically distinct AOA cultures were 91

enriched from freshwater sediments in Ohio, USA and their growth was characterized under 92

different conditions and compared with that of a freshwater AOB enrichment culture. Factors of 93

interest include the NH4+ concentration, pH, O2 concentration, and light wavelength and 94

intensity. These factors have strong effects on the physiology and niche differentiation of AOB 95

(7, 24, 46, 56) and are, therefore, also very likely to influence the physiology and niche 96

differentiation between AOA and AOB. 97

98

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Material and Methods 100

Sampling: Near shore sediment samples were taken from Lakes Acton (AC) (39°57’N, 101

84°74’W) and Delaware (DW) (40°39’N, 83°05’W) in Fall 2008. Additional sediment core 102

samples were collected from Lake Acton in Summer 2009. 103

Medium: Mineral salts medium (MS medium) used to enrich and cultivate AOA and AOB 104

contained 10 mM NaCl, 1 mM KCl, 1 mM CaCl22H2O, 0.2 mM MgSO47H2O, and 1ml l-1 105

trace elements solution (9, 63). HEPES buffer was added in a four-fold molar ratio to the NH4+ 106

concentration, and the pH was adjusted to 7.5 before autoclaving. After autoclaving, sterile 107

KH2PO4 solution was added to obtain a final concentration of 0.4 mM (9, 63). 108

Enrichment of the AOA (AOA-AC2, AOA-AC5 and AOA-DW): Sediment samples (1 g) 109

were inoculated into 50 ml MS medium with 0.25 mM NH4+ immediately upon arrival in the 110

laboratory. The enrichments were incubated at 27°C in the dark. NH4+ levels were monitored 111

weekly using a colorimetric assay (9, 29). When the cultures reached late logarithmic growth 112

phase (depletion of around 80% of the initial NH4+ concentration) they were transferred to fresh 113

medium using 10% v/v inoculum. The cultures were passed through 0.45 µm filters for the first 114

five to six transfers to exclude AOB (Annika Mosier, personal communication; 9). In addition to 115

filtration, the enrichment cultures from DW were also treated with 100 µg ml-1 streptomycin to 116

eliminate AOB. After several transfers, when the cultures depleted NH4+ in regular intervals, 20 117

ml was collected on 0.1 µm nitrocellulose filters for molecular characterization. The filters were 118

stored at -20°C. 119

AOB culture: We used the previously described AOB freshwater enrichment culture G5-7 120

(AOB-G5-7) to compare the growth of AOA to AOB (6, 7). The culture belongs to the 121

Nitrosomonas oligotropha cluster and is adapted to low NH4+ concentrations (6, 7). Members of 122


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this AOB cluster have been found in many freshwater environments around the world (11, 12, 123

14, 18, 23, 55). 124

Growth experiments: All growth experiments were conducted in MS medium with 0.5 mM 125

NH4+ at pH 7.5 in 125 ml Erlenmeyer flasks with cotton stoppers unless otherwise noted. We 126

tested the influence of different factors (NH4+ concentration, O2 concentration, pH, and light) on 127

the rate of NO2-/NO3

- production of the three AOA enrichment cultures (AOA-AC2, AOA-AC5 128

and AOA-DW) and the AOB enrichment culture (AOB-G5-7). All cultures were inoculated with 129

10% v/v of a conditioned late log phase culture and incubated in the dark at 27°C. Samples (1 130

ml) were taken at regular intervals and centrifuged at 16,000 rpm for 20 min. The supernatant 131

was stored at -20°C for further chemical analysis. To investigate the influence of different NH4+ 132

concentrations, media with 15 µM - 5 mM NH4+ were prepared with the corresponding HEPES 133

concentrations. The influence of pH was investigated by adjusting the initial pH in the medium 134

to values between 6 and 9. The influence of O2 concentration was investigated by equilibrating 135

the medium in serum bottles under anaerobic conditions overnight. After equilibration the bottles 136

were sealed with rubber stoppers. Different calculated O2 concentrations in the headspace were 137

achieved by exchanging the corresponding volume of the headspace with sterile filtered air. The 138

influence of light was investigated by incubating the cultures 18 cm above LED panels emitting 139

30 µmol photons m-2 s-1 at the wavelength 5000 - 7000 K (white light), 623 ± 3 nm (red light), 140

and 470 ± 5 nm (blue light); and 3 µmol photons m-2 s-1 at the wavelength 470 ± 5 nm (blue 141

light). The light intensity inside the glass bottles was 25 µmol photons m-2 s-1 (high light) and 2.5 142

