UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO ...repositorio.ul.pt/.../10451/10434/1/ulfc103211_tm_catarina_gouveia.pdf · UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA VEGETAL

Nitrification Control. How do the Natural

ecosystem do it?

Catarina Andreia Ricacho Gouveia

Tese de Mestrado

Dissertação

Mestrado em Microbiologia Aplicada

2012/2013

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA VEGETAL


ecosystem do it?

Dissertação orientada pela Profª Dr.ª Maria Manuela Carolino (FCUL)


Mestrado em Microbiologia Aplicada

2012/2013


ecosystem do it?


2012/2013

This thesis was fully performed at the Department of Plant Biology in the

University of Lisbon under the direct supervision of Prof. Dr. Maria Manuela

Carolino in the scope of the Master in Applied Microbiology of the Faculty of

Science University of Lisbon.

Master in Applied Microbiology 2012-2013

Master thesis Catarina Gouveia

Personal Acknowledgments……………………………………...…….………………………………………… I

Summary/Resumo (Portuguese)………………………………………………………………………………….. II

Abstract………………………………………………………………………………………………………………. 1

Introduction……………………………………………………………………………………………………..…….2

Materials and methods…………..……………………………………………….…………………………………8

Study site …………………………………………………………………………………………8

Experimental design …………………………………………………………………………….8

Soil and Root sampling …………………………………………………………………………9

AOA and AOB enrichments cultures ………………………………………………………...10

Ammonia and pH susceptibility ……………….................................................................10

Effect of organic compounds in AOB nitrite production…………………………………….10

AOM Indirect Growth Measurement …………………………………………………………10

DNA extraction …………………………………………………………………………………11

PCR conditions ………………………………………………………………………………...12

PCR-RFLP ……………………………………………………………………………………...12

Statistical analysis ……………………………………………………………………………..13

Results and Dicussion ……………………………………………………………………………………………13

Where does the ammonia oxidizing community prosper? ………………………………...13

Nitrite production by bacteria ………………………………………………………13

Nitrite production by archaea ………………………………………………………18

How would the AOB population respond to an increase in N input…….. ……………….21

Ammonia oxidizing activity by AOB from soils with and without N addition …..21

AOB community structure ………………………………………………………….27

Ammonia oxidizing activity by AOA from soils with and without N addition …. 29

How does the AOB population responds to abiotic factors? ……………………………...32

Ammonia susceptibility in AOB cultures from soils with and without N-

addition………………………………………………………………………………..32

pH susceptibility in AOB cultures from soils with and without N- addition

…………………………………………………………………………………….......33

Effects of organic compounds in AOB cultures from soils without N

addition………………………………………………………………………………..35

Conclusion ………………………………………………………………………………………………………….37

Acknowledgements ……………………………………………………………………………………………….38

References ………………………………………………………………………………………………………….38

Annex…………………………………………………………………………………………………………………40

Nitrification Control. How do the Natural ecosystem do it?



Personal Acknowledgments

I would like to thank the Department of Plant Biology (DBV) of the Faculty of Sciences,

University of Lisbon for hosting and supporting the research project for the development of my

Master thesis.

My greatest appreciation to Prof. Dr. Maria Manuela Carolino, who directly supervised

the project, for the mentorship and help through my question filled work, for the friendship and for

the interesting and fun talks that allow me to work easier in longer days. I also thank prof. Dr.

Cristina Cruz and Dr. Sandra Chaves for their patience and support in supervising my Master

thesis laboratory work, for teaching me how to write, not only a good scientific report, but also a

story, and for the inquisitive question that have allowed me to improve my work and myself. My

thanks, also, to Dr. Teresa Dias, whose ideas and work in Arrábida were the bases for my thesis

and helped create my scientific questions.

I would also like to thank Prof. Dr. Rógerio Tenreiro and Dr. Luís Carvalho for the given

help in a troublesome statistical analysis. To prof. Dr. Ana Reis for the stimulating discussions

that raised some surprizing questions, ideas and conclusions in my work. To prof. Dr. Lélia

Chambel for the help choosing the best DNA fingerprinting method when I could not make my

mind. To prof. Dr. Margarida Barata and Dr. Patricia Correia for support and motivating talks. To

prof. Dr. Francisco Dionisio for helping me in data analysis. My thanks to Manuela Lucas,

Herculana, Teresa Granja, Célia Lima, Rute Miguel, for the indispensable help around the lab

and for their teachings. To my lab collegues, Maria Calado, Raquel Costa, Marta Delgado, Inês

Meleiro, Juliana Melo, Frederico Eutrópio and Florian Uhlm, whose good humour make the lab

into a warm and fun environment to work.

I thank my family and friends, whose curiosity and misunderstanding to subject in the

academic field and about my research made me think in a simpler and easier way to explain

science. To my aunt whose strength made keep going forward. To my sister for enduring my

anxiety and stress in a way only she could do. And to my parents, for their endless love and

dedication that helped me follow my dreams, for the debates and discussion that pushed me to

be grow as a person and for their understanding of who I am as a person and a scientist.

Nitrification Control. How do the Natural ecosystem do it? I



Summary/Resumo

O ciclo do N, composto por diversas etapas, é mediado principalmente por

microrganismos. Os microrganismos diazotróficos fixam Azoto (N2), introduzindo-o na biosfera.

Os compostos orgânicos azotados são depois mineralizados, em ambientes aeróbios, a amónia.

