IDENTIFICATION, ENUMERATION AND DIVERSITY OF -OXIDIZING IN THE LAURENTIAN GREAT LAKES

Maitreyee Mukherjee

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 2013

Committee:

Dr. George S. Bullerjahn, Advisor

Dr. William H. O’ Brien Graduate Faculty Representative

Dr. Robert M. McKay

Dr. Scott O. Rogers

Dr. Zhaohui Xu © 2013

Maitreyee Mukherjee

All Rights Reserved iii

ABSTRACT

George Bullerjahn, Advisor

Nitrification is a major process of the global in which ammonia is oxidized

to nitrate. Previously it was assumed that bacteria (e.g., Nitrosomonas and Nitrobacter) are

responsible for this process. Over the past decade, researchers have discovered different groups

of nitrifying archaea, specifically Groups I.1a, and I.1b, in both marine and

freshwater environments. The physiological characteristics of these organisms are best suited to

the levels of ammonia found in aquatic systems. Since 1900, Lake Superior has seen a steady

increase in nitrate, yielding a severe stoichiometric imbalance of nitrogen to phosphorus. Stable

isotopic analyses indicate that nitrate increases result from in-lake biological processes. By

contrast, mesotrophic Lake Erie is prone to transient hypoxia, yielding nitrogen losses to the

atmosphere via denitrification and anammox. In this study, I examined these unique nitrifiers

contributing to in both lakes. of amoA sequences from lake seston

reveals that ammonia oxidizing archaea (AOA) dominate the Superior pelagic microbial

community, although AOA diversity in Lake Superior was observed to be low, indicating only a

few key ecotypes can survive the extreme oligotrophic conditions prevalent in the lake. CARD-

FISH probes specific for AOA, ammonia oxidizing bacteria (AOB) and nitrite oxidizers (NOB) allow their enumeration in samples obtained from 2010-12. During the stratified period in

Superior, AOA and NOB are detectable in the hypolimnion and oxic sediments (up to 7 x 104

mL-1), and absent in the epilimnion. AOB are not detectable (<101 mL-1) in Superior, but

dominate in Erie samples. For summer 2010, AOA abundance reflects parallel assays of

nitrification rates. This study concludes that Thaumarchaeota (AOA) dominate the ammonia- iv

oxidizing microbial population in Lake Superior, whereas, the ammonia oxidizing bacterial

population is negligible in the lake. In contrast, Thaumarchaeota are almost absent or negligible

in Lake Erie, whereas, the ammonia oxidizing bacterial are the dominant nitrifiers in the lake,

indicating towards a possible association of the AOA in environments with lower ammonium

concentrations, whereas that of the AOBs with environments with higher ammonium

concentrations. Thaumarchaeota (AOA), in spite of being so abundant in Lake Superior, tend to

be less diverse in the lake, indicating that only few species of these ammonia-oxidizers can

survive the extreme oligotrophic conditions of the lake. All the ammonia oxidizers, irrespective

of whether they are bacteria or archaea, tend to be absent from surface waters, whereas their abundance increases with depth of water column in the lakes. v

Dedicated to my dad, the teacher, philosopher, and guide of my life; to my mom, who made every possible sacrifice to see this day of my life; to the memory of my grandfather, who would have been proud; to my grandmother, for always believing in me; and last but not the least, to

my husband, without whose immense support, this work wouldn’t be completed.

| েতামায় িদলাম | vi

ACKNOWLEDGMENTS

First of all, I want to express my gratitude and thanks to my advisor Dr. George

Bullerjahn, without whose immense support, guidance, and valuable advice, this work would not be possible. I am grateful to him for taking me in his lab in the first place, making science so much fun, and giving me enough independence and freedom to do this research. In addition to being the best scientist and guide I’ve come across, I particularly admire his character of being able to provide a considerable sense of freedom to his students; to allow them to do their work in their own time and space. It was an absolute honor being his PhD advisee, and his student in the past five years, and I cannot thank him enough for making my scientific journey at Bowling

Green State University such amazing and memorable.

I would like to extend my special thanks to my co-advisor Dr. Robert Michael McKay for all his invaluable advice throughout this work. I also want to thank all the members of my PhD committee Dr. Scott Orland Rogers, Dr. Zhaohui Xu and Dr. William O’Brien for all their suggestions and advice towards this work. I also want to take this opportunity to thank all the members of the Bullerjahn-Mckay lab group for their helpful advices, trainings, and the wonderful science that we did together in the group. I want to thank all the staff of the

Department of Biological Sciences and the BGSU Graduate College for accepting me in their graduate program, and for making my PhD journey so wonderful and memorable.

Finally, I want to thank my family: my parents and my grandparents, for making every sacrifice to see this day of my life, for believing in me and taking immense pride in me forever. I want to thank all my great friends in Bowling Green and in India for supporting my work throughout these five years. And finally, I want to thank my husband without whose immense support and sacrifices, I wouldn’t have seen this day of my life. Thank you all. vii

TABLE OF CONTENTS

Page

CHAPTER I. INTRODUCTION ...... 1

1.The Nitrogen cycle...... 1

2.Nitrification...... 3

3.Denitrification...... 5

4.Chemolithotrophy...... 6

5.The Nitrifiers...... 7

6.The anaerobic Ammonia-oxidizers: Anammox Organisms...... 11

7.The Ammonia Oxidizing Archaea (AOA) or the Thaumarchaota...... 13

7.1. Habitat Diversity of the AOA...... 18

7.2. Cell structure, Physiology, Growth of the AOA...... 21

7.3. Stoichiometry and Kinetics of ammonia oxidation of AOA...... 23

7.4. The biochemistry of ammonia oxidation and

production by the AOA...... 25

7.5. Autotrophic, Mixotrophic and Heterotrophic growth in AOA...... 29

8.Nitrification in the Laurentian Great Lakes: two lakes,

two different stories...... 31

8.1. Structure and characteristics of Lake Superior vs. Lake Erie...... 32

8.2. Nitrification and trends in nitrate levels in Lake Superior

vs. Lake Erie...... 33

9. Objective and significance of the current study...... 36

REFERENCES...... 37

CHAPTER II. MATERIALS AND METHODS ...... 52 viii

1. Sampling ...... 52

2. DNA Extraction Procedures...... 57

3. Catalyzed Reporter Deposition Fluorescence in-situ Hybridization

(CARD-FISH)...... 58

4. PCR Methods...... 71

5. Bioinformatics Tools...... 75

REFERENCES ...... 76

CHAPTER III. RESULTS ...... 79

1. Sampling site characteristics ...... 79

2. Temperature Profiles...... 84

3. Diversity of ammonia oxidizing archaea (AOA) in Lake Superior

and Lake Erie...... 85

3.1. PCR analysis of 16S rDNA of environmental samples in

Lake Superior...... 86

3.2. Phylogenetic Analysis of 16S rDNA sequences

from Lake Superior...... 88

3.3. PCR analysis of amoA sequences from environmental samples

from Lake Superior and Lake Erie...... 91

3.4. Phylogenetic analysis of amoA sequences from Lake Superior

and Lake Erie...... 92

4. CARD-FISH Enumeration of Lake Superior AOA...... 95

4.1.2010 Samples...... 95

4.2.2011 Samples...... 100 ix

4.3.2012 Samples...... 105

5. CARD-FISH Enumeration of Lake Erie AOA...... 105

6. Total Bacterial Counts using CARD-FISH in

Lake Superior: 2010 – 2012...... 108

7. Nitrification rate measurements and total AOA cell comparison...... 111

8. Per cell Nitrification-Rate in AOA in Lake Superior...... 112

REFERENCES...... 113

CHAPTER IV. DISCUSSION AND CONCLUSIONS...... 115

1. Abundance of Ammonia-oxidizers in Lake Superior...... 116

2. Abundance of Ammonia-oxidizers in Lake Erie...... 120

3. Diversity of Ammonia-oxidizers in Lake Superior vs. Lake Erie...... 121

4. Conclusions and Future Directions...... 122

REFERENCES...... 124

APPENDIX A. ABBREVIATION...... 129 x

LIST OF FIGURES

Figure Page

1 The Nitrogen cycle...... 3

2 The Process of nitrification ...... 5

3 Ammonia-oxidation pathway ...... 10

4 Nitrite-oxidation pathway ...... 11

5 Biochemical pathway of anammox: enzymatic machinery of anaerobic ammonia

oxidation...... 13

6 Phylogenetics of the ammonia-oxidizing archaea ...... 16

7 Microscopic images of AOA...... 17

8 Distribution of amoA and 16S gene copies of AOA vs. nitrate and ammonium

concentrations...... 19

9 I. Distribution of inorganic nitrogen, ammonium, oxidation rates and archaeal

amoA and 16S rRNA in Guaymas basin...... 20

II. Vertical distribution of amoA, nitrate and ammonium concentrations in

Lake Kivu...... 20

10 Electron micrograph of maritimus strain SCM1...... 22

11 Stoichiometry of Nitrosopumilus maritimus...... 25

12 amoA gene clusters in ammonia oxidizing bacteria and archaea...... 28

13 Proposed pathways for ammonia oxidation in AOA vs. AOB...... 29

14 Modified 3-hydroxypropionate/malyl-CoA pathway in AOA...... 31

15 Nitrate increases in Lake Superior over the past century...... 35

16 Locations of A. Lake Superior and B. Lake Erie stations sampled...... 56 xi

Figure Page

17 Illustration showing the basic steps in CARD-FISH...... 60

18 Illustration showing the principle of CARD amplification step in

CARD-FISH...... 61

19 Temperature Profiles ...... 84-85

20 Gel image of PCR product targeting 16S rDNA of AOA...... 87-88

21 Neighbor-joining phylogenetic tree of 16S rDNA obtained from water column

and sediment samples from different stations in Lake Superior...... 90

22 Gel images from PCR diversity study from Lake Superior and Lake Erie...... 92

23 Neighbor-joining Phylogenetic tree of amoA sequences from AOA obtained from

Lake Superior and Lake Erie...... 94

24 Depth profile of abundance of AOA in Lake Superior using

CARD-FISH in 2010...... 99

25 Depth Profile of abundance of AOA in Lake Superior using

CARD-FISH in 2011 April samples...... 101

26 Depth profile of abundance of AOA in Lake Superior using

CARD-FISH in July 2011...... 104

27 Depth profile of abundance of AOA, and NOB (Nitrobacter and Nitrospira)

in Lake Superior Station WM 2012...... 105

28 Depth Profile of abundance of AOA in Lake Erie using

CARD-FISH in July 2011...... 107

29 Total AOA, AOB and NOB numbers in Lake Erie Station

880(84)...... 107 xii

30 Total bacterial cells per mL water sample across multiple depths

in Lake Superior...... 109-110

31 Comparison of nitrification rates versus total AOA number in Lake Superior...... 111 xiii

LIST OF TABLES

Table Page

1 Year, month, and depth each station was sampled from

Lake Superior and Lake Erie ...... 53

2 Lat/Long of the sampling stations of Lake Superior and Lake Erie...... 55

3 Oligonucleotide probe sequences used for FISH in this study...... 63

4 Percentage formamide concentration in hybridization buffers and NaCl

concentrations in washing buffer based on formamide concentrations in

the respective probes used in this study...... 68

5 20X PBS Stock Solution Recipe ...... 68

6 Primer pairs and their sequences used in PCR studies in this dissertation...... 74

7 Available ammonium, nitrate, DOC and chlorophyll a concentrations in

Lake Superior and Lake Erie between 2010-2011 ...... 81 Chapter I: INTRODUCTION| 1

CHAPTER I

INTRODUCTION

1. The Nitrogen Cycle:

The nitrogen cycle is the most complex of all the biogeochemical cycles. It is the fourth

most common element found in the cells largely as a constituent of amino acids and nucleic acids, comprising almost 12% of cellular dry weight. The element nitrogen can exist in several

+ - oxidation states ranging from +5 in ammonium (NH4 ) to -3 in Nitrate (NO3 ). It exists in its

+ - - organic form, as NH4 , Nitrite (NO2 ), Nitrate (NO3 ), nitrogen dioxide (NO2), nitrous oxide

(N2O), Nitric oxide (NO), and the most abundant form, dinitrogen gas (N2). Due to its existence in all these different oxidation states, it can function as an electron donor or an acceptor in several microbiologically-mediated biochemical processes. Although dinitrogen (N2) gas, the inert form of nitrogen, comprises of a large part of the atmosphere (78%), it has to be converted to fixed forms of nitrogen, in order to be biologically available. Of the bioavailable reservoirs of nitrogen, the ionic and organic forms of nitrogen comprise a very small fraction (Ward, Arp, and

Klotz, 2011). Chapter I: INTRODUCTION| 2

The first step of the biological cycling of nitrogen involves the step called nitrogen fixation. A group of over hundred different species of nitrogen-fixing microorganisms called diazotrophs, mostly bacteria and cyanobacteria, are responsible for conducting this first step of

the nitrogen cycle. Since the fixed form of nitrogen is essential to all living forms, these

nitrogen-fixing organisms are found almost everywhere on the planet. These organisms harbor

the nitrogenase enzyme complex, comprising two protein components, the iron protein

dinitrogenase reductase and the molybdenum iron protein dinitrogenase, which combines

nitrogen and hydrogen to produce ammonia in an energy intensive process (Dixon and Kahn,

2004; Cheng, 2008). The ammonium produced in this way is then assimilated by the cells of

these microorganisms into amino acids, protein cell wall components such as N-acetyl muramic

acid, as well as the nucleic acids. This process in the nitrogen cycle is referred to as the

ammonia assimilation or ammonia immobilization. The process in the nitrogen cycle that

reverses this assimilation, i.e., releases ammonia into the environment is known as the process of

ammonification or mineralization. This usually occurs when ammonia is released from dead or

decaying cells, which release ammonia usually due to degradation of nitrogen-containing

molecules such as peptidoglycan, proteins, nucleic acids, or , with the help of enzymes that

include nucleases, ureases and lysozymes (Galloway et al., 1998, 2008; Moir, 2011; Bernhard,

2012) (Fig. 1).

Chapter I: INTRODUCTION| 3

Fig. 1. The Nitrogen Cycle (Francis et al., 2007).

2. Nitrification:

The process of nitrification is very important with respect to nitrogen cycling, linking the

- + most oxidized form (NO3 ) and the most reduced form (NH4 ). Essentially, nitrification is a process performed by aerobic chemolithotrophic ammonia-oxidixing microorganisms, both bacteria (AOB) and archaea (AOA) in which ammonia is converted to nitrite, then nitrite oxidizing bacteria oxidize nitrite to nitrate. In the first step of the process, ammonia is first converted to hydroxylamine with the help of the enzyme ammonia monooxygenase (Amo) present in the ammonia-oxidizers, which is then converted to nitrite with the help of hydroxylamine oxidoreductase (HAO). In the next step, nitrite is converted to nitrate by nitrite-

Chapter I: INTRODUCTION| 4

oxidizing bacteria (NOB) with the help of the enzyme nitrite oxidoreductase (NO) (Kowalchuk et al., 2001) (Fig. 2). The energetics of this process is discussed later in this dissertation.

+ - - NH4 NH2OH NO2 NO3 AOA and AOB AOB NOB

Ammonia Hydroxyl Nitrite monooxygenase amine oxidoreductase

oxidoreductase During nitrification, energy is produced, which in turn is used by the autotrophic nitrifiers

to fix . This process is essentially an aerobic process, occurring in aphotic

environments, because ammonia oxidation has been found to be inhibited by light (Church et al.,

2010; French et al., 2012; Merbt et al., 2012). Ammonia-oxidizers and the nitrite oxidizers that

consume nitrite produced by AOA and AOB are most often are found in the same environmental

niches. Hence, nitrite usually does not accumulate in the environment due to these tightly-

coupled procedures.

Another important process linked to nitrification is the processes of denitrification in

which certain microorganisms reduce the nitrate produced from nitrification in multiple steps

under low-oxygen conditions. In anoxic areas or low-oxygen content zones of the environment,

when nitrate is supplied, the denitrifiers use it as an electron acceptor to perform respiration, by

reducing nitrate to nitrite, nitric oxide, nitrous oxide, and subsequently to dinitrogen gas in

several steps involving multiple enzymes. As a result, nitrification and denitrification are

dependent on one another, occurring in oxic-anoxic interfaces, and the stoichiometry of N

species greatly influences the fluxes of loss and gain of fixed nitrogen in the environment. For

example, a low level of fixed nitrogen in soil can determine the amount of fertilizers required for

agriculture. At the same time, very high levels of nitrate in a water body could be responsible for

Chapter I: INTRODUCTION| 5

the formation of harmful algal blooms causing eutrophication of an entire aquatic system (Lake

Erie LaMP. 2011; Davis, 1964) High nitrate levels are also of health concerns to human

population due to the risks of conditions such as methanoglobinemia and formation of

nitrosamines (Fewtrell et al., 2004; Kleinjans et al., 1991)

Fig. 2. The Process of Nitrification (Schleper et al., 2010).