µmol photons m-2 s-1 (low light conditions) as measured with a LI-250A light meter (LI-COR 143

Biosciences, Lincoln, NE) indicating that the glass filtered approximately 15% of the light. To 144

investigate the influence of light-to-dark and dark-to-light transitions on the growth of AOA and 145


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AOB, cultures were incubated in the dark until 50% of the NH4+ was consumed and then 146

transferred to the light. At the same time, cultures that were incubated in the light were 147

transferred from the light to the dark. Controls were incubated for the complete cycle in the dark. 148

Evaluation of growth experiments: NO2- and NO3

- concentrations were determined in the 149

supernatants using colorimetric assays (9, 51). NO2-/NO3

- concentrations were log transformed 150

and plotted against time (Fig. S1). Growth rates were calculated from the linear increase (slope) 151

of the log-transformed NO2-/NO3

- concentrations over time, assuming that NO2-/NO3

- production 152

in the cultures is correlated with the growth of AOA and AOB (3, 9, 31). The increase in NO2-153

/NO3- production was linear for several days to one week and the correlation coefficients were 154

always ≥ 0.97 but in most cases even ≥ 0.99. 155

156

Molecular analysis 157

DNA isolation from the AOA enrichment cultures: DNA was isolated from the nitrocellulose 158

filters using the Qiagen DNeasy Blood and Tissue Kit (Valencia, CA) with the following 159

modifications. Acid-washed zirconium beads (1g) and 500 µl high salt buffer (1 M NaCl, 5 mM 160

MgCl22H2O, 10 mM Tris, pH 8) (2) were added to the nitrocellulose filters. The filters were 161

homogenized using a bead beater (Biospec Products, Bartlesville, OK) at 4800 rpm for 30 s. This 162

was repeated three times and the samples were stored in between cycles on ice for 10 min. After 163

bead beating, 500 µl Qiagen Buffer AL and 50 µl proteinase K were added and the mixture was 164

incubated at 56°C for 30 min. The reaction mixture was spun down at 8000 rpm for 1 min and 165

transferred to spin columns supplied by the manufacturer. The spin columns were treated 166

according to the manufacturer’s recommendations and the DNA was eluted with 100 µl elution 167

buffer AE. 168


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PCR: GoTaq Green Master Mix (Promega, Madison WI) was used for all standard PCR 169

according to the manufacturer’s recommendations using the primers and protocols summarized 170

in Table S1. 171

Cloning and sequencing: PCR products were cleaned using the Wizard SV Gel and PCR 172

product Clean up system (Promega, Madison, WI) and cloned into the pGEM-T easy vector 173

system (Promega, Madison, WI). Transformants were screened for inserts using PCR with M13 174

primers and the PCR products were cleaned up and sequenced using the BigDye Terminator 175

V3.1 cycle sequencing kit (Life Technology Corporation, Carlsbad, CA) on an Applied 176

Biosystems 3730x1 DNA analyzer (Life Technology Corporation). 177

DNA sequence analysis: All sequences were edited with 4Peaks (A. Griekspoor and T. 178

Groothuis, The Netherlands Cancer Institute). The sequences were aligned using ARB (37). 179

Phylogenetic trees were constructed using the neighbor-joining algorithm in ARB, and 180

parsimony and maximum likelihood methods using PHYLIP (16). Trees constructed with all 181

three methods showed the same overall grouping, therefore only the tree constructed with 182

neighbor-joining method has been presented. All sequences were deposited in Genbank under 183

the numbers: JQ669389-JQ669394. 184

Fluorescence in-situ Hybridization (FISH): The CARD-FISH protocol (47, 50) was used with 185

the following modifications: the hybridization temperature was 46°C, the first wash was 186

performed at 48°C, followed by an amplification step at 46°C. All probes (Table S2) were 187

labeled at their 5’ end with horseradish peroxidase and used at a final concentration of 50 ng µl-1. 188

All filters were counterstained with DAPI for total cell counts. Direct microscopic counts by 189

fluorescence microscopy (Zeiss Axiophot HB0100, Carl Zeiss Inc, North America) were 190

performed at 1000X magnification. 191


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Results 192

Enrichment of AOA: AOA were enriched from the sediment of Lakes Acton (AOA-AC2 and 193