Esta é rapidamente oxidada a nitrito (NO2-) e nitrato (NO3

-) pelos microrganismos oxidantes de

amónia (AOM) e oxidantes de nitrito (NOM), processo denominado de nitrificação. Em ambientes

anaeróbios, pode ocorrer a desnitrificação com a redução do nitrato a óxido nítrico (NO), óxido

nitroso (N2O) e azoto molecular (N2) (Bock and Wagner, 2006).

Um equilíbrio da forma e quantidade de N nos ecossistemas é vital, pois o ecossistema

pode perder fertilidade com reduzida quantidade de N, mas pode sofrer eutrofização com

elevadas concentrações de N. O ciclo do Azoto pode não ocorrer linearmente, sendo a resposta

da comunidade microbiana responsável pelos processos de oxi-redução do azoto dependente

das condições do ecossistema, tal como, a taxa de conversão de cada processo dependente do

controlo da atividade microbiana, afeta a disponibilidade e a forma de azoto no ecossistema.

Com excesso de N na forma orgânica, deverá haver um aumento na atividade de mineralização,

seguido de nitrificação e desnitrificação, resultando na libertação de N para a atmosfera. Esta

situação ocorre, se existirem condições ótimas para todos os processos, no entanto, o aumento

da concentração de amónia, nitrito, nitrato, ou outros compostos resultantes dos processos oxi-

redutivos do ciclo do azoto, no solo, pode ocorrer se as condições necessárias para um dos

processos não se verificarem.

A nitrificação é o processo de oxidação da amónia a nitrito e nitrito a nitrato, catalisado

por 2 grupos de organismos distintos (Norton and Stark, 2011; Fienck et al. 2005; Sinha and

Annachhatre, 2007), sendo a oxidação da amónia considerado o passo limitante da nitrificação.

A oxidação da amónia é catalisada por diversos grupos, sendo as bactérias e árqueas

quimiolitotróficas oxidantes de amónia (AOB e AOA respetivamente) os principais intervenientes

em ambientes terrestres aeróbios (Norton and Stark, 2011; Sinha and Annachhatre, 2007).

As bactérias oxidantes de amónia (AOB) quimiolitotróficas são membros das classes β

ou γ de Proteobacteria. Nas β-proteobacteria existem 4 géneros diferentes, Nitrosomonas,

Nitrosospira, Nitrosolobus e Nitrosovibrio. O género Nitrosococcus pertence às γ-Proteobacteria

(Purkhold et al. 2000). As AOB estão distribuídas por diversos ambientes costeiros, marinhos e

ambientes polares (maioritariamente 2 clusters, Nitrosomonas e Nitrosospira), ambientes salinos

ou hipersalinos (Nitrosomonas), condições com temperaturas elevadas (Nitrosospira) e

ambientes acídicos (Nitrosomonas) (Junier et al. 2010; Prosser and Nicol, 2008).

AOB são bactérias litoautotróficas, tendo como substrato principal a amónia (NH3), e

como dador de eletrões a hidroxilamina. A oxidação da amónia é catalisada pela enzima amónia

monoxigenase (AMO), produzindo hidroxilamina, sendo esta oxidada a nitrito pela enzima

hidroxilamina oxiredutase (HAO) (Bock and Wagner, 2006; Hatzenpichler, 2012).

As árqueas oxidantes de amónia (AOA), pertencentes ao novo filo Thaumarchaeota

(Norton and Stark, 2011; Hatzenpichler, 2012) aparentam estar presentes em diversos ambientes

mesofilicos (marinhos, água-doce e terrestres) assim como em condições extremas (ambientes

Nitrification Control. How do the Natural ecosystem do it? II



acidófilos, termófilos, águas profundas e fontes termais) (Junier et al. 2010; Nicol and Schleper,

2006). A via metabólica das AOA é pouco conhecida, não tendo sido ainda identificado nenhum

homologo da enzima HAO (Hatzenpichler, 2012; He et al. 2012). No entanto, parecem ter a

capacidade de utilizar fontes orgânicas de energia como aminoácidos (He et al. 2012; Tourna et

al. 2011; Hatzenpichler, 2012). A enzima AMO das AOA, comparativamente à AMO das AOB,

tem uma maior afinidade para a amónia, podendo ter as AOA uma maior contribuição para a

atividade nitrificante em ambientes oligotróficos (He et al. 2012; Martens-Habbena et al. 2009).

A importância da nitrificação baseia-se no facto de ser o único processo biológico

oxidativo que liga fontes de azoto inorgânico reduzido (amónia) e oxidado (nitrito e nitrato)

(Martens-Habbena et al. 2009; Norton and Stark, 2011; Fienck et al. 2005; Sinha and

Annachhatre, 2007). Nitrito, o produto da oxidação da amónia, é normalmente encontrado em

baixas concentrações nos solos devido à sua toxicidade, sendo essencial manter uma baixa

concentração (Bock and Wagner, 2006; Norton and Stark, 2011; Cleemput and Samater, 1996).

Para além do nitrito, o óxido nítrico e óxido nitroso também podem ser produzidos através da

redução do nitrito pela nitrito redutase (NIR) presente em algumas bactérias oxidantes de amónia

(Bremner, 1997; Burns et al. 1996). Além de promoverem a acidificação do solo com a formação

de ácidos, o óxido nitroso age como um gás com efeito de estufa, 310x mais potente que o

dióxido de carbono (Yamanaka, 2008; Bock and Wagner, 2006; Hatzenpichler, 2012). O nitrato

pode ser utilizado como fonte de azoto por diversos organismos, e em excesso pode causar

eutrofização desse ecossistema. Sendo facilmente lixiviado pode causar a contaminação de

águas subterrâneas (Fienck et al, 2005).