3. Denitrification:

Denitrification is a process of nitrate reduction in which nitrate is reduced in several steps

into dinitrogen gas (Delwiche et al., 1976). This process essentially takes place in an anaerobic

condition. The denitrifying organisms are facultative anaerobes and typically belong to the

members of the group Proteobacteria. In the first step of denitrification, nitrate reductase, a molybdenum-containing membrane-integrated enzyme, aids the conversion of nitrate to nitrite in a two electron transfer process. Next the nitrite is reduced to nitric oxide with the help of the enzyme nitrite reductase. Nitric oxide is further reduced to nitrous oxide with the help of the

enzyme nitric oxide reductase, and finally the nitrous oxide is reduced to dinitrogen gas by the

Chapter I: INTRODUCTION| 6

enzyme nitrous oxide reductase. To reduce one mole of nitrate to ½ N2, a total of five electrons flow in and out of the cell membrane (Delwiche et al., 1976).

4. Chemolithotrophy:

Chemolithotrophy was first discovered by the Russian scientist Sergei Winogradsky, he

discovered two types of chemolithotrophy, namely ammonia oxidation and sulphur oxidation

(Winogradsky, 1949).

Chemolithotrophy is a process by which microorganisms can derive their energy by

utilizing inorganic substances as their electron donor. Chemolithotrophy is usually oxygen-

dependent in nature, where oxygen acts as the terminal electron acceptor in the electron transport

chain. Sources of these inorganic ions in a chemolithotroph can come from anthropological,

geological or biological sources. Chemolithotrophs usually use carbon dioxide as their carbon

source, and hence are autotrophic in nature. Organisms such as a hydrogen oxidizing bacteria can derive their energy from both chemoautrophic and a chemoorganotrophic mode of energy metabolism. That means, when organic carbon sources such as glucose or its derivatives are present, these organisms can use this glucose as their carbon source (through glycolysis and TCA cycle, and eventually into the ETC), whereas in absence of such an organic carbon source, they can use carbon dioxide directly as their carbon source, and convert it to cell material through the

Calvin cycle.

If we look at the energetics of chemolithotrophy, a reduced inorganic electron donor, e.g., ammonium, nitrite, hydrogen sulfide, or ferrous iron (Fe+2), donate electrons into an electron

transport chain in the membrane of the chemolithotroph (sometimes with the help of specialized

enzymes present in their membranes e.g., rusticyanin in iron oxidizers, hydrogenase in hydrogen

Chapter I: INTRODUCTION| 7

oxidizers, ammonia monooxygenase in ammonia oxidizers, sulphite oxidase or a reversed APS reductase in sulfate oxidizers). Electron(s) are transferred through membrane bound proteins that bind quinones, iron sulphur centers and hemes (cytochromes) depending on the type of chemolithotrophy, before reduction of oxygen, as the terminal electron acceptor. During this

process, a proton gradient is generated between the outside and inside of the cell, driving

production of ATP through ATP synthase in the membrane of the organism. During this process,

reducing power NADH is also formed from NAD+ either through direct electron donation or by

reverse electron flow depending on the reduction potential of the inorganic ion pair concerned.

For example, due to low reduction potential of H2, no energy requiring reverse electron flow is

needed in hydrogen oxidizing organisms, whereas in sulphur oxidation and ammonia oxidation a

reverse electron flow is needed due to the comparatively high redox potential of the electron

donors involved. (Dortch et al., 1990; Kowalchuk et al., 2001).

5. The nitrifiers:

The nitrifying microorganisms are those chemolithotrophs that are capable of converting

ammonium into nitrite. They consist of two types of microorganisms: those that convert

ammonia to nitrite called the ammonia oxidizing microorganisms, and the ones that converts

nitrite to nitrate known as the nitrite-oxidizing microorganisms. It has been assumed for decades

that only bacteria belonging to groups of Beta- and (Purkhold et al., 2003,

2000) are the only ammonia oxidizing taxa. In the last few years, researchers have discovered

different groups of nitrifying archaea, specifically Groups I.1a, and I.1b, recently

designated to a newly proposed Thaumarchaeota (Brochier-Armanet et al., 2008; Spang

et al., 2010). To date, it is known that both Bacteria and Archaea can perform ammonia oxidation

Chapter I: INTRODUCTION| 8

since they harbor the genes for ammonia monooxygenase, the enzyme that converts ammonia to hydroxylamine in the first step for ammonia oxidation, whereas, only limited groups of bacteria called the nitrite-oxidizers, harboring the genes for nitrite oxidoreductase, can convert nitrite to

nitrate. As a result of this, both these groups of organisms are found to be living in a consortium

in various environmental niches. (Schleper and Nicol, 2010).

5.1. Energetics of the nitrifiers and key N cycle microbes: Respiration, ATP

production and Carbon metabolism:

In ammonia oxidation, a two step process occurs. At first ammonia is oxidized to nitrite by ammonia oxidizing bacteria (e.g., Nitrosomonas) or archaea (e.g., Nitrosopumilus). Then this

nitrite is further oxidized to nitrate by a nitrite oxidizing bacteria (e.g., Nitrobacter, Nitrospira).

The total process of conversion of ammonia to nitrate is an 8 electron transfer process. The fact

that nitrite does not accumulate in most ecosystems suggests that these two processes are tightly

coupled in time and space.

5.1.1. Conversion of ammonia to nitrate:

This process is accomplished in again two steps:

Ammonia monooxygenase oxidizes ammonia to hydroxylamine. This is a two electron requiring

step, consuming 1 mol O2 in the process.

+ - 2H + NH3 + 2e + O2 NH2OH + H2O

In the next step, hydroxylamine is converted to nitrite by the periplasmic enzyme hydroxylamine

oxidoreductase (Fig. 3).

Chapter I: INTRODUCTION| 9

The enzymology of hydroxylamine to nitrite in AOB is well characterized; however in

AOA this process remains enigmatic (Kowalchuk et al., 2001; Stahl et al., 2012). Nonetheless, nitrite is the oxidized end product of both AOA and AOB metabolism.

- + NH2OH + H2O HONO + 4e + 4H

+ - 2H + 0.5O2 + 2e H2O

Therefore the overall reaction is:

- + NH4 + 3/2 O2 NO2 + 2H + H2O ΔG0 = -65.7 Kcal/ mol

This process is a 4 electron generation process, two of which are donated to a quinone

(ubiquinone) and is used in the reaction mentioned above, i.e., conversion of ammonia to hydroxylamine.

5.1.2. Nitrite oxidation:

With the help of the enzyme nitrite oxidoreductase, nitrite oxidizing bacteria convert nitrite into nitrate. In this process, at first electron from the conversion of nitrite to nitrate is donated to cytochrome c.

From here a part of the electrons go to ubiquinone oxidoreductase enzyme present in the membrane, where NAD+ is converted into reducing power NADH, which in turn is capable of converting carbon-dioxide to cell material through Calvin Cycle. This happens through a reverse electron flow since the reduction potential of NO3/ NO2 pair is very high (about 0.77 V), whereas

the reduction potential of H2O/ 1/ 2O2 is 0.84 V. NADH production is done at the expense of the proton motive force (Fig. 4).

- - NO2 + ½ O2 NO3 ΔG0 = -17.5 Kcal/ mol

Chapter I: INTRODUCTION| 10

In forward electron flow to oxygen, ATP is generated due to the proton motive force across the membrane with the help of ATP synthase in the membrane.

Fig. 3. Ammonia-oxidation Pathway (Brock Biology of Microorganisms, Madigan Martinko and

Parker).

Chapter I: INTRODUCTION| 11

Fig. 4. Nitrite-oxidation Pathway (Brock Biology of Microorgansims, Madigan Martinko and

Parker).

6. The anaerobic ammonia-oxidizers: Anammox organisms

A group of organisms can convert ammonia into dinitrogen anaerobically by using nitrate

or nitrite as an electron acceptor. These organisms, discovered very recently are called anammox, for anaerobic ammonia oxidation. They possess a specific organelle in their cell called the

anammoxosome where the anammox reaction occurs. A well studied anammox organism is

Brocardia anammoxidans. These organisms fall into the unique group of organisms (bacteria)

Chapter I: INTRODUCTION| 12

called the Planctomycetes, which contain membrane bound cellular organelles, including a

membrane-bound nucleoid similar to a nucleus (Kartal et al., 2011).

The anammox organisms obtain their nitrate or nitrite source from aerobic ammonia

oxidizers such as Nitrosomonas, and hence they have been found to inhabit environments

together with these organisms. They are capable of forming aerobic-anaerobic pockets in a

sediment or soil environment, so both these groups can coexist. The enzymatic pathway of

anammox is outlined in Fig. 5., adapted from Kartal et al., 2011.

These organisms use nitrite as an electron donor and are using carbon dioxide as a sole carbon source.

- + ’ NO2 + 2H + e- = NO + H2O (E0 = +0.38 V)

+ - ’ NO + NH4 + 2H + 3e = N2H4+ H2O (E0 = +0.06 V)

+ - ’ N2H4 = N2 + 4H + 4e (E0 = - 0.75 V)

+ 2- -1 NH4 + NO = N2 + 2H2O (ΔG0’ = -357 kJ mol )

Chapter I: INTRODUCTION| 13

Fig. 5. Biochemical Pathway of anammox: enzymatic machinery of anaerobic ammonia oxidation (Kartal et al., 2011).

7. The Ammonia Oxidizing Archaea (AOA) or the Thaumarchaota

Nitrification is crucial process in the biogeochemical nitrogen cycle. In this process,

ammonia is converted into nitrite and consequently into nitrate. Ammonia-oxidizing

microorganisms harboring ammonia monooxygenase (encoded by the genes amoABC) control

the first and rate-limiting step of this biochemical process. It has been assumed for decades that

only bacteria belonging to groups of Beta- and Gammaproteobacteria (Purkhold et al., 2003,

2000) are the only ammonia oxidizing taxa, whereas, Archaea have been considered to be a

composed largely of extremophilic organisms. In the last few years, researchers have discovered

Chapter I: INTRODUCTION| 14

different groups of nitrifying archaea, specifically Crenarchaeota Groups I.1a, and I.1b, recently designated to a newly proposed Phylum Thaumarchaeota (Brochier-Armanet et al., 2008, Spang et al., 2010). The initial reports of the abundance of marine planktonic archaea came in the 1900s discovered by culture-independent methods of studies (Venter et al., 2004; Beja et al., 2002) as well as by PCR-based 16S rRNA analyses (Delong et al., 1992, 1994; Furham et al.,

1992, 1993). Ammonia oxidizing archaea have sometimes been found to be the dominant group

responsible for nitrification in several marine and freshwater environments such as the Black Sea

(Lam et al,. 2007), Pacific Ocean (Mincer et al., 2007), Atlantic Ocean, North Sea (Wuchter et al. 2006), Southern California Bight (Beman et al., 2010), central California current (Santoro et

al., 2010), Lake Superior (Small et al., 2013) among others (Weidler et al., 2008, Hatzenpichler

et al., 2008, Auguet et al,. 2008, Pouliot et al., 2009, Lliros et al., 2010). All this evidence

together identified existing gaps of our understanding in ammonia oxidation pathways, and

towards the significance of nitrifying archaea in the biogeochemical cycling of nitrogen. Thus

understanding the physiology, and biochemistry of these organisms is of high importance

(Schleper et al., 2005; Prosser and Nicol, 2008; Stahl et al., 2012).

In 2005, the first archeon Nitrosopumilus maritimus strain SCM1 was brought into pure

culture, isolated from a marine aquarium (Könneke et al., 2005). The availability of this strain opened the doors to the possibilities of studying the genetics, biochemical activities and ecology

of these organisms, and also the possibilities of isolation and characterization of more such

nitrifiers from various other environments (Tourna et al., 2011; Hatzenpichler et al., 2008; Jung

et al., 2011). The strain SCM1 is found to be physiologically suited to grow well under lower

ammonium concentrations (Martens-Habbena et al., 2009). Such capacity of the ammonia-

oxidizing archaea to be able to scavenge ammonium from a very low ammonium concentration

Chapter I: INTRODUCTION| 15

environment is consistent with the abundance of Crenarchaeota found in such environments

(Massana et al., 1997; Mincer et al., 2007; Varela et al., 2008; Karner et al., 2001).

The strain SCM1 is phylogenetically designated to the Group I.1a Crenarchaeota (Fig. 6).

Metagenomics, phylogenetic and PCR studies reveal that the ammonia-oxidizing archaea is

distributed within the Group I Crenarchaeota clade (Schleper et al., 2005; Francis et al., 2005;

Treusch et al., 2005). Most of the mesophilic soil and marine ammonia-oxidizing archaea have

been found to be distributed into two phylogenetic groups within the group I Crenarchaeota

(Spang et al., 2010) : 1) The Group I.1a mainly comprising of the marine, water column and

sediment group, with the cultivated representative N. maritimus (Könneke et al., 2005); and 2)

the Group I.1b, usually representatives from soil and sediments with the moderately thermophilic

cultured representative gargensis (Hatzenpichler et al., 2008). was first enriched from a hot spring in Siberia in 2008. Nitrososphaera viennensis is

another such recent AOA enrichment culture from soil belonging to Group I.1b AOA (Töurna et

al., 2011).

Chapter I: INTRODUCTION| 16

Fig. 6. Phylogenetics of the ammonia-oxidizing archaea (Pester et al., 2011).

Chapter I: INTRODUCTION| 17

I

II

Fig. 7. Microscopic images of AOA.

I: Nitrosopumilus maritimus strain SCM1 (Könneke et al., 2005), II: Nitrososphaera viennensis (Töurna et al., 2011).

Chapter I: INTRODUCTION| 18

7.1. Habitat diversity of the AOA

In the past 10 years, with the help of PCR studies of 16S ribosomal rRNA and the

ammonia monooxygenase subunit-A (amoA) gene along with quantifying the archaeal

ammonia oxidizers, researchers have demonstrated the ubiquity of these nitrifers. They

have been found to be present in several marine and freshwater environments such as the

Black Sea (Lam et al., 2007), Pacific Ocean (Mincer et al., 2007), deep north Atlantic

Ocean (Agogue et al., 2008), North Sea (Wuchter et al., 2006), Southern California Bight

(Beman et al., 2010), central California current (Santoro et al., 2010) thermal springs in

the Austrian Central Alps (Weidler et al., 2008), hot springs (Hatzenpichler et al., 2008),

(Auguet et al., 2008), high Arctic Lakes (Pouliot et al., 2009), Lake Kivu in Congo

(Lliros et al., 2010), oligotrophic Alpine Lakes (Auguet et al., 2011), high mountain lakes

(Auguet et al., 2008), the rhizosphere of a freshwater macrophyte (Herrmann et al.,

2008), cold water (Radax et al., 2012), cold sulphidic marsh waters (Koch et al.,

2006), Lake Superior (Small et al., 2013), among others.

The ammonia oxidizing archaea have been found to survive extreme low

ammonia concentrations (Martens-Habbena et al., 2009). Therefore in oligotrophic

environments, where there are limited nutrients and organic carbon present, these

chemolithotrophic organisms are assumed to thrive better. (Könneke et al., 2005; Karner

et al., 2001; Dawson et al., 2000). In several studies conducted so far, the ammonia

oxidizing archaea have been found to occupy the deeper waters of the seas and oceans,

and there has been a positive correlation between their abundance and the increases of

nitrate levels and depletion of ammonium levels in these habitats (Beman et al., 2008;

Lliros et al., 2010; Lam et al., 2007; Wuchter et al., 2006) (Fig. 7, Fig. 8).

Chapter I: INTRODUCTION| 19

Fig. 8. Distribution of amoA and 16S gene copies of AOA vs. nitrate and ammonium

concentrations (Wuchter et al., 2006).

Chapter I: INTRODUCTION| 20

I.

II.

Fig. 9. I. Distribution of inorganic nitrogen, ammonium, oxidation rates and archaeal amoA and

16S rRNA in Guaymas basin (Beman et al., 2008); II. Vertical distribution of amoA, nitrate and

ammonium concentrations in Lake Kivu (Lliros et al., 2010).