AOA-AC5) and Delaware (AOA-DW) under autotrophic conditions with NH4+ as the sole 194

electron donor in the medium. Based on the AOA amoA sequences, all enrichment cultures 195

belong to the water column/sediment group I.1a of the Thaumarchaeota (Fig. 1). AOA-AC2 was 196

81-81.7% (amoA) and 92.8-93.1% (16S rDNA) identical to the other two enrichment cultures, 197

while AOA-AC5 and AOA-DW were 87.1% (amoA) and 97.9% (16S rDNA) identical to each 198

other. The amoA sequences of AOA-DW were 98.2-98.5% identical to clones from the sediment 199

of Lakes Acton, Delaware and Pleasant Hill (36), 98.5% to clones from the freshwater sediment 200

in the San Francisco Bay (40), and 98.1% to clones from a drinking water distribution system in 201

the Netherlands (61). The amoA sequences of AOA-AC5 were 99% identical to a clone from a 202

paddy soil in Japan (19). The third enrichment culture AOA-AC2 is closely related to Ca. 203

Nitrosoarchaeum koreensis (99.8% identity on amoA basis and 99.6% identity on 16S rDNA 204

basis) and Ca. Nitrosoarchaeum limnia (94.3% identity on amoA basis and 98.5% identity on 205

16S rDNA basis) (5, 28). In contrast to AOA-AC2, AOA-AC5 and AOA-DW were not closely 206

related to described AOA isolates or enrichment cultures such as N. maritimus and 207

Nitrososphaera viennensis among others (70-82% identity on amoA basis and 81-93% identity 208

on 16S rDNA basis) (Table 1). 209

Fluorescent in situ hybridization (CARD-FISH) was used to determine the proportion of AOA in 210

the enrichment cultures at the end of the logarithmic growth phase. AOA-DW contained 85% 211

AOA, AOA-AC2 91% and AOA-AC5 81% (Table 2). AOB and NOB (nitrite-oxidizing bacteria) 212

were not detected as tested by PCR amplification with AOB-specific 16S rRNA and amoA 213


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primers (Table S1) (results not shown) and FISH using AOB- and NOB-specific 16S rRNA 214

probes (Table 2 and Table S2). 215

Influence of NH4+ concentration on the growth rates of AOA and AOB. During stratification 216

in the summer the NH4+ concentration in Lake Acton increases up to 400 µM (43), which falls 217

within the tested range of NH4+ concentrations of 15 µM and 2 mM NH4

+. Increasing NH4+ 218

concentrations up to 1 mM NH4+ doubled the growth rate of AOB-G5-7, while the growth rates 219

of the AOA enrichment cultures decreased or remained constant (Fig. 2). The growth rate of 220

AOA-DW at the lowest NH4+ concentration (15 µM) was significantly higher than the growth 221

rate at higher NH4+ concentrations (Table S3). The same tendency was observed for the other 222

two cultures, although the statistical support was less strong (Fig. 2; Table S3). The AOA 223

enrichment cultures exhibited different tolerances to high NH4+ concentrations; AOA-DW grew 224

at NH4+ concentrations up to 1 mM, AOA-AC2 up to 2 mM and AOA-AC5 up to 5 mM (Fig. 2). 225

The lag phase of AOA and AOB differed; AOB-G5-7 became active 1-3 days after inoculation at 226

all tested NH4+ concentrations, whereas the lag phase of the AOA cultures increased with 227

increasing initial NH4+ concentrations up to more than two weeks before logarithmic growth 228

could be detected at NH4+ concentrations between 1 mM and 5 mM NH4

+ (Fig. S2; Table S4). 229

Influence of O2 concentration on the growth of AOA and AOB: Lake Acton, stratifies during 230

the summer and has an anaerobic zone as well as a zone with low oxygen availability (1 mg l-1 231

O2) (43). We therefore investigated the response of our enrichment cultures to 0.5-2% O2 232

(calculated) in the headspace, which corresponded to 0.2-0.8 mg l-1 O2 in the medium. The 233

growth rate of AOB-G5-7 decreased with decreasing O2 concentration and the growth rates at all 234

different O2 concentrations were significantly different from each other (Fig. 3; Table S5). The 235

AOA enrichment cultures grew at all O2 concentrations in the headspace with the exception of 236