Tendo a nitrificação uma forte influência no fluxo de azoto nos sistemas terrestres e

marinhos, e os seus produtos importantes na determinação da qualidade de solos e águas, o

estudo da influência dos fatores bióticos e abióticos no controlo da atividade nitrificante e

crescimento dos organismos nitrificantes é essencial.

Existem diversos fatores estudados que influenciam o crescimento e atividade das

comunidades oxidantes de amónia. Um dos principais fatores é a concentração de substrato,

neste caso, amónia. Como foi referido, o aumento da concentração de N no ecossistema pode

levar a um aumento da atividade nitrificante, dependendo da suscetibilidade das estirpes de AOB

ao substrato e a sua adaptação ao aumento da amónia. Este aumento de atividade é

problemática se houver acumulação de nitrito, nitrato ou outros produtos resultantes dessa

atividade. A acumulação de nitrito ocorre em condições favoráveis à oxidação da amónia

desfavoráveis à oxidação de nitrito e desnitrificação, como, ambientes bem oxigenados e

condições alcalinas ou com pH neutro (Fienck et al. 2005), sendo as AOB muito suscetíveis a

alterações no pH. Logo, a oxigenação dos solos e pH são outros fatores a ter em conta. Em

ambientes pouco oxigenados (condições de microaerofilia) a formação de óxidos nitroso e nítrico

é estimulada (Fienck et al. 2005; Bock and Wagner, 2006). Em solos carbonatados a produção

de óxidos é reduzida pela interação química com os carbonatos, formando nitrito e nitrato. (Bock

and Wagner, 2006). A produção de nitritos pode ser inibida pela presença de compostos

orgânicos (Adair and Schwartz, 2008), no entanto algumas espécies de AOB podem crescer

Nitrification Control. How do the Natural ecosystem do it? III



mixotroficamente (amónia e C orgânico como fonte de energia, podendo haver produção de

nitritos) com compostos como piruvato ou acetato, ou organotroficamente (C orgânico como fonte

de energia, sem produção de nitritos) com açúcares. Contudo a nitrificação pode ser importante

e benéfica para algumas plantas que utilizem preferencialmente o nitrato (Bock and Wagner,

2006), logo em ambientes pobres em nitrato a nitrificação pode ser estimulada pelas plantas.

Assim, fatores a considerar para o estudo de populações de microrganismos oxidantes de

amónia são nicho (solo rizosférico ou superfície das raízes), a adaptação à adição de N no solo,

a concentração de substrato, pH das culturas e presença de matéria orgânica.

Tendo em vista o estudo da comunidade oxidante de amónia (Bactérias e Árqueas) num

ecossistema mediterrânico os principais objetivos deste trabalho foram:

1. Estudar a localização preferencial da comunidade oxidante de amónia (solo

rizosférico ou superfície das raízes de Cistus ladanifer)

2. Estudar o efeito da adição de N no solo, na atividade e estrutura das

comunidades oxidantes de amónia

3. Estudar o efeito da concentração de substrato (amónia), pH e presença de

compostos orgânicos azotados/ glucose / acetato em culturas de AOB

provenientes de solos com e sem adição de N

Num ecossistema mediterrâneo, pobre em nutrientes, os nichos preferenciais para o

crescimento de microrganismos oxidantes de amónia deverão estar localizados perto da raiz das

plantas, ou na superfície das raizes, proporcionando uma maior troca de nutrientes entre planta-

microrganismos (Ochua-Hueso et al, 2011), além de facultar condições de oxigenação, pH e

conteúdo em água favoráveis para o crescimento e atividade de oxidantes de amónia.

Sendo os solos pobres em azoto e matéria orgânica (Dias et al. 2012), espera-se

encontrar populações de AOB mais adaptadas a ambientes oligotróficos. No entanto, em solos

com frequente adição de N ao longo de 6 (desde 2007), o aumento da disponibilidade de N na

forma de amónia, deverá determinar adaptação das populações de AOB, aumentando a

atividade oxidante de amónia, e alterando a comunidade ao nível estrutural.

Em relação ao efeito da concentração de amónia nas culturas de AOB, populações de

AOB adaptadas a baixas concentrações de amónia no solo, deverão ser mais suscetíveis a

elevadas concentrações de amónia, comparativamente com populações de AOB adaptadas a

maiores concentrações de amónia, ou seja provenientes de solos com frequente adição de azoto.

Relativamente ao efeito do pH, espera-se que populações com maior atividade nitritante sejam

mais tolerantes à acidificação, uma vez que o aumento da atividade leva a uma diminuição do

pH, podendo resultar ou não na acidificação do solo, dependendo da composição deste. No

efeito de compostos orgânicos nas culturas de AOB, espera-se que haja menor atividade com

fontes energéticas orgânicas (para crescimento mixotrófico ou organotrófico), uma vez que AOB

adaptadas a ambientes oligotróficos prefiram fontes energéticas e de carbono inorgânicas, como

amónia e CO2.

Para o estudo de microrganismos oxidantes de amónia, amostras de solo foram

recolhidas de um campo experimental, localizado no Parque Natural da Serra da Arrábida.

IV Nitrification Control. How do the Natural ecosystem do it?