Chapter I: INTRODUCTION| 21

7.2. Cell structure, Physiology, Growth of the AOA

Nitrosopumilus maritimus is the single isolated pure-culture strain of an

ammonia-oxidizing Group I.1a Thaumarchaeon till date (Könneke et al., 2005). The cells

of this organism are rod-shaped, very small in size, ranging between 0.5 µm to 0.9 µm in

length and 0.25 µm in width ((Fig. 7). Electron microscopic evaluation of its cellular

structure reveal the absence of cellular structures such as flagella, carboxysomes,

glycogen granules, polyphosphate particles or invaginations of the cytoplasmic

membrane (Fig 10). The cells have been at times found to contain an area of high

phosphorus content possibly functioning in phosphorus storage (Urakawa et al., 2011).

As expected for an archaeon, the cells do not contain an outer membrane or a

peptidoglycan cell wall. The cytoplasmic membrane is surrounded by a S-layer

containing a dense array of surface proteins. The membrane lipid Glycerol dialkyl

glycerol tetra-ether (GDGT) is present in the membranes of these organisms, the most

abundant core membrane lipid being (Schouten et al., 2008).

The culture of SCM1 grows up to 1.4 X 107 cells/ mL at 28ᵒC when grown at a

concentration of 500 µM ammonium. Addition of organic compounds inhibits the growth

of this organism (Könneke et al., 2005). The strain SCM1 can grows at the maximum rate

of 0.027/ h (Td ~ 26 h) at 30ᵒC, which is comparable to the ammonia oxidizing bacteria.

However, the SCM1 strain grows only at the temperature range of 20ᵒC – 30ᵒC. The

optimum pH for its growth is found to be between 7.0 and 7.8, and it exhibits no

ammonia-oxidation activity at pH lower than 6.7 (Könneke et al., 2005; Urakawa et al.,

2011). SCM1 has a low tolerance for higher ammonium concentrations, its growth ceases

at an ammonium concentration between 2mM and 3mM. The maximum per cell

Chapter I: INTRODUCTION| 22

-1 ammonium oxidation rate observed in this strain thus far is about 0.56 fmol NH3 cell hr

-1 (Könneke et al., 2005; Martens-Habbena et al., 2009).

Fig. 10. Electron Micrograph of Nitrosopumilus maritimus strain SCM1 (Urakawa et al.

2011). Rib: Ribosomes, Nuc: Nucleus, CM: Cell Membrane, SL: Slimy Layer.

Chapter I: INTRODUCTION| 23

7.3. Stoichiometry and Kinetics of ammonia oxidation of AOA

The ammonia oxidation kinetics and the conditions at which the ammonia

oxidizing archaea vs. the ammonia oxidizing bacteria dominate is of great importance in

order to be able to understand the function these organisms perform in nature.

Nitrosopumilus maritumus is the model organism used so far to study the kinetics and

stoichiometry of the AOA. The overall kinetics of ammonia oxidation in N. maritimus

has been found to be similar to that of the AOBs (Martens-Habbena et al., 2009) (Fig.

11).

N. maritimus is an extreme oligotroph, having an extremely low half saturation

+ constant (Km) value of approximately 132 nM for total ammonia (NH4 + NH3) (Martens-

Habbena et al., 2009). Nitrososphaera gargensis, the I.1b thermophilic Thaumarchaeota

also exhibits adaptations to lower ammonium concentrations (Hatzenpitchler et al.,

2008). By contrast, the cultured representatives of the AOB show an almost 100 fold

higher requirement for ammonium concentration having Km values ranging between 46

µM to 1780 µM total ammonium (Martens-Habbena et al., 2009; Koper et al., 2010).

Addition of only 200 nM ammonium to a resting culture can raise the rate of ammonia-

oxidation by the strain SCM1 by more than 50%. In fact, the SCM1 strain has been

observed to be intolerant to ammonium concentrations over 1mM. Similar properties of a

lower ammonium affinity have been reported by cultured representatives of AOA in

contrast to cultured AOB isolated from two Ohio lakes (French et al., 2012). Thus it is

likely the AOA can be viewed as the ammonia oxidizing taxa in oligotrophic

environments worldwide. Furthermore, the ammonium affinity of the strain SCM1 has

been found to be higher by 30 fold than the most oligotrophic organisms known so far

Chapter I: INTRODUCTION| 24

responsible for ammonia assimilation. This indicates towards a possible greater role of the AOA in nitrogen transformations in the nitrogen cycle (Urakawa et al., 2011). For example, considering the conditions in a soil environment, the Km in soil ranging between

2 µM and 42 µM (Martens-Habbena et al., 2009), both AOA and AOBs can thrive in soil depending on ammonia availability and competition between them to scavenge available ammonia. In a soil environment where the ammonia concentrations are less than 15 µg ammonium g-1 dry weight of soil, the AOA thrive better (Pratcher et al., 2011), whereas, the ammonia oxidizing bacteria are found to be the dominant nitrifiers in soil containing more than 100 µg ammonium/ g dry weight of soil (Pratscher et al., 2011; Xia et al.,

2011). Moreover, it has been observed that in a soil environment, where the soil is unfertilized, the AOA has been found to predominate over the AOBs (Lehninger et al.,

2006), whereas in fertilized soils with a higher ammonium concentrations, the AOBs are the dominant nitrifiers (Lehninger et al., 2006; Jia and Conrad, 2009). The AOA has been so far found to be aerobic, requiring concentrations of oxygen to approximately 4

µM, exhibiting no growth anaerobically (Stahl et al., 2012).

Chapter I: INTRODUCTION| 25

Fig. 11. Stoichiometry of Nitrosopumilus maritimus (Urakawa et al., 2011).

7.4. The biochemistry of ammonia oxidation and Nitrous oxide production by the

AOA

Annotation of the sequences of AOA suggests a marked difference in the

biochemical pathway ammonia oxidation in these organisms when compared to their

bacterial counterparts. Most genes for ammonia oxidation found in ammonia-oxidizing

bacteria are found to be absent in the AOA except the genes coding for the ammonia

monooxygenase enzyme (AMO) (Fig. 12), although they also are found to be

evolutionarily divergent. A homolog of the HAO (hydroxylamine oxidoreductase), the

enzyme responsible for the conversion of hydroxylamine to nitrite, is absent in AOA.

Moreover, they also lack bacterial c- type cytochrome genes in their . However,

the genome of N. maritimus encodes an array of plastocyanin-like copper proteins. This

suggests that instead of the iron-dependent system of electron transfer in the bacterial

ammonia-oxidation mechanism, the AOA uses a copper-dependent system of electron

transfer (Walker et al., 2010, Schleper et al., 2010, Urakawa et al., 2011, and Stahl et al.,

Chapter I: INTRODUCTION| 26

2012). Due to these differences of the genomic structure of AOA for ammonia-oxidation vs. their bacterial counterparts, the biochemical pathway for ammonia oxidation is still considered to be incomplete. Two pathways have been proposed to describe the completed ammonia oxidation pathway to nitrite (Walker et al., 2010, Schleper et al.,

2010, Stahl et al., 2012).

The first pathway suggests hydroxylamine being oxidized by a novel enzyme through a unique biochemical pathway through a copper hydroxylamine oxidoreductase

(CuHAO). In this case, the model suggests that the four electrons produced by the oxidation of hydroxylamine to nitrite is transferred to small copper containing plastocyanin electron carriers. Later, with the help of a membrane-bound quinone reductase, the electrons are further transported into the quinone pool, two of which are further transported into the electron transport chain, and the remaining two are recycled back to AMO (Walker et al., 2010; Stahl et al., 2012).

A second pathway proposes that nitric oxide (NO), produced by the reduction of nitrite through a proposed copper-dependent nitrite reductase plays an important role in the transport of electrons to AMO in the ammonia-oxidation pathway of the AOA, eliminating the need for recycling of electrons from the quinone pool in the first step for ammonia oxidation, as seen in the previous pathway. This pathway suggests the role of nitroxyl as an intermediate (Fig. 13), an alternative to hydroxylamine, with nitroxyl oxidoreductase (NxOR) as the enzyme mediating the conversion of nitroxyl to nitrite in the AOA (Walker et al., 2010). Therefore, in this pathway (Fig. 13), NO serves as a redox shuttle for delivering electrons to AMO, whereas the two electrons generated by the oxidation of nitroxyl to nitrite is shuttled into a quinone pool directly through a quinone

Chapter I: INTRODUCTION| 27

reductase (Walker et al., 2010; Stahl et al., 2012). Additionally, a NO reduction pathway yielding N2O may be present paralleling NO detoxification seen in AOB.

Indeed recent isotopic studies reveal the production of nitrous oxide (N2O) by enrichment cultures of AOA from Pacific Ocean (Santoro et al., 2011) as well as from pure culture N. maritimus and in tropical ocean areas (North Atlantic ocean and South

Pacific Ocean) (Löscher et al., 2012). In fact these isotopic studies also suggest that the ammonia-oxidizing archaea are largely responsible for the oceanic nitrous oxide production (Santoro et al., 2011). These studies relating archaeal nitrification with nitrous oxide production may provide key ingredients to the understanding of the biochemical pathway for nitrification in AOA in future (Stahl et al., 2012).

Chapter I: INTRODUCTION| 28

Fig. 12. amoA gene clusters in ammonia oxidizing bacteria and archaea (Urakawa et al., 2011).

Chapter I: INTRODUCTION| 29

Fig. 13. Proposed pathways for ammonia oxidation in AOA vs. AOB (Stahl et al., 2012).

7.5. Autotrophic, Mixotrophic and Heterotrophic growth in AOA

Our present knowledge of how AOA grow is incomplete. It is not completely

known whether they are strict autotrophs. For chemolithoautotrophic growth, unlike the

AOBs that use a Calvin cycle, the AOA use a modified 3-hydroxypropionate/ 4-

Chapter I: INTRODUCTION| 30

hydroxybutyrate pathway, a modified 3-hydroxypropionate/malyl-CoA pathway (Fig.

14). Although the fact that they can grow completely on inorganic medium has been established, their genome sequences reveal that they may have genes responsible for utilization of organic sources of nitrogen, phosphorus and carbon. Potential transporters of various organic substances such as different amino acids, glycerol, sulfonates, dipeptides/oligopeptides has been identified in the genomes of N. maritimus, C. symbiosum and on Thaumarchaeota fosmids. Moreover, a possibly incomplete TCA cycle is also present in N. maritimus, which is putatively used for biosynthesis purposes and not for . (Walker et al., 2010; Martin-Cuadrado et al., 2008; La

Cono et al., 2010; Pester et al., 2011; Urakawa et al., 2011). Therefore, based on this evidence as well as isotopic studies revealing the presence of radiocarbon in archaeal membrane lipids (Ingalls et al., 2006), a mixotrophic capacity of growth cannot be ruled out in these organisms. Some studies also show that soil AOA can grow heterotrophically when nitrification is inhibited (Jia et al., 2009). Hence, it can be assumed that further studies and research on the physiological characteristics of the pure culture N. maritimus will reveal in future the various modes of growth possibilities in AOA.

Chapter I: INTRODUCTION| 31

Fig. 14. Modified 3-hydroxypropionate/malyl-CoA pathway in AOA (Urakawa et al., 2011).

8. Nitrification in the Laurentian Great Lakes: two lakes, two different stories

This study is mainly based on two Laurentian Great Lakes: Lake Superior and

Lake Erie. Although these lakes are connected as parts of the same waterway, the

physiological and geochemical and biological characteristics of these two lakes vary

greatly, and in most respects they exhibit distinctly different characteristics. Therefore,

they serve as the perfect sites to study microbial community structures and compare their

functions and role in biogeochemical cycling of nitrogen.

Chapter I: INTRODUCTION| 32

8.1. Structure and characteristics of Lake Superior vs. Lake Erie

Lake Superior is the largest freshwater lake in the world having the largest

surface area ranging to approximately 82,100 km2. Of all the Laurentian Great Lakes,

Lake Superior is also the deepest with a maximum depth of 406 m, and an average depth

of 147 m. It is also the coldest with an average temperature of 4ᵒC. Lake Superior is oxic

throughout the water column; oxygen concentration is at near-saturation from the surface

to several cm into the sediments. It is considered to be ultraoligotrophic in due to its

extreme low phosphorus concentrations (Munawar et al., 2009; Ostrom et al., 1997). The

two biogeochemical parameters considered to be extreme in Lake Superior are the

3- concentrations of dissolved phosphorus and nitrogen. Phosphorus (PO4 ) concentrations

in the lake are extremely low (nM), whereas the nitrate concentrations are very high (25

µM), which creates a major N: P stoichiometric imbalance in the lake (Sterner et al.,

2007; Sterner, 2011) (Fig. 15). Ammonium concentrations average is 210 nM (Kumar et

al., 2007).

Lake Erie on the other hand is the shallowest of all the Great Lakes with an

average depth of 20 m, and is also the warmest of the lakes. The lake exhibits seasonal

hypoxia zones and anaerobic sediments.. Lake Erie is mesotrophic in nature, having the

highest production rate among all the Great Lakes. Lake Erie is mainly divided into three

separate basins: western, central, and eastern. The western basin of Lake Erie is

eutrophic, the central basin is mesotrophic, whereas the eastern basin is oligotrophic.

Therefore, Lake Erie as a whole can be seen as three different lakes due to the varying

trophic conditions as we move from west to east of the lake (Matisoff et. al., 2005). At

present the western basin of the lake experiences periodic seasonal toxic algal blooms,

Chapter I: INTRODUCTION| 33

mainly caused due to Microcystis spp., which causes concerns of water contamination

and health hazards due to toxic microcystin production. It is also very high in nutrient

content, due to nutrient loadings into the lake by agricultural and industrial runoff from

the highly populated watershed of the lake (Munawar and Weisse, 1989).

8.2. Nitrification and trends in nitrate levels in Lake Superior vs. Lake Erie

Lake Superior is extremely limited in phosphorus content whereas is extremely

high in nitrate levels. Ammonium concentration in the lake is found to be low,

averaging to about 0.21 µM. However, the nitrate levels in Lake Superior have been

increasing over the past hundred years. Over a century, the nitrate levels have been

found to increase from 6 µM in 1905 to 26 µM in present day amounting to about a

five-fold increase (Sterner et al., 2007; Sterner, 2011) (Fig. 15). According to Alfred

Redfield (1958), the elemental composition of is uniform, and changes in C,

N, and P concentrations in an aquatic system determined the rates of synthesis or

decomposition. This ratio called the “Redfield ratio” between C: N: P is 106:16:1 and

can be used to help predict production rates in limnology and oceanographic studies. In

modern days, N-limitation and P-limitation is differentiated in an aquatic system by

using the N:P ratio of 16:1 (Geider and La Roche, 2002). Due to the very high nitrate

levels in Lake Superior (26 µM) compared to its very low phosphate concentrations

- 3- (3nM), the NO3 : PO4 ratio in the lake estimates to 8700, which is orders of magnitude

higher than Redfield. Total P: Total N ratios exceed 300, also reflecting the extreme

stoichiometric imbalance (Sterner, 2011). Therefore, an investigation of the sinks and

sources of nitrogen in the lake can provide us with a better understanding of underlying

Chapter I: INTRODUCTION| 34

mechanisms contributing to N buildup. The watershed around Lake Superior is mostly forested, very scarcely populated, with little human, agriculture and industrial activities, therefore there is less possibilities of nutrient loadings into the lake due to these factors.

Atmosphere-derived nitrogen accumulations have been shown to be insufficient to account for the nitrate build-up in the lake (Sterner et al., 2007). Moreover, N and O stable isotopic studies have revealed a strong differences between nitrate obtained from in-lake processed versus precipitation or in-flow processes, providing strong evidence for in-lake nitrification processes in the nitrogen cycle to be responsible for this nitrate increases in the lake (Finlay et al., 2007).

Chapter I: INTRODUCTION| 35

Fig. 15. Nitrate increases in Lake Superior over the past century (Sterner et al., 2007).

Chapter I: INTRODUCTION| 36

9. Objective and significance of the current study

The largest of the Great Lakes, Lake Superior has been found to increase in nitrate level

several fold over the past century with more increases observed in recent years (Sterner et al.

2007). This has yielded a major stoichiometric imbalance of N: P in the lake which is of likely

importance in structuring the planktonic community in the lake. Many such freshwater lakes all

over the world have been found to have high levels of nitrate, and hence, this study will also

contribute to a broader aspect of understanding this phenomenon worldwide. Therefore, the

objective of this dissertation is to study the ammonia-oxidizing community in Lake Superior, and compare it with Lake Erie based on their physiological differences. Over a period of several research cruises completed in 2010, 2011 and 2012, multiple sets of water samples from Lake

Superior and Lake Erie has been collected from several different stations on these lakes at various depths in different seasons. These samples have been processed using CARD-FISH, PCR and phylogenetics to generate a detailed and comparative analysis of the structure and diversity

of the AOA and AOB community and their role in the nitrification rates in these two lakes. This

study also gives us insights into changes in AOA structure based on the differences in thermal profiles of the lakes at different season in different times of the year at various depths.