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AOA-AC2 at 0.5% O2. The decrease of the growth rates with decreasing O2 concentration in the 237

AOA cultures was less steep than the decrease of the growth rates in the AOB enrichment 238

culture. However, the growth rates at low O2 concentrations in the AOA-AC2 and AOA-AC5 239

were significantly lower than the growth rates at 21% O2 (Fig. 3; Table S5). 240

Influence of pH on the growth of AOA and AOB: We investigated the growth of all cultures 241

at pH 6-9, the range at which non-acidophilic ammonia oxidizers grow (32, 33, 34). The growth 242

rates of all cultures showed bell-shaped curves in relation to the pH with maximum growth rates 243

at pH 7-7.5 (Fig. 4). AOA-AC2 did not grow at pH 6, while the other AOA and AOB cultures 244

did. The growth rates of AOA-AC5 and AOA-DW at pH 9 were similar to the growth rates at pH 245

7.5, while the growth rates of AOA-AC2 and AOB-G5-7 differed significantly to their respective 246

rates at pH 7.5 (Fig. 4; Table S6). 247

Influence of light on the growth of AOA and AOB: The investigated intensities represent a 248

range at which phytoplankton in freshwater systems is able to grow, but below light saturation 249

(53). White light (30 µmol photons m-2 s-1) strongly inhibited the growth of AOA-DW, but had 250

no effect on AOB-G5-7 (Fig. 5). The AOA did not grow in white light and did not begin to grow 251

after being transferred from the light to the dark. However, growth continued when the AOA 252

cultures were transferred from the dark to the light. To get a better insight into which wavelength 253

of light had the strongest influence on the growth of AOA and AOB, we conducted similar 254

experiments with red (623±3 nm) and blue (470±5 nm) light. Both cultures grew in the red light, 255

but while the growth of AOB-G5-7 was not influenced by the red light, the growth rates of 256

AOA-DW were significantly lower in the red light and after transfer from the light to the dark 257

(Fig. 5; Table S7 and S8). Blue light at 30 µmol photons m-2 s-1 had the strongest effect on the 258

growth of both cultures (Fig. 5). In the blue light none of the cultures grew, and growth of AOA-259


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DW did not recover after transfer from the light to the dark. In contrast, AOB-G5-7 recovered 260

immediately after transfer from the light to the dark, but the growth rate was significantly lower 261

than the growth rate in the continuous dark (Table S7). Transfer of the cultures from the dark 262

into blue light stopped growth immediately. Both cultures grew in the less intense blue light (3 263

µmol photons m-2 s-1) but the growth rate of AOA-DW was significantly lower in the low blue 264

light than in the dark (Fig. 5; Table S8). 265

266

Discussion 267

Enrichment of AOA cultures AOA-DW, AOA-AC2, and AOA-AC5: We enriched and 268

characterized the growth of three different freshwater AOA enrichment cultures belonging to the 269

Thaumarchaeal group I.1a within the newly described phylum Thaumarchaeota (10, 54). One of 270

the cultures, AOA-AC2, is closely related to Ca. Nitrosoarchaeum koreensis, while the other two 271

strains, AOA-AC5 and AOA-DW, are only 70-82% (amoA) and 81-93% (16S rDNA) identical 272

to other cultivated isolates and enrichment cultures such as N. maritimus and Nitrososphaera 273

viennensis (Table 1). This finding indicates that these two enriched AOA belong to a new genus 274

of the ammonia-oxidizing Thaumarchaeota, assuming that the identity between two genera is on 275

average 96.4% based on the 16S rRNA gene (68). This new genus/group includes many 276

ribotypes from non-salt water systems such as freshwater (36, 40) and drinking water systems 277

(61) as well as soil and hot spring environments (69), as indicated by highly identical clones (Fig 278

1). 279

Pure cultures: In this study no pure cultures of the AOA have been obtained thus far. It is safe 280

to assume that the heterotrophic satellite community is providing some compound that enabled 281

the AOA to grow in the enrichment culture. Similar observations have been made with other 282


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AOA as well as for AOB. Potential compounds that positively influence the growth of AOA 283

could be small organic compounds such as pyruvate, which improved growth and enabled 284

isolation of N. viennensis (58). However, the addition of pyruvate during serial dilution did not 285

lead to isolation of any of these strains to date, indicating that different compounds might be 286

important for different AOA. Further research will be necessary to elucidate the interactions 287

between AOA (and AOB) and the heterotrophic satellite bacteria in ammonia oxidizing 288

enrichment cultures. 289

Growth of AOA and AOB: Overall the growth experiments showed that the growth rates of the 290