Plantas de Cistus ladanifer de solos sem adição de azoto foram recolhidas juntamente com solo

rizosférico a 5-6 cm de profundidade, em julho para estudar o nicho preferencial das AOM. Solo

rizosférico e raízes lavadas com água destilada foram utilizadas como inoculo para crescimento

de AOB. Para o estudo do efeito da adição de N no solo, amostras de solo foram retiradas a 1

cm da raiz primária de Cistus ladanifer a 5-6 cm de profundidade dentro dos talhões dos 4

tratamentos: controlo (solo sem fertilização) e três diferentes adições de azoto: 40 e 80 kg de N

/ha /y na forma de NH4NO3 (40AN e 80AN) e 40 kg de N/ ha /y na forma de NH4+ (40A). O solo

foi utilizado como inoculo no crescimento de AOB e AOA. As populações de AOM foram

seletivamente cultivadas por 30 (AOB) /60 (AOA) dias com 3 enriquecimentos sucessivos em

meio SFC (Synthetic Freshwater Crenoarchaeota) (Konneke et al. 2005; De La Torre et al. 2008),

de modo a assegurar a diluição de microrganismos heterotróficos e de matéria orgânica no

inoculo de solo inicial. O meio foi suplementando de acordo com a especificidade para bactérias

e/ou árqueas oxidantes de amónia (3 antibióticos diferentes). No 3º enriquecimento os meios e

condições de incubação foram suplementados/alterados de acordo com o objectivo (fonte de

azoto, carbono, concentração de amónia, pH). A atividade das comunidades oxidantes de

amónia foi avaliado regularmente pela quantificação do produto final de nitrito, permitindo

distinguir a cinética da produção de nitrito de cada cultura. A atividade de cada cultura foi

estudada no 3º enriquecimento de modo a ter-se culturas de AOB sem contaminantes. A

presença de árqueas e bactérias oxidantes de amónia foi confirmada pela amplificação do gene

16S rRNA e amoA, especifico para cada grupo. A estrutura das comunidades bacterianas

oxidantes de amónia foi avaliado por PCR-RFLP fingerprinting, com as enzimas de restrição HinfI

e HaeIII. A análise estatística dos dados foi efetuada em SPSS, versão 20 para Windows.

Comparando o número de culturas ativas, taxa de produção de nitrito e concentração

máxima de nitrito, uma localização preferencial de atividade das AOB foi observado no solo

rizosférico de Cistus ladanifer. No entanto, não foi detetada a presença de AOA nas culturas.

Relativamente ao efeito da adição de azoto no solo, verificou-se uma tendência para o

aumento da atividade oxidante de amónia das populações de AOB. No entanto, a resposta da

população ao aumento de N no solo depende da forma e quantidade de N aplicado. O controlo

da atividade dependerá também da disponibilidade da amónia nos solos, havendo uma

competição pelo substrato entre plantas e AOB (Verhagen et al. 1994), principalmente nos

tratamentos com maior cobertura vegetal como o 40AN (Dias et al, 2011). Embora a presença

de AOA não seja confirmada, as culturas de AOA provenientes de solos sem adição de azoto

apresentaram uma maior atividade oxidante de amónia, indicando uma resposta das populações

de AOA diferente das populações de AOB. Por PCR-RFLP verificou-se que a estrutura das

populações de AOB é alterada com a adição de azoto no solo, tendo os resultados também

demonstrado a heterogeneidade dos solos com o mesmo tratamento. Comparando os perfis de

PCR-RFLP realizados in-silico para as espécies conhecidas do género Nitrosomonas e

Nitrosospira e os perfis obtidos a partir das populações de AOB, Nitrosospira tenuis foi

identificada nas populações de AOB de solos com adição de azoto.

V Nitrification Control. How do the Natural ecosystem do it?



Nas populações de AOB foram observadas diferentes suscetibilidades a concentrações

crescentes de amónia, tendo as comunidades AOB provenientes de solo sem adição de N uma

maior suscetibilidade à amónia, comparativamente a populações de AOB de solos com adição

de N. A atividade oxidante de amónia, com produção cumulativa de nitrito, das culturas de

populações de AOB presentes em solos sem adição de N foi inibida pela presença de compostos

orgânicos como peptona, glucose e acetato. No entanto, as populações de AOB parecem ter a

capacidade de utilizar ureia como fonte energética em substituição da amónia.

Considerando a influência dos fatores estudados: nicho preferencial, adição de N no

ecossistema, concentração de substrato, pH e presença de compostos orgânicos na atividade

das populações de AOB, estas demostraram ser suscetíveis a alterações, não são resilientes e

não aparentam ter redundância funcional (não têm o mesmo nível de atividade). Mas a influência

que os fatores exercem sobre a população nitrificante é necessária para controlar essa atividade,

de modo a que não haja perda de N no sistema. A adição de N no solo não implica

necessariamente um aumento da atividade nitritante, se este N não estiver em excesso. No

entanto, N em excesso aumentará a atividade nitrificante, com consequente diminuição da

quantidade de N inorgânico no solo. Este aumento de atividade levará a uma diminuição no pH,

particularmente em solos sem um forte efeito tampão, o que leva a uma inibição da nitrificação.

O aumento de nitrito leva também ao aumento de nitrato (oxidação do nitrito a nitrato), e ao

consequente aumento da biomassa e compostos orgânicos no solo, inibindo a atividade de AOB

sensíveis a matéria orgânica. Diminuindo a oxidação da amónia haverá novamente o aumento

de N inorgânico no solo, de modo a não haver perda de N e consequente perda de fertilidade do

solo. Esta regulação é mantida devido à sensibilidade apresentada pelas populações de AOB a

estes fatores, sendo essencial para o equilíbrio de N no ecossistema.