Hypothesis:

Given our understanding of nitrate increases in Lake Superior, I hypothesize that:

Nitrate increases in Lake Superior is driven by biological nitrification by a unique ammonia- oxidizing archaeal population.

Chapter I: INTRODUCTION| 37

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Chapter II: MATERIALS AND METHODS| 52

CHAPTER II MATERIALS AND METHODS

1. Sampling

Water samples were collected on Great Lakes research cruises between Spring

2010 and Fall 2012. Water samples were collected both for DNA extractions and CARD-

FISH analysis. For DNA analysis, water samples were filtered through Sterivex

cartridges (Millipore). Up to 4 liters of lake water was filtered through Sterivex cartridges

to obtain appropriate biomass inside the cartridge. Filters were sealed with clay, frozen in

liquid nitrogen and stored at -80ᵒC. Triplicates of 10 mL water samples from each station

depths were collected for CARD-FISH analysis. Water samples were fixed with 2%

formaldehyde solution, and then passed through a 0.2 µm polycarbonate filter, using a

supporting filter of 5 µm pore size. Three filters from each sample were prepared, and

stored at -20ᵒC for further processing.

In 2010, water samples were collected from Lake Superior during the isothermal

mixing in May, and during stratification in August and October. In 2011, Lake Superior Chapter II: MATERIALS AND METHODS| 53

was sampled in April when the lake was also isothermally mixed. Lake Superior and

Lake Erie were also sampled extensively in July 2011 during a seventeen day research

cruise during the stratified period. In 2010, Lake Superior stations WM and CD1 were

sampled, while in July 2011, Lake Superior stations CD1, EL0, EL2, Michipicoten,

Whitefish Bay, and Black Bay were sampled at several depths. Lake Erie was sampled at

various stations at different depths: Maumee Bay in the western basin, CCB3, 1326, and

880(84) in the central basin, and EC23 and 91M in the eastern basin. Sampling sites, year

and months sampled, and depths sampled from each station in the lakes are listed in

Table 1 and Fig. 16, as are the Latitude/Longitude coordinates in Table 2.

Table 1. Year, month, and depth each station was sampled from Lake Superior and Lake Erie.

YEAR MONTHS LAKE STATIONS DEPTHS SAMPLED (m)

2010 May Superior CD1 5, 235

WM 2, 155

2010 August Superior WM 2, 5, 20, 40, 60, 80, 100,

130, 155, sediment overlay

CD1 2, 8, 15, 20, 35, 150, 244,

sediment overlay

2010 October Superior WM 5, 20, 155, sediment

overlay

CD1 10, 150, 245, sediment

overlay

Chapter II: MATERIALS AND METHODS| 54

2011 March Superior Sterner A, B, C, D Surface water (2m)

2011 April Superior CD1 5, 15, 30, 50, 100, 150,

200, 245

WM 5, 15, 155

2011 July Superior EL0 2, 7, 12, 25, 40, 50, 75,

100, 145, 160

EL2 5, 10, 30, 50, 60, 75, 100,

150, 185, 195

Michipicoten 5, 15, 78

White Fish Bay 15, 60

Black Bay 2, 8

Sleeping Giant 5, 240, 272

2011 July Erie Maumee Bay 1, 7

1326 1, 5, 10, 12, 17, 20

CCB3 5, 9, 11, 15, 18, 20, 22

880(84) 1, 5, 10, 15, 20

EC23 10, 20, 30, 42, 50, 60

91M 1, 4, 8

Chapter II: MATERIALS AND METHODS| 55

Table 2. Lat/Long of the sampling stations of Lake Superior and Lake Erie.

LAKE STATION LATITUDE LONGITUDE

Superior CD1 47.0080 -91.43200

WM 47.33300 -89.80000

EL0 47.75000 -87.50000

EL2 47.00000 -85.50000

White Fish Bay 46.66700 -84.83300

Black Bay 48.50000 -88.60766

Michipicoten 47.12300 -84.65500

Sleeping Giant 48.22282 -88.90600

Lake Erie CCB1326 41.73300 -81.69800

91M 41.84100 -82.91700

880(84) 41.91700 -81.63300

EC23 42.30000 -85.63300

CCB3 42.10090 -81.27714

Chapter II: MATERIALS AND METHODS| 56

A

B

Fig. 16. Locations of A. Lake Superior and B. Lake Erie stations sampled.

Chapter II: MATERIALS AND METHODS| 57

2. DNA Extraction Procedures:

2.1. DNA Extraction from environmental samples:

For DNA extractions from Sterivex cartridges, a standard DNA Extraction

technique using a phenol-chorloform method was followed (Ilikchyan et al. 2009).

Sterivex cartridges were injected with 2mL of STET buffer containing 50mM Tris-

HCl, 50mM EDTA, 5% Triton X-100, and 8% sucrose (pH. 8.0), amended with 10

mg ml-1 of lysozyme, and incubated for an hour at room temperature. After removal

of the liquid by a syringe and transfer to a 15 mL Falcon tube, one third the volume of

10% SDS was added to the solution and incubated at 65ᵒC for about 40 minutes. Next,

one third volume of 5M NaCl was added to the same tube, and incubated at 65ᵒC for

another 20 minutes, followed by a phenol-chloroform-isoamyl alcohol extraction

(25:24:1).

2.2. DNA Extraction from cultures:

For DNA extraction from cultures, the UltraClean Microbial DNA Isolation Kit

from MoBio Laboratories Inc. (Catalog No. 12224-50) was used. Approximately 1.8

ml of archaeal culture was centrifuged at 10,000 g for 30s. The supernatant was

decanted and more culture was added to repeat the process for at least 3-5 times until

a faintly visible pellet was obtained (Mike Schlais, personal communication). The

pellet was subjected to the procedure described in the manufacturer’s instructions. At

the end of the procedure, approximately 10 ng of DNA was obtained and stored at -

20ᵒ C.

Chapter II: MATERIALS AND METHODS| 58

3. Catalyzed Reporter Deposition Fluorescence in-situ Hybridization (CARD-FISH):

Catalyzed Reporter Deposition Fluorescence in-situ Hybridization, or in short

“CARD-FISH” is a DNA-based cultivation-independent method that is widely used to

identify, enumerate, and characterize the composition and dynamics of specific microbes

in a microbial community.

3.1. FISH Principle:

Since the discovery of the use of the highly conserved ribosomal RNA as a

method of taxonomic species differentiation between bacteria, archaea, and eukarya

(Woese et al., 1978 and 1990), the use of SSU rRNA sequencing and subsequent

formation of a large database of these sequences is now the most popular method of

identifying uncultured microorganisms from environmental sources, given the fact that

characterization by culturing of microorganisms is cumbersome, sometimes impossible,

and may not cover the entire diversity of a particular environmental niche. The use of

SSU rRNA sequences as an identification tool for specific organisms has gained

momentum and today there are more than 500,000 SSU rRNA database entries. The

technique of FISH was introduced first in the 1980s (DeLong et al 1989 and Amann et al.

1990), generally targeting the ribosomal RNA sequences within a cell using

complementary oligonucleotide probes. It is also referred to as a form of phylogenetic

staining (DeLong et al. 1989), and the principle of CARD-FISH method is outlined in

Fig. 17.

3.2. CARD FISH:

CARD-Amplification is a process of increasing the sensitivity the technique of

FISH by increasing the fluorescence yield from hybridized cells (Pernthaler et al 2002).

Chapter II: MATERIALS AND METHODS| 59

Here, covalently crosslinked Horseradish Peroxidase (HRP) labeled oligonucleotide probes are used followed by a secondary signal amplification step with fluorescently- labelled tyramide (Trebesius et al. 1994 and Schönhuber et al. 1997). The HRP-labelled probe catalyzes the deposition of multiple fluorescently labeled tyramide molecules, providing with a staining with a significantly enhanced signal (Fuch and Amman 2008,

Thielle et al. 2011). The principle behind the tyramide amplification step is outlined in

Fig. 18. For CARD amplification, two important modifications needs to be made from

FISH, such as: 1) embedding in agarose to minimize loss of cells, and 2) incubation in fluorochrome labeled tyramide at a temperature of 46ᵒC (Ishii et al. 2004).

Chapter II: MATERIALS AND METHODS| 60

Fig. 17. Illustration showing the basic steps in CARD-FISH (Thiele et al. 2011).

Chapter II: MATERIALS AND METHODS| 61

Fig. 18. Illustration showing the principle of CARD Amplification step in CARD-FISH

(Thiele et al. 2011)

3.3. Probes:

The probes were labeled at their 5’ end with HRP and HPLC purified (ordered

from Biomers, Germany). Depending on their specificity and stringency of

hybridization, different formamide concentrations were used for the hybridization

buffer of each probe (Table 4). The various probes used in this study and their

respective sequences are listed in the Table 3. The probes were prepared in sterile

MilliQ water at a final concentration of 50 ng mL -1.

3.3.1. Probe preparation and testing:

Probe stocks were delivered from the manufacturer in a lyophilized solid

condition. Exact probe concentrations were determined at first by adding 100 µL

Chapter II: MATERIALS AND METHODS| 62

of PCR-grade water to the probe stock for each OD (1 OD = 100 µL) with no

-1 vortexing or shaking, assuming that 1 OD260nm = 20 ng µL DNA (Thiele et. al.

2011). The OD value is provided by the manufacturer. The vial was then kept

overnight in dark at 4ᵒC, taking care not to freeze probes so that the enzymatic

activity could be retained. Typically, small amounts of working concentrations of

probes were prepared and kept at 4ᵒC for use. OD was measured at a nanodrop

spectrophotometer (Model Nanodrop1000, Thermo Scientific). To measure exact

probe concentration, it was important to consider the fact that the HRP enzyme

also has a heme-dependent broad absorption maximum at 404 nm which also

contributed to the measured absorbance at 260 nm (Thiele et al. 2011, and

personal communication with Dr. Bernhard Fuchs, Mina Bizic Ionescu and Stefan

Thiele). To do this, the UV-VIS mode was selected on the nanodrop

spectrophotometer and absorbance was measured at 206 nm (HRP and DNA) and

404 nm (HRP).

The exact concentration of the probe was determined using the following

formula:

OD260 (Oligo) = OD260 – OD404 X 0.276

[0.276 = correction factor for the contribution of HRP to the absorption

maximum at 260 nm] (Thiele et al. 2011, and personal communication with Dr.

Bernhard Fuchs and Mina Bizic Ionescu).

3.3.2. Probes used in this study:

Specific probes targeting total bacteria were used in this study on each

sample using an equal concentration mixture of Eub338I, Eub338II and

Chapter II: MATERIALS AND METHODS| 63

Eub338III. A non-hybridizing negative control probe, NON338 was used on each

sample to target and minimize visible false positive signals. The probe Cren554,

initially designed to target the Marine Group I.1a Crenarchaeota was used to

target the ammonia-oxidizing archaeal community in the Great Lakes. Probes

specific to total ammonia-oxidizing bacteria and total nitrite oxidizing bacteria

were also used in a separate study on the same samples to be able to obtain a total

picture of the entire nitrifying microbial community in these lakes. (see Table 3

for probe detail and references).

Table 3. Oligonucleotide probe sequences used for FISH in this study.

FISH TARGET SEQUENCE(5’-3’) REFERENCE

PROBE ORGANISM

Eub338I Bacteria GCTGCCTCCCGTAGGAGT Amann et al, 1990

Eub338II Supplement to GCAGCCACCCGTAGGTGT Daims et al, 1999

Eub338I

Eub338III Supplement to GCTGCCACCCGTAGGTGT Daims et al, 1999

Eub338I

NON338 control ACTCCTACGGGAGGCAGC Wallner et al. 1993

Cren554 Crenarchaeota TTAGGCCCAATAATCMTCCT Massana et al, 1997

3.4. Controls:

In order to identify a false positive signal, the non-hybridizing probe NON338

was used on every sample. Usually there was no signal visible under NON338. If any

Chapter II: MATERIALS AND METHODS| 64

were observed, they were counted and subtracted from the total values to subtract

false positive signals. As positive controls, a total bacterial probe made from an equal

concentration mixture of Eub338I, Eub338II and Eub338III (Table 4) was used on

each sample. All the signals on Eub338I-III were counted each time, and the total

value was compared to the average bacterial counts observed on Lake Superior vs.

Lake Erie. This also helped to target errors in hybridization. Each filter from each

sample was also counter-stained using the fluorescent nuclear stain DAPI (4',6-

diamidino-2-phenylindole). All DAPI signals were counted on each sample on ten

fields and compared with flow cytometry counts to obtain an average estimate of total

cells on each sample. This also gave a provision of calculating the percentage signals

of total cells present in a particular sample (Pernthaler et al. 2003).

3.5. CARD-FISH Procedure:

The process of CARD-FISH started with sample fixation and filter preparation,

followed by embedding, permeabilization, hybridization, washing, CARD

amplification, and finally DAPI staining and mounting. After preparation, slides were

observed by fluorescence microscopy using specific filter sets for FITC and DAPI

(outline in Fig. 17). Cells were counted to obtain a total cell count per mL of water

sample. The standard protocol for CARD-FISH as mentioned in Sekar et al. 2002 was

followed with some modifications. Methods used in this study are outlined below:

3.5.1. Sample fixation and filter preparation:

Depending on the amount of biomass usually found in the lakes sampled

(5 mL standard for Lake Erie and 10 mL standard for Lake Superior), 5-10 mL of

Chapter II: MATERIALS AND METHODS| 65

water sample was at first fixed using formaldehyde solution at a final working

concentration of 1% (Thiele et. al. 2012). The samples were kept at 4ᵒC overnight

or at room temperature for two hours before filtration. The samples were prepared

in triplicates, passing them through polycarbonate filters of 0.1 µm pore-size and

25 mm in diameter. A supporting filter of 5 µm pore size was used every time to

ensure an even distribution of the biomass in the water samples. The filters were

dried on a tissue paper for 30 minutes, and then stored at -20ᵒC. Filters at this step

were stored at -20ᵒC for months (Sekar et. al. 2003).

3.5.2. Embedding:

In order to ensure minimum loss of cells after the various washing steps,

the filters were embedded in freshly-prepared 0.1% low gelling point agarose.

The filters were dipped into the molten agarose, and placed face-down on a flat

parafilm sheet on a glass plate, and kept in a 37ᵒC oven until completely dry (30

minutes approximately). Next, 96% ethanol was added to soak the dried filters,

allowing their removal from the parafilm, and transferred to a tissue paper and

dried. At this stage, the filters could be be stored at -20ᵒC for several weeks until

further processing (Sekar et. al. 2003).

3.5.3. Permeabilization:

After embedding, the filters were permeabilized using freshly prepared

lysozyme solution. The lysozyme was prepared at a concentration of 10mg ml-1 in

a buffer containing 0.05 M EDTA, and 0.1 M Tris-HCl (pH 8.0). The filters were

incubated in the lysozyme solution separately in 1.5 mL eppendorf tubes at 37ᵒC

for 1 hour. After this, the filters were washed in excess MilliQ water. After

Chapter II: MATERIALS AND METHODS| 66

permeabilization, the filters were incubated in 0.01 M HCl for about 20-30

minutes, followed by washing in excess MilliQ water for one minute. Filters were

then washed in 96 % ethanol for one minute, dried completely and then stored at -

20ᵒC until further processing.

3.5.4. Hybridization: Hybridization is the most important and longest step in the

procedure of CARD-FISH. The process is outlined below.

3.5.4.1. Hybridization buffer preparation: Each probe requires a specific

hybridization buffer based on the formamide concentration at which the

probe’s specificity has been previously tested (see Table 4 for references).

Hybridization buffers for each probe were prepared separately and can be

stored at -20ᵒC for future use. Twenty mL of the hybridization buffer

contained 3.5 mL 5 M NaCl, 0.4 mL of 1 M Tris-HCl (pH 8.0), 20 µL of 20 %

SDS, x mL of formamide (depending on the formamide concentration

required by each probe, see Table 4), 2 mL of Blocking reagent (10%

concentration, purchased from Roche, prepared before according to

manufacturer’s instructions), 2.0 g of dextran sulfate and MilliQ water up to

the 20 mL volume. The mixture was heated to 60ᵒC and stirred until the

dextran sulfate dissolves. The buffer was then dispensed in 0.5 mL Eppendorf

tubes in 400 µL volumes, and stored at -20ᵒC (MPI CARD-FISH course script

by Dr. Fuchs).