AOA were almost always lower than the growth rates of the AOB. All our experiments have 291

been conducted under strict chemolithoautotrophic conditions. The results indicate that AOB-292

G5-7 had an advantage over the three tested AOA strains under the conditions investigated. In 293

nature, however, conditions are often less defined with respect to energy generating processes. It 294

has been suggested that not all Thaumarchaeota are chemolithoautotrophic ammonia oxidizers; 295

some carry the amoA gene but are not actively oxidizing NH4+ and others utilize mixotrophic or 296

heterotrophic lifestyles in pure and enrichment cultures (41, 58, 67). Based on these observations 297

and our data one could speculate that AOA in natural samples utilize mixotrophic and/or 298

heterotrophic rather than a completely autotrophic life style, which could explain their success in 299

nature compared to the laboratory. 300

Increasing NH4+ concentrations have different influence on the growth rates and lag phases of 301

AOA and AOB with AOB growing faster and having shorter lag phases than AOA (Fig. 2; Fig. 302

S2; Table S3 and S4). After comparing these results with data provided by other studies that 303

determined the Km of AOA for NH3/NH4+ to be approximately 1000 times lower than Km of AOB 304

(28, 38, 45) we suggest that AOB have an advantage over AOA at higher NH4+ concentrations (> 305


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10µM). This assumption is supported by the detection of high abundances of AOB environments 306

with higher NH4+ input due to fertilization and other processes, while AOA are more abundant in 307

low NH4+ and unfertilized environments (17, 22, 25, 64, 66). 308

The enrichment cultures AOA-DW and AOA-AC5 showed lower tolerance to high NH4+ 309

concentrations than AOA-AC2, with the highest concentrations supporting growth at 1 mM 310

NH4+ (AOA-DW) and 2 mM NH4

+ (AOA-AC5). These concentrations are lower than the highest 311

tolerances towards NH4+ observed for N. viennensis (15 mM), Ca. Nitrosoarchaeum koreensis 312

(10 mM) and enrichment AOA-AC2 (5 mM), a strain closely related to Ca. Nitrosoarchaeum 313

koreensis (28, 58). These results indicate that AOA-DW and AOA-AC5 are less tolerant to high 314

NH4+ concentrations when compared with other AOA isolates and enrichment cultures. Similar 315

observations have been made for AOB; members of the Nitrosomonas oligotropha cluster, which 316

are also commonly found in freshwater environments, are less tolerant to high NH4+ 317

concentrations and better adapted to low NH4+ concentrations, while members of the 318

Nitrosomonas europaea/eutropha cluster are found primarily in environments with high NH4+ 319

concentrations (6, 7, 32, 33, 34). 320

AOA and AOB responded differently when cultured over a range of O2 concentrations. AOA-321

AC5 and AOA-DW grew at all tested O2 concentrations at the same rate, while AOA-AC2 did 322

not grow at 0.5% O2, and the growth rate of AOB-G5-7 decreased with decreasing O2 323

concentrations (Fig. 3; Table S5). Environmental surveys often detected AOA at the oxic-anoxic 324

interface (4, 13, 17, 49) indicating an adaptation to low oxygen conditions. The low Km for O2 325

found for N. maritimus as well as other AOA (28, 38, 45) and the environmental data support the 326

hypothesis that AOA are very likely better adapted to low O2 than AOB and may therefore have 327


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a competitive advantage at the oxic-anoxic interface while AOB are active under more aerobic 328

conditions. 329

AOA and AOB grew at most of the tested pH values, with AOA growing at almost the same rate 330

over a wide pH range and AOB showing a more bell-shaped curve with the highest growth rate 331

at pH 7-7.5 (Fig. 4; Table S6). AOA are found over a wide pH range in different environments 332

such as soils and hot springs (15, 20, 22, 42, 48), but most cultivated AOA such as N. maritimus, 333

N. viennensis, Ca. Nitrosoarchaeum koreensis and Ca. Nitrosotalea devanaterra have rather 334

narrow pH ranges for growth and activity compared with the tested AOA enrichment cultures 335