VI Nitrification Control. How do the Natural ecosystem do it?



Nitrification control. How do the Natural ecosystem do it?

Catarina A.R. Gouveia Department of Plant Biology, University of Lisbon, Lisbon, Portugal Master in Applied Microbiology, Faculty of Sciences, University of Lisbon, Lisbon, Portugal

Nitrification is a fundamental and central step in the nitrogen cycle, linking the

reduced and oxidized nitrogen pools, which processes are mostly accomplished by

microorganisms. The first step is mainly done by ammonia oxidizing bacteria (AOB) and

by ammonia oxidizing archaea (AOA). Regulation of nitrification activity is essential in

ecosystem to maintain N balance, since N excess can cause eutrophication and

production of toxic compounds, and N limitation can reduce soil fertility. Mediterranean

ecosystem soils are poor in terms of N therefore microbial communities can be more

susceptible to change when confronted with N input, pH change or presence of organic

matter. This study aims to understand the niche preference, the impact of N addition in

soils, pH, substrate (ammonia) concentration and presence of organic compounds in

ammonia oxidizing populations from Mediterranean soils. Rhizospheric soil and root

samples from Cistus Ladanifer were collected from an experimental field in which has been

added 3 different N - treatments since 2007, in Serra da Arrábida Natural Park. By using

serial culture enrichments and nitrite quantification through incubation time it was

possible to quantify and study nitrifying activity. The presence of AOB and AOA in each

enrichment was confirmed by PCR. AOB structural community’s change by N-addition in

soils was assessed by PCR- RLFP. AOB susceptibility to ammonia, pH and organic

compounds was studied by testing several ammonia concentrations in the medium,

altering pH and supplementing the medium with organic compounds. As a preferential

niche for AOB activity, AOB populations appear to be more abundant in rhizospheric soil

of Cistus ladanifer. Soils with N-addition had altered AOB population with higher ammonia

oxidizing activity and a different community structure. By comparing in silico PCR-RFLP

profiles and the AOB profiles it was possible to identify Nitrosospira tenuis in the AOB

population. Though the presence of AOA was not confirmed in any of the treatments,

activity of possible AOA from soils without N –addition was higher, than of AOA from soils

with N-addition. AOB activity appeared to be influenced by the substrate concentration,

with AOB from soils with N-addition being less susceptible to change in substrate

concentration. AOB cultures appear to be susceptible to pH, having an activity inhibition

with pH above 7 and under 6. The populations are also sensible to the presence of organic

matter, however, being capable of using urea as a substrate for ammonia in litotrophic

growth and nitrite production activity. It was observed that ammonia oxidation activity can

be stimulated by N-addition, altering not just the activity but also the population structure

to have a higher tolerance to the substrate. While ammonia oxidising activity is inhibited

by pH changes and presence of organic matter.

1 Nitrification Control. How do the Natural ecosystem do it?



Introduction

Nitrogen Cycle

Nitrogen is one of the important elements for life, as it is needed to synthesize major

molecules in cell components, as aminoacids and nucleotides being two of many examples. The

biogeochemical cycle of nitrogen comprises the biotic and abiotic conversion of the nitrogen

compounds between their oxidised and reduced forms. These conversions are done in the

atmosphere, in the biosphere and the interaction between both (Bock and Wagner, 2006). In the

biosphere, most of the reactions are catalysed by microorganisms. Dinitrogen (N2) can be fixed

and introduced in the biosphere by diazotrophs, in the form of ammonia (NH3) or organic nitrogen

(amine group). Ammonia, the most frequently found form of nitrogen in the biosphere can be

released from organic compounds by mineralization. In aerobic environments ammonia is quickly

oxidised to nitrite (NO2-) and nitrate (NO3

-) by ammonia-oxidizing microorganisms (AOM), and by

nitrite-oxidizing microorganisms (NOM) in a process called nitrification. In anoxic conditions

denitrification can occur. The nitrate is used by denitrifying microorganisms, producing dinitrogen

(N2), nitrous oxid (N2O) and nitric oxid (NO) (fig.1). (Bock and Wagner, 2006; Fiencke et al. 2005)

The cycle is not entirely linear. Depending on the ecosystem conditions the microbial community

responses change. So, the key processes stated above can directly affect the availability and

form of N within the ecosystems. The increase of nitrogen can augment the concentration of toxic

products, such as ammonia, nitrite, nitrous acid and nitric acid. It can also cause eutrophication

of that ecosystem, and the contamination of ground water supplies (Fiencke et al. 2005). The

decrease nitrogen can affect soil fertility and plant production (Bock and Wagner, 2006).

Strategies of the Natural ecosystem to

balance the nitrogen availability

The natural ecosystems have

developed mechanisms that involve

multiple paths to nitrogen conservation

(Dias et al. 2012). Paths to balance the

inorganic and organic nitrogen, and to

compensate the nitrogen increase or

decrease in soil are essentials to the

ecosystems. If there is an excess of

nitrogen in soils, it can be reduced by

mineralization (if the nitrogen appears as

an organic form) followed by nitrification

(directly if the nitrogen is in the form of

ammonia or urea) and denitrification

(directly with nitrate), resulting in the

formation of atmospheric nitrogen. If the

Fig. 1 Nitrogen Cycle Scheme 1) Nitrogen fixation; 2) Ammonia assimilation; 3) Ammonia mineralization; 4) Ammonia oxidation (1st step in Nitrification); 5) Nitrite oxidation (2nd step in Nitrification); 6)Nitrate reduction 7) Nitrite reduction; 8) Nitrate leaching; 9)Nitrate assimilation; 10) Denitrification




system has limited nitrogen there is a need to increase nitrogen fixation and diminished

nitrification and denitrification.