3.5.4.2. Hybridization procedure and washing: Depending on the total number of

probes to be used, each filter was first cut into small sections and labeled

Chapter II: MATERIALS AND METHODS| 67

accordingly. Labeling was always done with pencil and not with markers.

Hybridization was performed in 0.5 mL Eppendorf tubes (personal communication, Mina Bizic Ionescu) containing 400 µL hybridization buffer and 4 µL of probe. The hybridization buffer: probe concentration was usually kept in the ratio of 100:1. Filters were then dipped into the hybridization solution, and incubated in a hybridization oven (Model Hybaid Micro-4,

Midwest Scientific) at 46ᵒC for at least 2 hours.

Washing buffers were prepared fresh each time at a final volume of 50 mL for each probe separately. Fifty mL of wash buffer contained 0.5 mL of 0.5 M

EDTA, 1.0 mL of of 1.0 M Tris-HCl, pH. 8.0, x µL of NaCl depending on probe (Table 4), 25 µL of 20% SDS, and MilliQ water to a final volume of 50 mL. The wash buffer was first pre-warmed at 37ᵒC. The filter pieces were transferred to washing buffer at 48ᵒC for 15 minutes. After washing, the filters were rinsed in 50 mL of 1X Phosphate Buffer Saline (20X PBS stock solution recipe: Table 5) at room temperature for another 15 minutes. At no point of the washing steps after hybridization, the filters were allowed to air dry.

Chapter II: MATERIALS AND METHODS| 68

Table 4. Percentage formamide concentration in hybridization buffers and NaCl concentrations in washing buffer based on formamide concentrations in the respective probes used in this study.

Probe Target organism % Formamide NaCl µL 5M Reference concentration in concentration NaCl hybridization (M) in 50 buffer mL Eub338I- Most bacteria plus 35 0.080 700 Amann et

III Planctomycetales al, 1990,

and Daims et al,

Verrucomicrobiales 1999

NON338 Non hybridizing 35 0.080 700 Wallner et

control al. 1993

Cren554 Crenarchaeota 0 0.900 9000 Massana et

al, 1997

Table 5. 20X PBS Stock Solution Recipe

Dissolved the following order per liter H2O:

1. 28.8 g Na2HPO4

2. 160.00 g NaCl

3. 4.80 g KH2PO4

4. 4.00 g KCl

Adjust to pH 7.2 with 10 M NaOH. Filter/autoclave and store at room temperature.

Chapter II: MATERIALS AND METHODS| 69

3.5.5. CARD Amplification:

For the CARD amplification step, a fresh solution of 0.15 % H2O2 in PBS

ᵒ was prepared by adding 1 µL of 30 % H2O2 to 200 µL of PBS at 4 C.

Amplification buffer is mixed with H2O2 solution in a ratio of 100:1.

Fluorescently labeled tyramide containing 1 mg dye mL-1 was added and mixed

well. The fluorescent dye FITC (fluorescein isothiocyanate) was used for

tyramide labeling in this study. The amplification mix was kept dark at all times.

The mix was then dispensed into separate 0.5 µL Eppendorf tubes and the filters

were transferred into the tubes. The tubes were then incubated at 46ᵒC for 30-45

minutes. The filters were thoroughly washed afterwards first with 1X PBS at

room temperature for 15 minutes, and then with MilliQ water at room temperature

for one minute, and a final rinse in 96% ethanol for one minute. The filters were

then dried thoroughly in the dark at least for 30 minutes before proceeding with

DAPI counterstaining.

3.5.6. DAPI staining:

Each section cut from a filter was counter-stained with DAPI. DAPI was

used at a final concentration of 1 µg mL-1. For the DAPI staining, a petri dish was

at first covered with a parafilm. 10 µL of the DAPI in working concentration was

placed on the parafilm, and then the filter piece was placed on top of the drop of

DAPI face-down for 3 minutes (the biomass facing the solution). Next, the filter

pieces were rinsed in MilliQ water for 1 minute, and then in 96 % ethanol for 1

Chapter II: MATERIALS AND METHODS| 70

minute. The filter pieces were then dried for 30 minutes in the dark before

mounting.

3.5.7. Mounting, slide preparation and cell counting:

The filters were mounted in the antifadant AF1 purchased from Citifluor

Ltd., London, UK. For the mounting procedure, 10 µL of mounting solution was

placed on a glass slide; the dried filters were placed on the solution face-up,

followed by applying another 10 µL of mounting solution to the top of the filter.

The slide was then mounted using a cover slip of size 22 X 60 mm. The mounted

slides were stored at -20ᵒC in the dark for several days before counting without

considerable loss of probe fluorescence.

The slides were observed under a fluorescence microscope (Zeiss

Axiophot HB0100) using specific filter sets for FITC. Although very time

consuming, manual cell counting is the most preferred method until now for

CARD-FISH due to several limitations observed in semi automatic and automatic

cell counting (Thiele et al. 2011). A 10 mm X 10 mm microscopic grid was used

to aid in the counting process. A total of at least ten fields (more for low-cell

density samples) were counted, with a minimum of 500 total cells counted. Total

cells stained by DAPI were counted on every filter set to obtain an average value

of total cells and this was compared to numbers obtained from the same samples

by flow cytometry to maintain uniformity of counts. DAPI counts were done at

least up to 1000 cells per sample to reduce counting errors. Both percentage DAPI

Chapter II: MATERIALS AND METHODS| 71

count measurements and total cell count measurements were performed. The

following formula was used to obtain total cell counts in a sample.

Average cell counts X Area of filter Total cell signals per mL = Area of grid X Volume of water filtered

4. PCR Methods:

PCR studies on the ammonia-oxidizing archaea from the two lakes were

performed using two different sets of primers. One set of primer was directed towards

targeting the 16S rRNA gene sequences from the AOA, whereas another set of primer

was used to target the amoA (ammonia monooxygenase subunit A) gene of the AOA.

Detailed methods and primers used are discussed below. Primer sequences and references

are listed in Table 6.

4.3. AOA 16S PCR:

For PCR targeting the 16S rRNA of the AOA population in Lake

Superior, a nested PCR approach was used (Lliros et al. 2008). From each sample

1-2 µL of environmental DNA sample (10 ng) was used to run the first PCR

reaction. The universal archaeal primer pair 21F/958R was used to obtain a 937

base pair fragment. The PCR- mastermix (50 µL final volume) contained 0.2 µM

dNTPs (Promega), 1.0 unit of GoTaq DNA Polymerase (Promega), and 1X PCR

buffer (Promega). This fragment was PCR purified using the Qiagen PCR

Chapter II: MATERIALS AND METHODS| 72

Purification Kit (Catalog no. 28104), and the PCR product was used to run the

nested PCR using the primer pair 344F/ 915R. For the PCR reaction with the

universal primer pair, an initial denaturation step at 94ᵒC was performed followed

by 30 cycles of denaturation at 94ᵒC, annealing at 56ᵒC, and elongation at 72ᵒC. A

final elongation was performed at 72ᵒC for 30 minutes. To visualize the PCR

product 10 µL of the product was run on a 1.5% ethidium bromide stained

agarose gel for 40 minutes at 67 V constant voltage before visualizing the gel by

UV transillumination. The nested PCR product was purified by using the Qiagen

PCR purification kit, followed by a TOPO-TA cloning and transformation as

described in sections 2.1.3.

4.4. AOA amoA PCR:

For PCR targeting the amoA gene from the AOA, the specific primer pair

amoAF/amoAR was used (Francis et al., 2005). An initial denaturation step was

performed at 94ᵒC, followed by 35 cycles of denaturation at 94ᵒC, annealing at

53ᵒC, and elongation at 72ᵒC. A final elongation was performed at 72ᵒC for 10

minutes. The PCR- mastermix (50 µL final volume) contained 0.2 µM dNTPs

(Promega), 1.0 unit of GoTaq DNA Polymerase (Promega), and 1X PCR buffer

(Promega). The PCR product was visualized in a 1.5% agarose gel and purified

using the Qiagen PCR Purification kit.

Chapter II: MATERIALS AND METHODS| 73

4.5. Cloning and Transformation:

The PCR purified products were cloned using the TOPO-TA Cloning Kit

from Invitrogen (Catalog No. K457501). The standard cloning procedure in the

kit was followed. Two µL of the PCR product, 1 µL salt solution, 2 µL of sterile

water and 1 µL of the pCR-2.1-TOPO vector were mixed together for 5 minutes

at room temperature and then transferred to ice. Two µL of this cloned vector

containing the PCR product as an insert was then transformed into one vial of

competent E. coli cells DH5α-T1. Two hundred and fifty µL of S.O.C. medium

provided in the kit was added, followed by incubation at 37ᵒC with shaking at 200

rpm for one hour. The cells were then spread onto LB agar media containing

ampicillin at a working concentration of 100 µg mL-1. The plates were incubated

at 37ᵒC for 18-24 hours depending on the appearance and size of colonies on the

plate.

4.6. Colony PCR of Transformants:

After isolated colonies of transformed E.coli cells containing the plasmid

appeared on LB Ampicillin plates, each of the transformed colonies was picked for

screening by colony PCR. The primer pair T3/T7 was used to obtain the insert. For

colony PCR, the colonies were obtained with sterile gel-loading pipette tip, and

dispersed in 5µL of sterile PCR water. Next, 45µL of PCR- mastermix containing 0.2

µM dNTPs (Promega), 1.0 unit of GoTaq DNA Polymerase (Promega), and 1X PCR

buffer (Promega) was added to the cells. An initial denaturation step was performed

at 94ᵒC, followed by 30 cycles of denaturation at 94ᵒC, annealing at 56ᵒC, and

Chapter II: MATERIALS AND METHODS| 74

elongation at 72ᵒC. A final elongation was performed at 72ᵒC for 10 minutes. The

PCR products were then purified using the Qiagen PCR Purification Kit. Sequencing

was performed off-site at the University of Chicago Sequencing Center.

Table 6. Primer pairs and their sequences used in PCR studies in this dissertation.

TARGET PRIMER SEQUENCE (5’-3’) REFERENCE

ORGANISM SET

USED

Archaeal universal 21F TTCCGGTTGATCCYGCCGGA DeLong et al. 1992

16S DeLong et al. 1992

(First PCR) 958R YCCGGCGTTGAMTCCAATT

Archaeal 16S 344F ACGGGGCGCAGCAGGCGCGA Raskin et al. 1994 specific to

Crenarcheaota 915R GTGCTCCCCCGCCAATTCCT Stahl & Amann

(Nested PCR) 1991

Archaeal amoA amoAF STAATGGTCTGGCTTAGACG Francis et al. 2005

amoAR GCGGCCATCCATCTGTATGT

Colony PCR T3 ATTAACCCTCACTAAAGGGA TOPO-TA Cloning

Primers T7 TAATACGACTCACTATAGGG Kit

Chapter II: MATERIALS AND METHODS| 75

5. Bioinformatics Tools:

Environmental sequences were retrieved from the NCBI nucleotide database by

using accession numbers of similar AOA 16S and amoA sequences. BLAST searches

were done on the sequences obtained by PCR and cloning using the NCBI nucleotide

blastn tool

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&

PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome). To target highly

similar sequences on the database, the tool megablast was chosen. After appropriate

BLAST searches, the sequences were aligned together along with other database

sequences obtained from NCBI. The alignment was done with the phylogenetics

software MEGA5 (Tamura el al. 2007 and 2011, Kumar et al. 2012) using

CLUSTALW pairwise and multiple alignment tools. Phylogenetic analysis was

performed on MEGA5 using neighbor-joining methods with 1000 bootstrap relicates.

Bootstrap values less than 70% were not included. Appropriate outgroup sequences

were used for all phylogenetic trees generated in this study. Occasionally the online

phylogenetic tool http://www.phylogeny.fr/ was also used for alignments and

phylogenetic analysis.

Chapter II: MATERIALS AND METHODS| 76

References:

Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. “Fluorescent-oligonucleotide Probing of Whole

Cells for Determinative, Phylogenetic, and Environmental Studies in Microbiology.” Journal of

Bacteriology 172 (2): 762–770.

Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. “Phylogenetic Identification and in-situ

Detection of Individual Microbial Cells without Cultivation.” Microbiological Reviews 59 (1):

143–169.

Amann, R.I., and B. M. Fuchs. 2008. “Single-cell Identification in Microbial Communities by

Improved Fluorescence in-situ Hybridization Techniques.” Nature Reviews Microbiology 6 (5):

339–348.

DeLong, E. F. 1992. “Archaea in Coastal Marine Environments.” Proceedings of the National

Academy of Sciences 89 (12): 5685–5689.

Ilikchyan, I. N., R. M. L. McKay, J. P. Zehr, S. T. Dyhrman, and G. S. Bullerjahn. 2009. “Detection

and Expression of the Phosphonate Transporter Gene phnD in Marine and Freshwater

Picocyanobacteria.” Environmental Microbiology 11 (5): 1314–1324.

Ishii, K., M. Mußmann, B. J. MacGregor, and R. I. Amann. 2004. “An Improved Fluorescence in-

situ Hybridization Protocol for the Identification of Bacteria and Archaea in Marine Sediments.”

FEMS Microbiology Ecology 50 (3): 203–213.

Chapter II: MATERIALS AND METHODS| 77

Kumar, S., G. Stecher, D. Peterson, and K. Tamura. 2012. “MEGA-CC: Computing Core of

Molecular Evolutionary Genetics Analysis Program for Automated and Iterative Data Analysis.”

Bioinformatics 28(20): 2685-2686.

Lliros, M., E. O. Casamayor, and C. Borrego. 2008. “High Archaeal Richness in the Water Column of

a Freshwater Sulfurous Karstic Lake along an Inter-annual Study.” FEMS Microbiology Ecology

66 (2): 331–342.

Pernthaler, A., J. Pernthaler, and R. I. Amann. 2002. “Fluorescence in-situ Hybridization and

Catalyzed Reporter Deposition for the Identification of Marine Bacteria.” Applied and

Environmental Microbiology 68 (6): 3094–3101.

Sekar, R., A. Pernthaler, J. Pernthaler, F. Warnecke, T. Posch, and R. I. Amann. 2003. “An Improved

Protocol for Quantification of Freshwater Actinobacteria by Fluorescence in-situ Hybridization.”

Applied and Environmental Microbiology 69 (5): 2928–2935.

Schönhuber, W., B. M. Fuchs, S. Juretschko, and R. I. Amann. 1997. “Improved Sensitivity of

Whole-cell Hybridization by the Combination of Horseradish Peroxidase-labeled

Oligonucleotides and Tyramide Signal Amplification.” Applied and Environmental

Microbiology 63 (8): 3268–3273.

Chapter II: MATERIALS AND METHODS| 78

Trebesius, K., R. I. Amann, W. Ludwig, K. Mühlegger, and K. H. Schleifer. 1994. “Identification of

Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-targeted Polynucleotide Probes.”

Applied and Environmental Microbiology 60 (9): 3228–3235.

Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. “MEGA4: Molecular Evolutionary Genetics

Analysis (MEGA) Software Version 4.0.” Molecular Biology and Evolution 24 (8): 1596–1599.

Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. “MEGA5: Molecular

Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and

Maximum Parsimony Methods.” Molecular Biology and Evolution 28 (10): 2731–2739.

Thiele S, B. M. Fuchs and R. I. Amann. 2011. Identification of Microorganisms using the Ribosomal

RNA Approach and Fluorescence in-situ Hybridization. Treatise on Water Science, Oxford

Academic Press 3: 171–189.

Woese, C. R., L. J. Magrum, and G. E. Fox. “Archaebacteria.” 1978. Journal of Molecular Evolution

11 (3): 245–252.

Woese, C. R., O. Kandler, and M. L. Wheelis. 1990. “Towards a Natural System of Organisms:

Proposal for the Domains Archaea, Bacteria, and Eucarya.” Proceedings of the National

Academy of Sciences 87 (12): 4576–4579.

Chapter III: RESULTS| 79

CHAPTER III

RESULTS

1. Sampling site characteristics

In Lake Superior the average ammonium concentrations between 2010-2012 was

low, averaging 0.281 µM (Table 7). Relatively higher ammonium values were observed

in many of the samples collected from the bottom of the lake (CD1 August 2010, EL0

and EL2 July 2011 samples), indicating possible efflux of ammonium from sediments in

these samples. These values compare favorably with previously reported values of 0.210

µM in Lake Superior averaged on over 160 samples (Kumar et al., 2007). In Lake Erie,

on the other hand, the average ammonium concentration was found to be much higher

compared to Lake Superior, averaging at 1.69 µM. Samples from the Western Basin of

Lake Erie (Maumee Bay and 91M), near the mouth of the Maumee river, exhibited the

highest concentrations of ammonium (Table 7).