(28, 31, 58, 60). 336

AOB-G5-7 was more tolerant to light than AOA-DW and also recovered faster after exposure, 337

while AOA-DW did not fully recover from light exposure (Fig. 5; Table S7 and S8). In the 338

environment, maxima of Thaumarchaeal amoA and 16S rRNA copies have been detected at 339

levels below where photosynthetically active radiation (PAR) in the water column dropped to 340

zero, indicating that no light was penetrating to this depth (4, 49). In the same study, AOB and 341

AOA were detected in low abundance in more shallow waters of the Pacific, indicating that AOB 342

as well as some AOA strains could be more tolerant to light than those that are most abundant in 343

the lower parts of the water column (49). The light response of AOA and AOB could be due to 344

differences in the reaction of the copper-containing enzymes to light. AOB are very sensitive to 345

blue near UV light (24, 52). The authors discussed that this inhibition could be attributed to the 346

absorption of light by the oxygenated state of the copper- containing ammonia monooxygenase, 347

which leads to inactivation of the enzyme (52). Genome studies of AOA showed a large number 348

of copper containing enzymes such as multi-copper oxidases and blue copper proteins (5, 65), 349

suggesting that some of the copper-containing enzymes in AOA could be sensitive to light as 350


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well, leading to inhibition of overall metabolism in AOA by light. During the preparation of this 351

manuscript Merbt et al. (2012) published a study investigating the response of two AOB 352

(Nitrosomonas europaea and Nitrosospira multiformis) and two AOA (N. maritimus and C. 353

Nitrosotalea devanaterra) to white light (39). The study confirmed our findings. 354

Conclusion: The results of this study show that AOB are able to outcompete AOA under almost 355

all tested conditions. These findings are in accordance with other cultivation-based studies, as 356

well as observations made in the environment using molecular approaches. Further investigation 357

must be done using other cultivation-based experiments such as continuous cultures, which 358

enable us to cultivate AOA and AOB under more stringently controlled conditions, and in situ 359

incubations which enable us to investigate the response of AOA and AOB to environmental 360

changes in conditions which allow AOA and AOB to utilize metabolic functions as they would 361

naturally in the environment. 362

363

Acknowledgements: We thank Annika Mosier (UC Berkeley) for helpful discussions at the 364

beginning of the project; Michael Vanni and Beth Mette (Department of Zoology, Miami 365

University) for support with sampling; Lynn Johnson (Instrumentation Laboratory, Miami 366

University) for construction of the light installations; Anne Bernhard (Connecticut College, New 367

London) for providing her AOA amoA ARB alignment file, and Anne Morris Hooke 368

(Department of Microbiology; Miami University) for critical reading of the manuscript. This 369

work was supported by startup funds of Miami University and by the National Science 370

Foundation grants no: DEB-1120443 to AB and OCE-0927277 to GB. This paper is dedicated to 371

the memory of Dr. John W. Hawes (Center for Bioinformatics and Functional Genomics and 372

Department of Chemistry and Biochemistry, Miami University). 373

374


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Figure legends: 594

Figure 1: Neighbor-joining phylogenetic tree of the AOA enrichment cultures based on amoA 595

gene sequences (595bp). Bootstrap values > 50 of 100 replicates are shown at the 596

nodes. 597

598

Figure 2: Influence of NH4+ concentration on the growth rates of the enrichment cultures AOA-599

AC2; AOA-AC5, AOA-DW, and AOB-G5-7 (mean ± SD; n=3). A: NH4+ 600

concentration linear scale; B: NH4+ concentration logarithmic scale 601

602

Figure 3: Influence of the calculated O2 concentration in the headspace of the bottle on the 603

growth rate of the enrichment cultures AOA-AC2; AOA-AC5, AOA-DW, and AOB-604

G5-7 (mean ± SD; n=3) 605

606

Figure 4: Influence of the pH of the medium on the growth rates of the enrichment cultures 607

AOA-AC2, AOA-AC5, AOA-DW, and AOB-G5-7 (mean ± SD; n=3) 608

609

Figure 5: Influence of white, red, and blue light with the intensity of 30 µmol photons m-2 s-1 and 610

blue light with the intensity of 3 µmol photons m-2 s-1 on the growth rates of the 611

enrichment cultures AOA-DW and AOB-G5-7 (mean ± SD; n=3) 612

613


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TABLE 1: Identities [%] of AOA in the enrichment cultures AOA-AC2, AOA-AC5, and AOA-614