Nitrification and ammonia oxidation

Nitrification is the biological conversion of reduced nitrogen, ammonia, ammonium (NH4+)

or organic N to oxidized N, nitrite and to nitrate successively. The importance of this process lies

on the fact that it is the only oxidative biological process linking reduced and oxidized pools of

inorganic nitrogen in nature (Martens-Habbena et al. 2009; Norton and Stark, 2011; Fiencke et

al. 2005; Sinha and Annachhatre, 2007), and therefore has a strong influence in the fate of N in

terrestrial systems.

The oxidation of ammonia by microorganisms can be done by several groups. In the

presence of oxygen by the chemolithotrophic bacteria and heterotrophic bacteria. In the absence

of oxygen by Planctomycetes. In aerobic soil environments the key participants in nitrification are

chemolithotrophic bacteria and archaea. Heterotrophic nitrification can be catalysed by fungi,

actinomycetes, and other bacteria, and seems to be significant in some forest and acidic pasture

soils. The anaerobic oxidation of ammonium is known as the anammox process and it is

widespread in marine and freshwater environments, but rarely found in soil. (Norton and Stark,

2011; Sinha and Annachhatre, 2007).

Ammonia-oxidizing bacteria (AOB)

Since the first chemolithotroph ammonia oxidizing bacteria isolation at the end of the

nineteenth century by Winogradsky (1890), 16 more species of AOB have been described,

accounting for a total of 25 culturable species (Sinha and Annachhatre, 2007). However, many

not described species can be found, in culture collections at least 15 additional genospecies (Bock

and Wagner, 2006) and more in natural environments.

These 25 species of chemeolitotrophic ammonia oxidizing bacteria (AOB) are comprise

in the Proteobacteria phylum. Comparative 16S rRNA sequence analysis has shown that all

recognized AOB are members of the β- or γ-subclass of Proteobacteria. In the β-proteobacteria

there is 4 genera Nitrosomonas, Nitrosospira, Nitrosolobus and Nitrosovibrio. The Nitrosococcus

genus belongs to the γ-Proteobacteria (Purkhold et al. 2000).

AOB are widespread in coastal, open ocean, polar environments (mainly 2 clusters,

Nitrosomonas and Nitrosospira), hypersaline enviroments (Nitrosomonas), high temperatures

(Nitrosospira) and acid environments (Nitrosomonas) (Junier et al. 2010; Prosser and Nicol,

2008). Most soil bacterial ammonia oxidizers belong to Nitrosospira genera (Purkhold et al.

2000). Ammonia oxidizing Bacteria rarely grow in soils with a pH lower tan 7, however there are

several niches for AOB were the pH is optimal for AOB (Junier et al. 2010).




For lithotrophic AOB ammonia (NH3) but not ammonium (NH4+) is essential as the primary

substrate and the intermediate hydroxylamine (NH2OH) as the real energy source. Organic

substances like urea or glutamine can be used as alternatives energy sources, when ammonia is

not available, by some strains of Nitrosomonas (Nitrosomonas ureae) (Bock and Wagner, 2006).

An ammonia oxidation schematic model of electron transport is shown in figure. 2. Ammonia

oxidation begins with the oxidation of ammonia to hydroxylamine by AMO. Oxygen (O2) is needed

to form the intermediate compound and water. Hydroxylamine is oxidize to nitrite by HAO

(hydroxylamine oxidoreductase). Two of the four electrons derived are required for AMO activity.

The other two are used for energy generation (Bock and Wagner, 2006; Hatzenpichler, 2012). As

most metabolic studies have been done in Nitrosomonas europaea, the metabolic pathways may

not be the same for the other genera.

AOB are autotrophic bacteria able to fix carbon through the Calvin-Benson-Bassham

(Calvin) cycle (Hatzenpichler, 2012; Sinha and Annachhatre, 2007). Some Nitrosomonas strains

have the capacity to grown mixotrophically with organic compounds as carbon source (Clark and

Schmidt 1967).

In presence of certain organic carbon compounds, such as pyruvate, the production of

nitric oxide and nitrous

oxide is enhance.

Nitrosomonas eutropha

and Nitrosomonas

europaea denitrify under

low oxygen concentration

conditions (Yamanaka,

2008; Bock and Wagner,

2006; Hatzenpichler,

2012).

Ammonia oxidizing

archaea (AOA)

The discovery of ammonia-oxidizing archaea (AOA) (Könneke et al. 2005) fundamentally

changed our view of nitrogen cycling in the environment. It was also the start of a new trend of

studies related to the importance of archaea in moderate ecosystems and in nitrification

processes. Since the first study by Kӧnneke (2005) several culture- independent studies with the

amoA gene have shown the presence of AOA in several diverse environments. They are present

in mesophyll environments (marine, freshwater, and terrestrial environments) as well as in

extreme conditions (acidophilic condtions, thermophile environments, deep-sea water and hot

springs) (Junier et al. 2010; Nicol and Schleper, 2006). In some ecosystems they can have a

higher contribution to nitrification, out-growing the AOB that are not adapted to the environmental

conditions (Junier et al. 2010; Nicol and Schleper, 2006). Anoxic conditions in river sediments

Fig. 2 Model of electron transport chain of Nitrosomonas europaea. Scheme from Fienck et al. (2005). AMO= ammonia monooxygnase; C= cytoplasmic side of membrane; Cyt= cytochrome; HAO=hydroxylamine oxireductase; NIR= nitrite reductase; P=periplasmic side of membrane; UQ= ubiquinone




(Liu et al. 2013), acidic soils or poor nutrient ecosystems (He et al. 2012) are examples, among

other.