Nitrate concentrations in Lake Superior were considerably higher than Lake Erie,

averaging 25.67 µM, across the various stations and depths profiled during 2010-2012

(Table 7). This value is consistent with previous reports of high and increasing nitrate Chapter III: RESULTS| 80

concentrations in the lake (averaging at 26 µM, Sterner et al, 2007, 2011). In Lake Erie, except for the samples from the Western Basin (Maumee Bay and 91M), where the

Maumee river empties itself into the lake, all other samples showed a much lower nitrate concentration than lake Superior, averaging at 7.59 µM. In several samples from Lake

Superior, the deeper water often contained higher nitrate compared to the surface waters

(Table 7, July 2011 CD1, Michipicoten, Sleeping Giant samples), possibly reflecting summer time drawdown of nutrients by phytoplankton during the stratified period (July-

Oct).

DOC and Chlorophyll concentrations were low in Lake Superior, when compared to Lake Erie (Table 7). DOC averaged at 1.69 mg L-1 in Lake Superior, and 2.314 mg L-1 in Lake Erie. The highest DOC in Lake Erie was observed once again in the western basin stations of Maumee Bay and 91M where the DOC averaged at a high of 3.14 mg L-

1. These observations can be attributed to the oligotrophic nutrient-limited conditions in

Lake Superior where the total microbial population is considerably lower (Fig. 29) compared to the mesotrophic Lake Erie.

Chapter III: RESULTS| 81

Table 7. Available ammonium, nitrate, DOC and chlorophyll a concentrations in Lake Superior and Lake Erie between 2010-2011 (core measurement data obtained at University of Minnesota and Bowling green laboratory). Ammonium Nitrate DOC Chl a Year Month Lake Station Depths (µM) (µM) (mg L-1) (µg L-1) 2010 May Super CD1 5 0.5289 - - 0.537 ior 235 0.5250 - - 0.113 WM 2 0.0992 - - 0.507 0.283 155 0.1297 - - 2010 Aug Super WM 2 0.2850 - 2.131 0.270 ior 5 0.2508 - 2.187 0.262 10 0.2849 - 2.299 0.368 20 0.2528 - 2.118 0.376 30 0.3104 - 2.329 0.636 40 0.2994 - 2.110 0.401 60 0.1506 - 2.170 0.157 90 0.0927 - 2.149 0.100 120 0.3148 - 2.100 0.068 155 0.1399 - 2.045 0.095 CD1 2 0.2868 - 2.162 0.390 0.389 8 0.2684 - 2.205 0.382 15 0.2399 - 2.133 0.293 35 0.2845 - 2.150 0.074 150 0.1846 - 2.105 0.089 244 0.4833 - 2.045 2010 Oct Super WM 5 0.1706 - 2.382 1.050 ior 20 0.1949 - 2.314 1.179 155 0.1483 - 2.075 0.234 CD1 5 0.2651 - 2.225 1.164 10 0.2549 - 2.484 1.314 0.132 150 0.2163 - 2.956 2011 April Super CD1 5 0.4460 25.428 - 0.458 ior 15 0.2234 26.500 - 0.568 30 0.2409 25.786 - 0.437 50 0.3364 27.214 - 0.560 100 0.2736 27/214 - 0.404 150 0.2206 26.928 - 0.205 200 0.2700 26.714 - 0.106 245 0.1438 27.000 - 0.127 WM 5 0.3189 27.500 - 0.429

Chapter III: RESULTS| 82

Ammonium Nitrate DOC Chl a Year Month Lake Station Depths (µM) (µM) (mg L-1) (µg L-1) 0.477 10 0.1905 20.000 - 0.143 155 0.2482 27.000 - 2011 July Super EL0 2 0.148 26.143 1.107 0.244 ior 5 0.107 26.714 1.190 0.293

0.351 12 0.087 26.214 1.590 20 0.076 25.786 2.010 0.538 30 0.091 26.071 1.181 0.718 50 0.291 26.428 1.212 0.215 75 0.369 26.500 1.357 0.186

100 0.423 26.714 1.173 0.181

0.085 145 0.440 26.500 1.220 160 0.362 26.500 1.280 0.154 EL2 5 0.150 25.857 1.271 0.161 10 0.132 26.214 1.298 0.210 30 0.167 26.071 1.134 0.358 0.424 50 0.209 26.143 1.157 60 0.185 26.214 - - 75 0.265 26.428 - 0.160 100 0.388 26.714 1.134 0.265 150 0.400 26.785 1.220 0.131

185 0.312 26.286 - -

0.133 195 0.419 26.357 1.174 CD1 2 0.503 24.214 1.282 0.422 5 0.191 25.857 1.463 0.471 10 0.238 25.714 1.288 0.767 20 0.147 25.571 1.501 0.640

35 0.221 26.428 1.191 0.482

50 0.270 26.214 1.147 0.240 100 0.702 26.643 1.317 0.129 150 0.433 26.571 1.205 0.219 200 0.407 26.428 1.141 0.211 245 0.742 26.500 1.336 0.190

Michipicoten 5 0.341 23.357 - 0.463

15 0.416 23.214 - 0.757 78 0.372 26.214 - 0.060 Black Bay 2 0.195 21.143 1.617 0.283 8 0.295 15.571 2.339 0.837

Chapter III: RESULTS| 83

Sleeping 0.444 Giant 5 0.402 22.643 1.517 240 0.363 26.500 1.166 0.059

272 0.307 26.500 1.157 0.100

2011 July Erie Maumee Bay 1 1.391 78.928 4.253 6.027 7 1.548 78.285 4.099 4.541 CCB3 5 0.346 - 2.067 0.774 9 0.346 6.142 1.927 0.663 11 0.648 4.857 - 0.688 15 2.621 - 2.102 0.829 18 2.173 5.571 2.213 0.788 20 2.378 5.785 1.795 0.991 22 2.311 5.643 2.077 0.865 CCB1326 1 0.535 - 2.042 0.345 5 0.466 - 1.953 0.386 10 0.775 - 1.930 0.502 12 0.940 - - - 17 2.143 - 4.948 0.646 20 2.434 - 2.006 0.755 880(84) 1 0.381 7.357 2.080 0.669 5 0.643 7.357 2.016 0.573 10 0.468 5.357 2.024 0.409 15 0.994 9.071 2.171 0.653 20 4.245 3.785 1.877 0.414 22 4.477 3.714 1.885 0.338 EC23 10 0.940 6.714 2.084 0.914 20 1.850 9.928 1.957 0.832 30 2.405 11.714 1.963 0.639 42 - 11.857 2.023 0.399 50 3.489 12.071 1.917 0.284 60 2.768 12.143 1.827 0.023 91M 1 1.917 39.142 2.544 1.150 0.705 4 1.678 38.143 2.509 0.472 8 1.855 38.428 2.508

Chapter III: RESULTS| 84

2. Temperature Profiles

Selected temperature profiles taken across multiple depths in Lake Superior and Lake

Erie station 880(84) collected during the sampling cruises of 2010 (August, October and May) and 2012 (July) are represented in Fig. 19. Parallel chlorophyll profiles in the lakes across the same depths and months are also plotted in the figures. During the months of August and

October 2010, and in July 2011, the temperature profiles shows stratification, whereas during the

month of May 2010, the temperature profile shows isothermal mixing. During stratification in

Lake Superior, the epilimnion extends to a depth of approximately 20 m.

I II

IV III A

Chapter III: RESULTS| 85

V VI

VII VIII

Fig. 19: I-VIII. Temperature profiles (in blue) and chlorophyll profiles (in red) at several depths at various Lake Superior stations during the stratified months of August 2010 (V, VI), October 2010 (VII, VIII), and July 2011 (II) at stations CD1 and WM, in Lake Erie station 880(84) (IV) in July 2011, and during the unstratified month of May in 2010 (I, III).

3. Diversity of ammonia oxidizing archaea (AOA) in Lake Superior and Lake Erie

To target the diversity of the ammonia-oxidizing archaeal population in Lake

Superior and Lake Erie, DNA samples from July 2011 cruise were extracted, and PCR

was run using two different sets of primers: 1) targeting partial 16S rDNA of AOA and 2)

targeting partial amoA gene sequence of the AOA. For Lake Superior amoA AOA

diversity, I used both water column sampled from CD1 at 150m depth on July 29, 2011,

Chapter III: RESULTS| 86

and sediment sampled during the June 2010 research cruise from the WM station.

Samples were also used from the surface water obtained from station Sterner C during the

May 2010 cruise. For Lake Erie AOA amoA diversity, I used the water column samples obtained from station CCB at 20 m depth on July 22, 2011.

3.1. PCR analysis of 16S rDNA of environmental samples in Lake Superior

For 16S rDNA PCR analysis, two sets of PCR amplicons were obtained. The first

PCR using the universal archaeal primers 23F/ 958R (see table 6 for primer list)

generated a 935bp PCR product (Fig. 20-I), faintly visible on a 1.5% agarose gel. The

PCR product was purified and reamplified with the same primers (Fig. 20-II). This

DNA was then used as the template for the second round of the PCR using the nested

primer pair 344F/915R which generated a 571 bp fragment (Fig. 20-III). Over 100

clones of PCR products were obtained following transformation and ampicillin

selection. The insert was analyzed using colony-PCR with primer pair T3/ T7. The

PCR products obtained were then sent to the University of Chicago Sequencing

Center for sequencing.

Chapter III: RESULTS| 87

I

II

Fig. 20. Gel image of PCR product targeting 16S rDNA of AOA.

I. Image from first PCR using universal primer pair 23F/958R using samples from stations WM and Sterner C. The two wells show different volumes of starting environmental DNA used: 1µL and 3 µL. The well designated as C is the negative control run for each sample consisting of the PCR amplification mix only with no DNA. The last lane consists of the 100bp DNA ladder.

II. Image of amplified PCR product from WM and Sterner C first PCR products as seen in image A. The second sample containing a 3 µL of DNA initially was used for the re-amplification reaction.

Chapter III: RESULTS| 88

III

Archaeal Primers Pair 1 344F/915R

C C

Fig. 20. (continued) Gel image of PCR product targeting 16S rDNA of AOA. III. Gel image of PCR products obtained after the second PCR using the first PCR product as the template with the nested primer pair 1 344F/ 915R. Two different volumes of template DNA was used for both samples: 3 µL and 5 µL. The well designated as C is the negative control run for each sample consisting of the PCR amplification mix only with no DNA. The last lane consists of the 100bp DNA ladder.

3.2. Phylogenetic Analysis of 16S rDNA sequences from Lake Superior

The sequences, on an average between 500 - 600 bp in length, were analyzed

using NCBI BLAST. Appropriate sequences were then aligned by MEGA5 software,

and a neighbor-joining phylogenetic tree was generated using the sequences obtained

in this study as well as database sequences, obtained from NCBI, using various

environmental clones and cultured representatives of AOA from several other studies

Chapter III: RESULTS| 89

(Fig. 21). Phylogenetic analysis of the cloned PCR products from the16S rDNA study of the AOA suggests that Lake Superior supports a unique group of ammonia oxidizing archaeal population (Fig. 21). The water column population is distinct from the sediment population since they are found to form separate groups within the

Thaumarchaeota Group I.1 a cluster (Fig. 21, and Annette Bollmann, personal communication). Sequences from enrichment cultures obtained from Lake Superior

(Mike Schlais dissertation work) also point out that the cultured representatives group within the Group I.1a Thaumarchaeota cluster and are grouped within the cloned representatives from the water column. This result is consistent with previous studies where other abundant microbes from Lake Superior, both cyanobacteria and

Actinobacteria have been found to be restricted to few specific unique phylogenetic clusters (Ivanikova et al., 2007; Sharma et al., 2009). This low diversity of microbial populations in Lake Superior, including the AOA, may be attributed to the cold, ultraoligotrophic low phosphate environment in the lake, where only microorganisms belonging to just a few ecotypes are able to adapt and survive the extreme nutrient conditions in the lake.

Chapter III: RESULTS| 90

Chapter III: RESULTS| 91

Fig. 21. Neighbor-joining Phylogenetic tree of 16S rDNA obtained from water column and sediment samples from different stations in Lake Superior. The tree also contains sequences obtained from enrichment cultures of AOA from the lake (Mike Schlais cultures). The tree was generated using bootstrap values of at least 1000 replications. The outgroup sequence used in the tree is an extremophilic archaea Thermoplasma acidophilum. Blue triangles represent samples from Lake Superior station WM water column (2m), pink triangles represent samples from Lake Superior station Sternec C water column samples (2m), the brown circles represent Lake Superior sediment samples from station CD1, the green rectangles represent enrichment cultures from Lake Superior cultured on a solid medium, and the red rectangles represent enrichment cultures from Lake Superior cultured on a liquid media (enrichment cultures are isolated by Mike Schlais).

3.3. PCR analysis of amoA sequences from environmental samples from Lake

Superior and Lake Erie

To target the amoA gene diversity of the AOA population in Lakes Superior and

Lake Erie, samples obtained from the July 2011 cruise were used. For PCR,

approximately 2 µL of environmental DNA obtained from both the lakes were used

(10 ng). Specific primer pair amoA-AF/ AR targeting amoA of AOA was used

(Francis et al., 2005). The PCR product obtained was approximately 650 bp in length

(Fig. 22-I). These products were purified, and cloned using a TOPO TA cloning kit,

following which a colony PCR was performed on the colonies obtained from

transformation (Fig. 22-II).

Chapter III: RESULTS| 92

I C

II C

C

Fig. 22. Gel images from PCR diversity study from Lake Superior and Lake Erie. I. Gel image of PCR product showing the 650 bp amoA amplicons obtained from Lake Superior and Lake Erie water column samples. II. Gel image of PCR product from colony PCR products of amoA amplicons obtained after transformation. The lane designated as C is the negative control run for each sample consisting of only the PCR amplification mix with no DNA. The last lane consists of the 100bp DNA ladder.

3.4. Phylogenetic analysis of amoA sequences from Lake Superior and Lake Erie

Phylogenetic analysis of the AOA amoA sequences obtained from Lake Superior

and Lake Erie shows a very low diversity of the population of these nitrifers in both

these lakes (Fig. 23). Despite the low diversity of the AOA in the two lakes, they are

found to form two completely distinct clusters within the Group I Thaumarchaeota.

The low diversity in Lake Superior (while compared to the high numbers of AOA in

the lake) may be attributed to the oligotrophic characteristics of the lake, where in

Chapter III: RESULTS| 93

spite of being the dominant nitrifier population, the AOA groups are restricted to few genera and species, suggesting that only certain groups of microorganisms are able to adapt to the cold, nutrient-depleted conditions in the lake. The Lake Superior clones are also found to form unique clusters within the freshwater groups, consistent with other similar observations on the lake’s unique microbial communities of picocyanobacteria and actinobacteria (Ivanikova et al., 2007; Sharma et al., 2009). By contrast, Lake Erie population is assumed to be low when the very low CARD-FISH counts are taken into account (see below). CARD-FISH counts on both AOA and

AOB (ammonia-oxidizing bacteria; Ray, 2012 thesis) demonstrate that in Lake Erie, the major nitrifier population is the AOB while AOA are almost negligible in the lake.

Chapter III: RESULTS| 94

Chapter III: RESULTS| 95

Fig. 23. Neighbor-joining Phylogenetic tree of amoA sequences from AOA obtained from Lake Superior and Lake Erie. All the 113 clones from Lake Erie and 90 clones from Lake Superior grouped into the Group I.1a cluster of Thaumarchaeota. Outgroups used in the tree were two AOBs Nitrosospira multiformis and Nitrosomonas sp. Cultured representatives from both Group I.1a and I.1b Thaumarchaeota are indicated with a red arrow in the tree. The tree was generated in software MEGA5 using 100 replicates of bootstrap analysis. The green circles represent amoA sequences from Lake Superior station CD1 at a depth of 150 m, and the pink diamonds represent amoA sequences from Lake Erie station CCB at a depth of 20 m.

4. CARD-FISH Enumeration of Lake Superior AOA

The AOA community in the lake was quantified using CARD-FISH. Depth-

resolved samples were obtained over a period of three years, during stratified and

isothermally mixed periods. For each sample analyzed by CARD-FISH, total cell counts

were performed using the nuclear stain DAPI targeting the entire population of

microorganisms present in a particular water/ sediment sample. Total bacterial cell counts

were also performed on each sample using an equivalent mixture of the total bacterial

probes Eub338I, Eub338II and Eub338III. A non-hybridizing control probe was used on

each sample as a negative control (See Table 3, Chapter 2 for probe detail). Below I

summarize the results from all the samples collected during 2010, 2011, and 2012 cruises

on Lake Superior.