DW in comparison with previously cultivated AOA. 615

AOA-AC2 AOA-AC5 AOA-DW

amoA 16S amoA 16S amoA 16S

Nitrosopumilus maritimus (31) 88.6 96.2 79.8 92.9 78.8 92.9

Nitrososphaera viennensis (58) 69.6 82.7 70.4 83.7 71.1 83.7

Ca. Nitrososphaera gargensis (21) 70.9 81.9 72.7 82.7 72.1 82.2

Ca. Nitrosocaldus yellowstonii (15) 71.1 80.4 71.1 81.8 70.0 81.0

Ca. Nitrosoarchaeum limnia (5) 94.3 98.4 81.9 92.6 81.5 92.9

Ca. Nitrosoarchaeum koreensis (28) 99.8 99.6 81.6 92.9 81.2 92.8

Ca. Nitrosotalea devanaterra (35) 77.9 88.5 76.7 89.7 76.1 89.3

616

Comparisons are based on 16S rRNA genes (794 bp; corresponding to 109 to 915 in Escherichia 617

coli numbering) and amoA genes (595 bp). 618

619


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TABLE 2: Quantitative analysis of the composition of the enrichment cultures AOA-AC2; 620

AOA-AC5 and AOA-DW. 621

AOA-AC2 AOA-AC5 AOA-DW

Crenarchaeota 91.0 81.2 85.4

Bacteria 9.5 3.3 9.2

Nitrospira (NOB) ND* ND ND

AOB ND ND ND

The cell numbers were determined using CARD-FISH [% of DAPI counts] (n=1). Samples were 622

taken at the end of the logarithmic phase. 623

*ND: not detected. 624

625

626


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Enrichment AOA-AC2

Candidatus Nitrosoarchaeum koreensis MY1DQ278585 (wastewater)EU651166 (freshwater sediment -SF bay)HQ317041 (wastewater - oil refinery)HQ317037 (wastewater - oil refinery)

EU651167 (freshwater sediment - SF bay)Candidatus Nitrosoarchaeum limnia

Clone BO-D07Clone BO-22

EU650806 (estuarine sediment - SF bay)EU651121 (estuarine sediment - SF bay)

Nitrosopumilus maritimus

Enrichment marine sediment Arctic (FJ656552)Enrichment marine sediment South Korea (FJ656572)

DQ148692 (Black sea)Enrichment marine water CN150 (JF521547)Enrichment marine water CN25 (JF521548)

EU651017 (freshwater sediment - SF bay)

EU852677 (drinking water distribution system)

Clone AC−30

Clone DW−41Clone PH−30

Enrichment AOA-DW

FJ543353 (groundwater)Clone PH−46

FJ543354 (groundwater)

AB516244 (paddy soil)Enrichment AOA-AC5

EU553412 (hot spring)EU553410 (hot spring)

Candidatus Nitrosotalea devanaterraDQ148879 (soil, non-contaminated)

DQ312267 (soil with high nitrogen)FJ227868 (estuarine sediment)

DQ501047 (estuarine sediment)Candidatus Nitrososphaera gargensis

EU651130 (estuarine sediment - SF bay)

EU239968 (hot spring - Yellowstone)Candidatus Nitrosocaldus yellowstonii

Nitrososphaera viennensis

0.1

54

59

61

>99

59

>99

98

92

>99

65

>99

>99

97

54

93

>99

>99

>99

59

63

76

96

73

>99

>99

98

67

>99

>99

soil/sed

imen

tw

ater colu

mn

/sedim

ent

]

]


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G5-7DWAC2AC5

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NH4

+ concentration [mM]

A

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0.01 0.1 1 10

G5-7DWAC2AC5

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-1]

NH4

+ concentration [mM]

B


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0.04

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G5-7DWAC2AC5

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O2 concentration in gas phase [%]


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0.06

5.5 6 6.5 7 7.5 8 8.5 9 9.5

G5-7DWAC2AC5

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pH value


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light light -> dark dark -> light dark

DWG5-7

Gro

wth

rat

e [h

-1]

light treatment

DWG5-7

White light

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

light light -> dark dark -> light darkG

row

th r

ate

[h-1

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light treatment

Red light

0

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0.05

0.06

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Gro

wth

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e [h

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light treatment

Blue light - low intensity

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Gro

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light treatment

Blue light


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Documents

Enrichment and characterization of three ammonia-oxidizing