While in an aquatic environment the majority of AOA belong to “mesophilic

crenarchaeaota” 1.1a lineage, representing approximately 20% of all prokaryotes found in these

ecosystems (Karner et al. 2001; Prosser and Nicol, 2008), most soils are dominated by

sequences of ammonia oxidizing archaea belonging to 1.1b lineage.

The attention and studies done in this group of archaea culminated in a proposal of a third

archaeal phylum named Thaumarchaeota (Brochier-Armanet et al. 2008; Spang et al. 2010), with

a set of genomic characteristics that make it and distinct phylum (Hatzenpichler, 2012; Spang et

al. 2010). Currently the only common physiological trait is the chemolithoautotrophic growth by

ammonia oxidation. (Norton and Stark, 2011; Hatzenpichler, 2012; He et al. 2012).

The metabolic pathways for ammonia oxidation in AOA are still unclear. No HAO

homologue has been found in any AOA genome, so AMO may not catalyse the same reaction as

in AOB (Hatzenpichler, 2012; He et al. 2012). In contrast to AOB, thaumarchaeotal populations

seem to have the genetic potential and metabolic ability to use amino acid as energy source

(Hatzenpichler, 2012). This metabolic ability can also be seen by the preference for organic

sources of nitrogen, e.g. aminoacids (He et al. 2012; Tourna et al. 2011). AOA also have a higher

affinity for ammonia (Martens-Habbena et al. 2009), being more adaptable to low nutrients

conditions, AOA can out-number the AOB in oligotrophic environments.

Some marine species of AOA, e.g. “Candidatus Nitrosopumilus maritimus” have shown

a growth inhibition in presence of organic carbon. However the terrestrial Nitrososphaera strains

growth can be enhanced by small amounts of pyruvate. So in some soil environments the AOA

diversity can be enhance by a rich organic carbon content (Liu et al. 2013), contrasting with most

AOB species that are sensible to the presence of organic carbon. So, the preferential nutritional

and environmental conditions of AOB and AOA can be distinct, allowing a separation of both

communities in distinct niches.

Importance of nitrification and regulation:

Through the nitrification process, several compounds can be produced. The intermediary

product of nitrification, nitrite is usually is found in trace amounts comparing with other forms of N

that can appear in relatively high concentrations (Bock and Wagner, 2006; Norton and Stark,

2011, Dias et al., 2012). The maintenance of a low nitrite concentration in aerobic habitats is

essential since nitrite is toxic to microorganisms, root plants and seeds (Bock and Wagner, 2006;

Norton and Stark, 2011; Cleemput and Samater, 1996). So, a sequential and almost immediate

nitrite oxidation to nitrate by nitrite oxidizing bacteria (NOB) is fundamental in maintaining a low

nitrite concentration. The sequential nitrite consumption and oxidation is possible since ammonia

oxidation to nitrite is done at low rates, being considered the rate-limiting step of nitrification (Bock

and Wagner, 2006; Norton and Stark, 2011; Fiencke et al. 2005).

Nitrite accumulation can be found at low oxygen partial pressure, e.g. soil with a water

content, under alkaline conditions where ammonia oxidation is enhanced, in environments with




high ammonia concentrations and in conditions with decreased or inhibited nitrite oxidation

activity. Nitrite oxidising organisms (NOB) can be inhibited by high ammonia concentrations

(Norton and Stark, 2011) or by nitrite, since high concentrations are toxic. Without nitrite oxidation

by NOB, it becomes difficult to regenerate the low nitrite state of the ecosystem.

In anoxic conditions, nitrite accumulation can lead to nitric oxide and nitrous oxide

production by heterotrophic bacteria denitrifcation, or by AOB denitrification.. It was though that

these oxides were produced as intermediaries in ammonia oxidation (Cleemput and Samater,

1996; Bremner, 1997). However it is now known that AOB are capable producing these oxides

by a denitrification process, with the reduction of nitrite by nitrite reductase (NIR) in anoxic

conditions (Bremner, 1997; Burns et al. 1996), as seen in some species of AOB e.g. Nitrosomas

eutropha and Nitrosomonas europaea (Yamanaka, 2008; Bock and Wagner, 2006;

Hatzenpichler, 2012). The production of these oxides is problematic since both oxides form acids

(nitrous acid and nitric acid) when interacting with water and may contribute to soil acidification

and root damage (Fiencke et al., 2005). Nitrous oxide also acts like a greenhouse gas with a

potential 310 times higher than carbon dioxide (Bock and Wagner, 2006; Fiencke et al., 2005).

Therefore regulation of nitrifying activity is essential to maintain the level of ammonia,

nitrite and nitrate so that there is not a loss of fertility with low N quantities, nor a system

eutrophication due to high N quantities.

As it is mentioned above the main factors that can regulate ammonia oxidation activity

and AOM growth are substrate concentration (ammonia concentration), pH, presence of organic

matter, oxygenation, water content, temperature and biotic regulation through plant-AOM

interaction which can be by substrate competition with plants, or effect of plant exudates.