4.1. 2010 Samples

In 2010, samples were collected during May, August and October. Mostly surface

and bottom depths of were profiled for May, whereas in August 2010, a detailed

depth profile sampling was performed.

Chapter III: RESULTS| 96

4.1.1. May 2010: Stations WM and CD1

In May, samples were obtained from two depths from stations WM and

CD1: surface and bottom. For Station WM, samples were collected from 2 m

and 155 m, and for station CD1, samples were from 2 m and 245 m. CARD-

FISH counts of total AOA were similar in numbers at both surface vs bottom

samples ranging between 1.5 X 104 – 2 X 104 cells mL -1 (Fig. 24). This can

be attributed to the fact that the lake is in an isothermal mixing condition in

May, causing an even distribution of microbial communities throughout the

lake.

4.1.2. August 2010: Stations WM and CD1

Samples from August were collected at depth 2 m, 8 m, 15 m, 20 m, 35 m,

150 m, 244 m, and sediment overlay samples from CD1 station. For WM

station, samples were collected at multiple depths of 2 m, 5 m, 20 m, 40 m, 60

m, 80 m, 100 m, 130 m, 155 m, and sediment overlays.

By contrast to the isothermal mixing during May 2010, the lake is in a

thermal stratified condition during August and October (Fig. 19), and

therefore microorganisms differentially distribute between the surface mixed

layer (epilimnion) and deep waters (hypolimnion). In both the stations WM

and CD1, AOA are found to be mostly restricted to the deeper waters (Fig.

24). Knowing that the thermocline is at approximately 20 m (Fig. 19) very

few AOA are detected in the epilimnion (0 - 20 m). The general trend is that

the number of AOA increases with depth. This observation parallels a similar

Chapter III: RESULTS| 97

study in the trends in the distribution of nitrite oxidizers in the August 2010 samples (Ray, 2012, MS thesis), indicating that the nitrifying organisms, both the ammonia-oxidizers and nitrite oxidizers, are absent on the surface and distributed mostly in deeper waters pointing out to the fact that these two groups remain in consortium with each other to complete the entire process of nitrification. It is important to mention here that the rates of nitrification observed at these stations at the same depths during August stratification are also in correspondence with the total AOA counts (Small et al., 2013) (see

Fig. 31). The sediment overlay samples from August 2010 showed a high number of AOA in both the stations WM and CD1, reaching up to 2.5 X 10 4 cells mL -1, paralleling observations in October from the sediments.

During the August 2010 sampling cruise, while sampling the CD1 station, an extraordinary and unprecedented southwest wind resulted into a massive storm on Lake Superior which yielded an upwelling and circulation event in the northern half of the lake (Austin, 2011 IAGLR Abstract). Interestingly, only the surface water samples that were processed from this particular date on station CD1 showed a very high number of AOA, likely arising from the upwelling of the AOA from the bottom of the lake due to the massive storm

(Fig. 24-II). Indeed the location of station CD1 near the north shore is consistent with the site of upwelling.

Chapter III: RESULTS| 98

4.1.3. October 2010: Stations WM and CD1

Samples were collected from depths 5 m, 20 m, 155 m at station WM and

at 10 m, 150 m, and 245 m at station CD1, including samples from sediment

overlays. In October 2010, similar trends in AOA numbers were observed as

found in the August samples. The nitrifiers were absent from the surface water

samples whereas the abundance increased in the deeper water column

samples. The sediment overlay sample in October 2010 from WM station

showed a very high number of AOA amounting up to 5.5 X 10 4 cells mL -1,

comparable to similar observations of the high AOA numbers on the sediment

overlay samples in August 2010 (Fig. 24). This observation of high AOA

numbers in sediments from Lake Superior is also consistent to a parallel study

on the AOA amoA clones obtained from the sediment samples on Lake

Superior (Annette Bollmann, personal communication).

Chapter III: RESULTS| 99

Total cells mL -1 III 0 20000 40000 60000 0 20 40 OCTOBER 2010 WM

60 AUGUST 2010 WM 80 MAY 2010 WM 100

Depths (m) Depths 120 140 160 180

Total cells per mL 0 50000 100000 150000 0

50

OCTOBER 2010 CD1 100 MAY 2010 CD1 150 AUGUST 2010 CD1 Depth (m) Depth 200

250

300

Fig. 24. Depth Profile of abundance of AOA in Lake Superior using CARD-FISH in 2010. Sediment overlay samples are marked with a sign.

I. Total AOA cells ml -1 in Station WM during May, August, and October 2010. II. Total AOA cells ml -1 in Station CD1 during May, August, and October 2010.

Chapter III: RESULTS| 100

4.2. 2011 Samples

4.2.1. April 2011

In April 2011, the AOA numbers showed a similar trend as seen during May

2010. AOA were also found on the surface water samples compared to

negligible AOA observed during the stratified months of August and October

(Fig. 25). The lake remains isothermally mixed during this time, therefore the

nitrifiers are found to be present throughout the water column. Higher AOA

numbers were observed in CD1 than in WM. This trend supports previous

observations that AOA tend to thrive better in lower ammonium

concentrations (Martens-Habbena et al., 2009). The AOA numbers also

parallels the nitrite-oxidizer numbers in these samples (Ray, 2012 thesis)

where nitrite oxidizers were also found in the surface waters in the April 2011

samples alongside the AOA, again pointing out to the fact that the AOA and

the NOBs tend to live together in the same niches to be able to complete the

process of nitrification.

Chapter III: RESULTS| 101

Total cells mL -1 0 5000 10000 15000 20000 25000 30000 35000 40000 0

50

100 WM April 2011 CD1 April 2011 150 Depths (m) Depths 200

250

300

Fig. 25. Depth profile of abundance of AOA in Lake Superior using CARD-FISH in 2011 April samples.

4.2.2. July 2011

In July 2011, water samples were collected from both the lakes: Lake Superior

and Lake Erie across several stations and depths. A seventeen day research

cruise was undertaken to complete this high-resolution sampling process on

the lakes. Overall, total average AOA cell numbers were lower in 2011 than in

2010, as were nitrification rates (Chip Small, personal communication).

Below I summarize the total AOA counts on the Lake Superior samples. The

total AOA counts observed on Lake Erie in July 2011 can be seen in section 4

of this chapter.

Chapter III: RESULTS| 102

4.2.2.1. Stations EL0, EL2, and CD1

Similar to the observations of AOA abundance in 2010 samples, the

offshore stations CD1, EL0, and EL2 in 2011 demonstrated a high

abundance of AOA. AOA were found to almost absent in the surface

samples from all these stations. As observed in all samples collected

during stratified periods on the lake in 2010, the July 2011 samples also

showed an increase in AOA abundance with depth in the lake. This may

be attributed to a photoinhibitory effect the ammonia monooxygenase

enzyme so that AOA thrive better in low light intensities (Merbt et al.,

2012; French et al., 2012). Highest numbers of AOA were observed in the

EL0 station where the average AOA numbers went up to 1.1. X 10 4 cells

mL -1 observed at 160 m, close to the bottom of the lake. Station EL2 also

showed high numbers of AOA between the depths of 30 m and 195 m,

with the highest numbers observed again on the sample collected near the

bottom of this site ranging up to 4 X 10 4 cells mL -1 at 195 m depth.

Station CD1 showed a similar pattern of AOA numbers along its depth,

close to the numbers seen in 2010 August, with the highest cell numbers

observed at 150 m with 6 X 10 4 cells mL -1 (Fig. 26-I).

4.2.2.2. Stations White Fish Bay, Michipicoten, Black Bay and Sleeping

Giant

Between the samples of White Fish Bay, Michipicoten, Black Bay and

Sleeping Giant, the 2 m and 8 m samples from Black Bay revealed < 10

Chapter III: RESULTS| 103

cells mL -1 AOA. In all the other stations, a similar trend of a high number of AOA was seen in the deeper water samples. In station Michipicoten, no

AOA were found in the surface samples of 5 m and 15 m, whereas the sample obtained from 78 m depth showed presence of AOA in the order of

1 X 10 3 cells mL -1. Sleeping Giant also showed a very similar trend of

AOA with no AOA found on the surface 2 m sample, whereas 3 X 10 3 and 4 X 10 3 cells mL -1 being present in the deeper samples of 240 m and

272 m respectively. Similar trends were again seen in the White Fish Bay sample where no AOA were found on the surface whereas; AOA numbers reached up to approximately 2 X 10 3 in the deeper water column sample of 60 m (Fig. 26- II).

Chapter III: RESULTS| 104

Total cells mL -1 0 2000 4000 6000 8000 10000 12000 0

50 EL-0 JULY 2011

100 EL-2 JULY 2011 150 CD-I JULY 2011

Depths (m) Depths 200

250

300

Total cells mL -1 0 1000 2000 3000 4000 5000 0

50 MICHIPICOTEN JULY 2011

100 SLEEPING GIANT JULY 2011 150 WHITE FISH BAY JULY 2011 Depths (m) Depths 200

250

300

Fig. 26. Depth profile of abundance of AOA in Lake Superior using CARD-FISH in July 2011.

I. Total AOA cells ml -1 at various depths in stations EL0, EL2, and CD1 during July 2011. II. Total AOA cells ml -1 at various depths in stations Michipicoten, Sleeping Giant and White Fish Bay during July 2011.

Chapter III: RESULTS| 105

4.3. 2012 Samples

In 2012, samples were collected from Lake Superior from station WM at various

depths in the month of June. A similar trend like the previous years was observed this

year too, where no AOA were observed on the surface samples, whereas, as depth

increases, the number of AOA became more prominent. NOB, either Nitrobacter or

Nitrospira was observed in all depths (Fig. 27).

Total cell mL-1 0 5000 10000 15000 20000 0

20

40

60 AOA WM 2012

80 Nitrobacter WM 2012 100 Nitrospira WM 2012

Depths (m) Depths 120

140

160

180

200

Fig. 27. Depth profile of abundance of AOA, and NOB (Nitrobacter and Nitrospira) in Lake Superior Station WM 2012.

5. CARD-FISH Enumeration of Lake Erie AOA

Lake Erie was sampled only in 2011 across multiple stations and depths. Stations

sampled were 91M and Maumee Bay from Western Basin, 880(84), CCB3, and 1326

from the Central Basin, and EC23 from the Eastern Basin. No AOA were seen at depths

Chapter III: RESULTS| 106

of 1 m, 5 m, 7 m, 12 m, 15 m, 17 m, 20 m, and 22 m on the CCB3 sample and at depths of 5 m, 10 m, 20 m, 30 m, 42 m, 50 m, and 60 m at station EC23. Station 880(84) reflected no AOA on any depth except at the bottom of lake at 20 m ranging up to 1.5 X

10 3 cells mL -1. The 8 m sample from shallow western basin station 91M contains surfacial sediments, known to contain a sediment AOA population (A. Bollmann, personal communication). It is important to mention here that in station 880(84), the

AOB numbers were very high reaching up to 1.5 X 10 4 cells mL -1, one order in magnitude higher than that that of the AOA numbers found at the same depth of the station (Fig. 29). Station 1326 also exhibited no AOA on the surface water samples, except for the depths of 10 m and 12 m (Fig. 28). Results from CARD-FISH enumeration of the total numbers of AOA observed at all stations in Lake Erie when compared to the

AOB numbers obtained from same depths on the same station (Fig. 29) reflects that AOB are the dominant nitrifiers in Lake Erie compared to Lake Superior, where the dominant nitrifiers were the AOA.

Chapter III: RESULTS| 107

Total cell per mL 0 5000 10000 15000 0 2 91M JULY 2011 4 1326 JULY 2011 6

880(84) JULY 2011 8 Maumee Bay July 2011 10 12 Depths (m) Depths 14 16 18 20

Fig. 28. Depth Profile of abundance of AOA in Lake Erie using CARD-FISH in July 2011. Samples shown here are from stations Maumee Bay, 91M, 1326 and 880 (84). Samples were also collected from other stations included CCB3, and EC23, where no AOA were found across several depths between surface to the bottom of the lake.

Total cells per mL 0 10000 20000 30000 40000 50000 0

5 Total AOA 880(84)

10 Total NOB 880(84) Total AOB 880(84) 15 Depth (m) Depth

20

25

Fig. 29. Total cells mL-1 of AOA, AOB and NOB in Lake Erie Station 880(84).

Chapter III: RESULTS| 108

6. Total Bacterial counts using CARD-FISH in Lake Superior: 2010 - 2012

Total bacterial abundance was enumerated in every station and depth profiled in

this study throughout the 2010, 2011 and 2012 cruise using specific bacterial CARD-

FISH probes (Table 3). A general trend of lower bacterial abundance was observed in all

of these years. On an average, in 2010, total bacterial counts averaged at 2.3 X 105 cells

mL-1 in station WM (Fig. 30-I), and at 1.4 X 105 cells mL-1 in station CD1 (Fig. 30-II). In

2011, the total bacterial counts averaged at 1.2 X 105 cells mL-1 in the farther off-shore

station of EL0 (Fig. 30-III). Similar results of lower bacterial abundance in Lake Superior

off-shore stations have been reported before, indicating towards the ultra-oligotrophic

extreme conditions within the lake (Sterner et al., 2004). Another off-shore station EL2

also exhibited lower bacterial abundance in 2011 averagin at 2.04 X 105 cells mL-1 (Fig.

30-III). In station CD1 the average bacterial abundance in 2011 was restricted to 0.93 X

105 cells mL-1(Fig. 30-III) across the various depths sampled. In 2012, the bacterial

population was once again observed to be low, averaging at 1.61 X 105 cells mL-1 at

station WM, sampled in June (Fig. 30-IV). These results are consistent with previous

studies (Sterner et al., 2004) wherein the total bacterial abundances in the oligotrophic

Lake Superior have been reported to be considerably lower than that observed in the

other Great Lakes. Incidentally, the highest bacterial numbers were observed on some of

the samples collected during the 2010 August cruise (Fig. 29-II), the time during which a

major storm across the northern shore of Lake Superior led to an upwelling an event

(Austin, 2011 IAGLR Abstract).

Chapter III: RESULTS| 109

Total bacterial cells mL -1 0 100000 200000 300000 400000 500000 0

20

40

60 May 2010 WM

80 August 2010 WM

100 October 2010 WM Depths (m) Depths 120

140

160

180

Total bacterial cells mL -1 0 100000 200000 300000 400000 0

50 May 2010 CD1 100 August 2010 CD1 October 2010 CD1 150 April 2011 CD1 Depths (m) Depths 200

250

300

Fig. 30. Total bacterial cells per mL water sample across multiple depths in Lake Superior samples collected in 2010-2012. Sediment overlay samples are marked with a sign. I. Station WM Samples across various depths in May, August, October 2010. II. Station CD1 Samples across various depths in May, August, October 2010 and April 2011.

Chapter III: RESULTS| 110

III Total bacterial cells mL -1 0 100000 200000 300000 400000 0

50 July 2011 EL0

100 July 2011 EL2

July 2011 CD1 150 July 2011 Sleeping Giant

Depths (m) Depths July 2011 Whitefish Bay 200

250

300

IV Total bacterial cells mL -1 0 50000 100000 150000 200000 250000 0

20

40

June 2012 WM 60

80 Depths (m) Depths 100

120

140

Fig. 30. (continued) III. Total bacterial cells per mL water sample in stations EL0, EL2, CD1, Sleeping Giant and Whitefish Bay across various depths in July 2011. IV. Total bacterial cells per mL water sample in station WM across various depths in June 2012.

Chapter III: RESULTS| 111

7. Nitrification rate measurements and total AOA cell comparison

A positive correspondence between nitrification rates and the total AOA numbers

is observed in Lake Superior (Fig. 31). In general, no nitrification is observed on surface

waters, which increases with depth (Fig. 31-I), correlating with the same trend of total

AOA numbers observed, where the AOA are also found to be negligible at the surface

waters from the lake and the number AOA increase with depth in the lake (Fig. 31-II).

I II

Fig. 31. Comparison of nitrification rates versus total AOA number in Lake Superior. I: Nitrification rate measurements (Small et al., 2013) across August 2010 samples in station WM. II: Total AOA counts in Lake Superior station WM across same depths (this dissertation).

Chapter III: RESULTS| 112

8. Per cell Nitrification-Rate Calculations in AOA in Lake Superior

The average nitrification rate in in Lake Superior in 2010 was on an average 24.1

nmol N L-1 d-1 (e.g., Fig. 31; Small et al., 2013). The average AOA numbers on Lake

Superior in station CD1 in 2010 was averaged 4.6 X 106 cells L-1 (Fig. 24). Therefore,

from these data, the average per-cell nitrification rate in Lake Superior is equivalent to

5.2 fmol cell-1 d -1. This rate is comparable to the oceanic nitrification rate of 4.6 fmol cell-

1 d-1, as seen in Atlantic Ocean (Wuchter et al., 2006). The rate is also comparable to the

per-cell nitrification rates in the pure culture of AOA, Nitrosopumilus maritimus, which

is calculated to be 0.53 mol cell-1 h-1 (Könneke et al., 2005; Urakawa et al., 2011). Thus,

we are confident that our cell enumeration and nitrification rate measurements reflect

values typical for oligotrophic aquatic environments.

Chapter III: RESULTS| 113

References:

Austin, J. A. and P. Cheng. 2011. An extraordinary upwelling event in Lake Superior during Summer

2010. International Association for Great Lakes Research, Conference Abstract.

Ivanikova, N. V., L. Popels, R. M. L. McKay and G. S. Bullerjahn. 2007. “Lake Superior supports

unique clusters of cyanobacterial picoplankton”. Applied and Environmental Microbiology 73:

4055-4066.

Kumar, S., R. S. Sterner, J. C. Finlay, and S. Brovold. “Spatial and temporal variation of ammonium

in Lake Superior.” Journal of Great Lakes Research, no. 33 (2007): 581–591.

Ray, A. 2012. “Identification, Enumeration and Diversity of Nitrifying Bacteria in the Laurentian

Great Lakes”. MS Thesis, Bowling Green State University.

(http://etd.ohiolink.edu/view.cgi?acc_num=bgsu1351276518)

Sharma, A. K., K. Sommerfeld, G. S. Bullerjahn, A. Matteson, S. W. Wilhelm, J. Jezbera, U. Brandt,

W.F. Doolittle and M.W. Hahn. 2009. “Widespread distribution of actinorhodopsin genes in

freshwater habitats and among diverse actinobacterial lineages”. ISME Journal 3: 726-737.

Small, G. E., G. S. Bullerjahn, R. W. Sterner, B. F. N. Beall, S. Brovold, J. C. Finlay, R. M. L.

McKay, and M. Mukherjee. 2013. “Rates and controls of nitrification in a large oligotrophic

lake”. Limnology and Oceanography 58(1): 276–286.

Chapter III: RESULTS| 114

Sterner, R. W., T. M. Smutka, R. M. L. McKay, Q. Xiaoming, E. T. Brown, and R. M. Sherrell. 2004.

“Phosphorus and trace metal limitation of algae and bacteria in Lake Superior”. Limnology and

Oceanography 49: 495–507.

Chapter IV: DISCUSSION AND CONCLUSIONS| 115

CHAPTER IV

DISCUSSION AND CONCLUSIONS

Lake Superior and Lake Erie are two very different lakes based on their nutrient content, trophic levels, operation of their biogeochemical cycles, as well as the structure of the microbial communities present. Lake Superior is an ultra-oligotrophic, and is considered to be one of the most P-limited lakes in the world (Munawar et al., 2009).

This extreme P limitation has resulted in a N: P stoichiometric imbalance in the lake

(Sterner et al., 2007, 2011). On the contrary, Lake Erie is a mesotrophic lake, consisting of three basins: the western, central and eastern basins. The western basin is meso- to- eutrophic, the central basin is mesotrophic whereas the eastern basin is oligotrophic.

Therefore, as we move from west to east of the lake, a strong difference in trophic levels is observed. In the western basin of Lake Erie, the incidence of periodic harmful algal blooms, mostly dominated by a population of Microcystis spp., is observed almost every year (Vincent et al., 2004; Rinta-Kanto et al., 2005; Chaffin et al., 2011). The watershed around Lake Superior is very sparsely populated, with very little agricultural and industrial activity, whereas, the watersheds around Lake Erie is heavily populated, and Chapter IV: DISCUSSION AND CONCLUSIONS| 116

dominated by agricultural activities (Munawar and Weisse, 1989). With regards to oxygen gradients, the two lakes also show very different characteristics. While Lake

Superior is oxic throughout its water column, even up to 5 cm into its bottom sediments,

Lake Erie is prone to hypoxia and is oxygen depleted in the bottom sediments (Matisoff et al., 2005). The central basin, due to its bathymetry, yields a thin hypolimnion after thermal stratification. Microbial respiration consumes oxygen so that hypoxic conditions occur at depth every July-September (Edwards et al., 2005; Matisoff et. al., 2005). These key differences make the two lakes an excellent system to study and compare microbial community structures and their physiologies, imparting to us a perspective on how these microorganisms contribute to the nutrient cycling and how community composition influences in biogeochemical cycles.

In this dissertation, I studied the AOA community structure, in Lakes Superior and Erie. In combination with a parallel study directed towards understanding the AOB present in the two lakes (Ray, 2012), this dissertation is the first study done towards identification, localization, quantification and diversity of the ammonia-oxidizing microbes in the Great Lakes, focusing on the different nitrifiers that inhabit oligotrophic

Lake Superior and mesotrophic Lake Erie.

1. Abundance of Ammonia-oxidizers in Lake Superior

This study revealed that in Lake Superior Archaea are the dominant ammonia- oxidizers. The AOA numbers in Lake Superior averaged approximately 106 cells per liter of lake water (Figures 24-27). On the contrary, the ammonia oxidizing bacterial population typically fell below detection limits (Ray, 2012). Of the total of 41 Lake

Superior samples in 2010 from various depths and stations analyzed for AOB, only 3

Chapter IV: DISCUSSION AND CONCLUSIONS| 117

showed detectable AOB, the total number of which averaged <10 cells per mL. These results are in agreement with a parallel study on the diversity and population of the Lake

Superior sediment AOA done in the lab of Dr. Annette Bollmann (personal communication), where qPCR studies using archaeal amoA primers in sediment samples from Lake Superior have shown that the AOA dominate the ammonia-oxidizing population in the lake, whereas the AOB population in the sediments of the lake is several orders of magnitude lower (Annette Bollmann, personal communication). The nitrite oxidizing bacterial (NOB) population, responsible for the final step of the nitrification process (the conversion of nitrite to nitrate), was also quantified at all the stations and depths profiled along with the AOA and AOBs (Ray, 2012). These results indicated the presence of the NOBs in all samples wherever the ammonia-oxidizing microbes were detected, irrespective of whether they are AOA or AOBs. This indicates that the nitrite oxidizers tend to live in a consortium with the ammonia-oxidizing microbial population in a physiologically tightly coupled community, reflected in the fact that nitrite is typically present at vanishingly low levels in most water samples. Such associations between the ammonia-oxidizers and the nitrite oxidizers have been reported previously indicating that the abundance of the ammonia-oxidizers in a particular niche determines that of the nitrite oxidizers in the same niche (Belser et al., 1979; Norton et al., 2011; Daims et al., 2011).

The reason why the AOA outnumber the AOB population in Lake Superior can be attributed to the ultraoligotrophic condition of Lake Superior. Previous kinetic characterization of the AOA culture of Nitrosopumilus maritimus have shown that these organisms are adapted to low ammonium concentrations with an extremely low half

Chapter IV: DISCUSSION AND CONCLUSIONS| 118

+ saturation constant (Km) of approximately 132 nM for total ammonia (NH4 + NH3)

(Martens-Habbena et al., 2009). In addition, Nitrososphaera gargensis, the I.1b thermophilic member of the Thaumarchaeota also exhibits adaptations to lower ammonium concentrations (Hatzenpitchler et al., 2008). By contrast, the cultured representatives of the AOB show a 100 fold higher requirement for ammonium concentration with Km ranging between 46 µM to 1780 µM total ammonium (Martens-

Habbena et al., 2009). Consistent with these findings, the average ammonium concentrations in Lake Superior has been found to be very low averaging at only 0.21

µM (Kumar et al., 2007). Similar observation in the sediments of Lake Superior as mentioned above (Annette Bollmann, personal communication) confirms this finding, wherein the AOB population was found to dominate the sediment nitrifying community. qPCR studies directed towards quantifying the amoA gene diversity of the nitrifying archaeal vs. bacterial populations in the sediments of Lake Superior at various stations indicate that the AOBs are the dominant nitrifiers in the sediments of Lake Erie, whereas the AOA population is very low (averaging at 106 AOA g-1 DW sediment in Lake Erie, compared to 109 AOA g-1 DW sediment in Lake Superior) compared to the AOB

(Annette Bollmann, personal communication and ASLO February 2013 talk).

In addition to the strong correlation observed between the very low ammonia concentrations and high AOA numbers in the lake, this study also indicates that the AOA tend to occupy the deeper waters of the lake, being selectively absent from the surface waters of the lake during the thermally stratified months of July, August and October.

Unsurprisingly, the nitrite oxidizers, obtaining energy from the AOA or AOB generated nitrite, follow the similar trend as of the AOA. They are found to be almost absent on the

Chapter IV: DISCUSSION AND CONCLUSIONS| 119

surface water; their abundance increasing with the depth in the lakes, as the AOA numbers increases (Ray, 2012). This observation parallels observation of the distribution of the AOBs in Lake Erie (Ray, 2012), where the AOBs are found to be restricted to the deeper water columns in the lake. This phenomenon of the AOA being completely absent from the surface waters of the lake during the stratified months can be attributed to the process of photo-inhibition of the AOA and AOB ammonia monooxygenase enzyme as suggested from other studies (Merbt et al., 2012; French et al., 2012). Such observations leave us with future scientific questions and exciting possibilities of looking into the enzymology, molecular biology, and crystal structure of AMO, in order to explain the biochemistry of the phenomenon of light inhibition observed in these organisms. Another explanation for the absence of the ammonia-oxidizers in the surface waters is attributed to greater ammonia-scavenging by heterotrophic bacteria abundant in the surface waters

(Small et al., 2013). This is less likely as the Km for ammonia uptake is typically higher than the Km for AOA-dependent ammonia oxidation (Verhagen et al., 1991). In any case, the stratification of ammonia oxidation leaves us with important questions regarding the biochemistry, adaptations and ammonia-scavenging processes by these organisms.

During the non-stratified months of April and May, the ammonia-oxidizers are found to be evenly distributed throughout the water column; no significant differences are observed in their numbers between surface and deeper water columns during these months. This can be attributed to the isothermal mixing condition observed in the lake during these months. The nitrite-oxidizers are found to follow the same pattern as the

AOA and AOB, being present in the surface waters during the isothermal mixing months,

Chapter IV: DISCUSSION AND CONCLUSIONS| 120

once again indicating the fact that the two groups of organisms tend to live together to complete the entire process of nitrification.

2. Abundance of Ammonia-oxidizers in Lake Erie

By contrast to what was observed in Lake Superior, the AOA population in Lake

Erie was typically found to be close to or below detection limits. Except for some very rare occurrences in the deeper waters, AOA were almost completely absent from the

Lake Erie nitrifier community. Contrasting to this observation, the ammonia-oxidizing bacterial population was found to dominate in the water samples analyzed by CARD-

FISH in Lake Erie at various depths and stations from the July 2011 cruise. Similar to the trend seen with AOA localizations in Lake Superior, AOB were also found to occupy the deeper water columns in Lake Erie, being completely absent from the surface samples, once again pointing out towards the light-inhibitory function of the ammonia mono- oxygenase enzyme. The very low ammonium concentrations in Lake Superior (0.21 µM) may contribute to the very low abundance of AOB in the lake, whereas due to the higher ammonium concentrations found in Lake Erie (2.64 µM in the eutrophic western basin, and 0.66 µM in the mesotrophic central basin), the AOB were found to be dominant

(Makarewicz et al., 2000; Richards and Baker, 2002).

The nitrite oxidizing bacteria (NOB) tend to follow the similar trend as observed in Lake Superior, that is, they tend to be present in every niche where the ammonia- oxidizers are present, irrespective of whether they are AOA or AOBs (Ray thesis). This again strongly points out to the fact that since the source of energy for these organisms is the nitrite, end product or ammonia-oxidation, they are likely to inhabit only the regions where the ammonia-oxidizers are present.

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3. Diversity of Ammonia-oxidizers in Lake Superior vs. Lake Erie

Based on PCR, sequence analysis and phylogenetic studies, the overall diversity of the AOA in Lake Superior was found to be very low. Mostly, both 16S and amoA-gene targeted diversity analysis of the AOA community in the lake falls within the Group I.1a

Thaumarchaeota cluster, grouping with the cultured marine representative

Nitrosopumilus maritimus. Almost all of the 16S and amoA environmental clones, obtained from water column samples in the lake at various stations including stations

Sterner C, WM, CD1, as well as from the sediments and various enrichment cultures from the lab (Mike Schlais dissertation work), fell into the Group I.1a Thaumarchaeota cluster (Figures 21, 23). Although belonging to the same group I.1.a Thaumarchaeota, the sediment clones were distinctly separate from the water column clones indicating a difference in the type of AOA species present in these two physiologically distinct habitats. Most clones obtained from water column as well as sediments from Lake

Superior, although belonging to Group I.1a Thaumarchaota, formed separate clusters distinct to Lake Superior in the phylogenetic trees, indicating that the Thaumarchaota or

AOA in the lake are endemic to the lake nitrifier populations. This observation parallels the diversity of the AOBs in the lake, where the less-dominating nitrifiers were also found to be restricted to a fewer distinct novel branches in the phylogenetic tree, most of the sequences grouping into a freshwater Nitrosospira cluster (Ray, 2012). The lower diversity of the AOA in contrast to their high abundance in the lake is consistent with similar observations of other abundant microbes in the lake, including cyanobacteria

(Ivanikova et al. 2007) and actinobacteria (Sharma et. al., 2009). These studies showed that the cyanobacteria and actinobacteria, although being abundant in the lake, were

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restricted to only a few specific phylogenetic clusters. The cold, oligotrophic conditions in the lake, with very low phosphorus availability, may be responsible for such lower diversity of microorganisms in the lake selecting for a few specific ecotypes that dominate the water column.

4. Conclusions and Future Directions:

This study concludes that Thaumarchaeota (AOA) dominate the ammonia- oxidizing microbial population in Lake Superior, whereas, the ammonia oxidizing bacterial population is negligible in the lake. In contrast, Thaumarchaeota are almost absent or negligible in Lake Erie, whereas, the ammonia oxidizing bacterial are the dominant nitrifiers in the lake, indicating towards a possible association of the AOA in environments with lower ammonium concentrations, whereas that of the AOBs with environments with higher ammonium concentrations. Thaumarchaeota (AOA), in spite of being so abundant in Lake Superior, tend to be less diverse in the lake, indicating that only few species of these ammonia-oxidizers can survive the extreme oligotrophic conditions of the lake.

All the ammonia oxidizers, irrespective of whether they are bacteria or archaea, tend to be absent from surface waters, whereas their abundance increases with depth of water column in the lakes. Future studies directed towards understanding the reasons for such niche separation may throw some more light into the reasons for this. Indeed, light inhibition of AOA or AOB may be one of the reasons. Such studies may aim at understanding the light sensitivity of AOA cultures isolated from the lakes, by growing them in presence and in absence of various light intensities, and subsequently measuring amoA expressions in the AOA cells using techniques such as qPCR or microarrays.

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Since most of the enzymes involved in the biochemistry of nitrification in these organisms are copper dependent, a possible effect of copper in the environment on the growth and amoA expression in the organisms cannot be ruled out. Therefore, another important experiment that may reflect further on the biochemistry of these nitrifiers is to study the effect of various concentrations of copper on the growth and expression of amoA in enrichment cultures of AOA from the lakes. Similarly, comparing and contrasting the protein structure of AMO in both organisms may also throw important light into the biochemistry as well provide us with a greater understanding of the reasons for the preferential niche separation between these two groups.

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APPENDIX A. ABBREVIATIONS

AMO Ammonia Monooxygenase amoA Ammonia Monooxygenase Subunit A

AOA Ammonia-oxidizing Archaea

AOB Ammonia-oxidizing Bacteria

CARD-FISH Catalyzed Reporter Deposition Fluorescence in-situ Hybridization

Chla Chlorophyll a

DAPI 4, 6-diamidino-2-phenylindole

DOC Dissolved Organic Carbon

DW Dry Weight

ETC Electron Transport Chain

HAO Hydroxylamine Oxidoreductase

IAGLR International Association for Great Lakes Research

Km Half Saturation Constant

NADP Nicotinamide Adenine Dinucleotide Phosphate

NCBI National Center for Biotechnology Information

NOB Nitrite-oxidizing Bacteria

NO Nitric Oxide

NTP Nucleotide tri-phosphate

PBS Phosphate Buffer Saline

PCR Polymerase Chain Reaction qPCR Quantitative Polymerase Chain Reaction APPENDIX| 130

rRNA ribosomal Ribonucleic Acid

TCA Tricarboxylic Acid Cycle