Mediterranean type ecosystem

This study focus on AOB and AOA communities found in Mediterranean-type ecosystem.

Mediterranean ecosystem can be characterized by a highly seasonal climate with hot dry

summers and mild wet winters. This asynchronous ecosystem with marked seasonal changes

greatly influences the N fluxes. In summer when soils become water-limited the microbial activity,

like decomposition and ammonia mineralization, tends to decrease, as should also occur to

nitrification. The microbial activity should be being higher in spring or/and autumn due to the

increase in water content (Ochua-Hueso et al. 2011; Dias et al. 2012). Concerning vegetation,

the study site, in Serra da Arrábida was characterized as being a dense maquis with Cistus

ladanifer as the dominant summer semi-deciduous plant (Dias et al. 2008). As for inorganic N

content, it is known to vary greatly through the year, being lower in August and higher in November

(Ochua-Hueso et al. 2011). Soil can be considered poor in N content, with the non-fertilized soils

(without any N addition) having a very low quantity of inorganic N even in spring when soils have

a greater nutrients turnover. It is an acidic soil with a pH of 5.8 ±0.2. (Dias et al. 2012). Microbial

communities from these types of ecosystem should be adapted to low water content, climate

changes, and low nutrient content. For microorganism adapted to low nutrient concentrations, as




the microbial communities found in Mediterranean soils, a nutrient input can cause abrupt and

drastic alterations in community structure and activity.

Aims of this thesis

The work presented in this thesis aims to understand the ammonia oxidizing community

in soils from a Mediterranean type ecosystem. The first objective was to know in which niche AOM

community’s had a higher ammonia oxidation activity. Two niches were studied the root surface

and rhizospheric soil of the most abundant plant species, Cistus ladanifer (Dias et al. 2008). After

assessing the niche with the higher ammonia oxidation activity, the second objective was to

compare the ammonia-oxidising communities from soils under nitrogen addition. The 3rd objective

was to assess the effect of ammonia concentration, pH and presence of organic matter in AOB

cultures.

Hypothesis:

In a poor nutrient ecosystem as the Mediterranean ecosystem, the preferential niche for

the AOM growth and activity should be near the plant root, to have a higher nutrient exchange

between the plant and AOM (Ochua-Hueso et al. 2011), besides giving conditions, as pH and

oxygenation, favourable to AOM. Microsite in the surface of the root may have the environmental

conditions to ammonia mineralization and production to occur, increasing the quantity of substrate

for AOM in that area (Cleemput and Samater, 1996), while rhizospheric soil around the root may

not have the same quantity of nutrients.

Bacterial or archaeal communities that are adapted to low nutrient conditions tend to

suffer great changes with nutrient addition. Although some microbial groups have shown a higher

degree of metabolic flexibility and physiological tolerance when changing the growth conditions

this case is not seen for AOB communities, as rapid growth rates and high abundances are

needed for the community’s strains to have that flexibility and adaptability (Allison et al. 2008).

Regarding the effect of N-addition in soils, the increase in ammonia-oxidation activity

when the ecosystem has an increase in N quantity is excepted since it can be a strategies to

counterbalance the nitrogen excess in the naturally low nutrient ecosystem. However, as AOM

populations in poor nutrient soils may be adapted to oligotrophic environments, a change in the

population structure may occur as adaptation to the higher N input.

As population adapted to low N content, the AOB population from soil without N addition

are expected to be more susceptible to higher ammonia concentrations. Concerning pH it is

described that AOB prefer pH basic to neutral (Bock and Wagner, 2006; Norton and Stark, 2011;

Hatzenpichler, 2012), however AOB population can have ammonia oxidation activity at a slightly

acid pH (>6, <7) (Hatzenpichler, 2012), so it is expected to have ammonia oxidation activity

between pH 6.5 and pH 7.8. As the soils are poor in organic matter (Dias et al. 2012), AOM

populations may prefer inorganic energy and carbon sources, though AOM can have a

mixotrophic or even organotrophic growth.




Materials and Methods:

Study site

The present study was conducted at Serra da Arrábida in the Arrábida Natural Park, a Natura

2000 site in south of Lisbon, Portugal (PTCON0010 Arrábida/Espichel). The study site (38º 29’ N

- 9º 01’ W) is located within a region belonging to the sub-humid thermo-mediterranean bioclimatic

domain (Clemente 2002). According to the climatic normal (2012) in July, when the first soil collect

was done, the mean maximum temperature was 28ºC, mean minimum temperature was 15ºC

and mean precipitation was 1 mm. In October, the second soil collect was done, the mean

maximum temperature was 22ºC, mean minimum temperature was 14ºC and mean precipitation

was 50 mm. Reported data refer to the nearest climatic station (Setúbal, 15 km distance –Instituto

Português do Mar e Atmosfera). The dominant plant species is Cistus ladanifer (Dias, 2008).

Experimental design

The experimental design was defined on the scope of FCT project “Spheres of ecosystem

response to nitrogen: a case Study in a Mediterranean-type ecosystem in the southern of Portugal

(SERN) PTDC/BIA BEC/099323/2008. It consists of 12 plots, of 400 m2 each. N availability was

modified by the addition of 40 and 80 kg N ha-1 yr-1 in the form of NH4NO3 (40AN and 80AN,

respectively) and 40 kg N ha-1 yr-1 as a 1:1 mixture of NH4Cl-N and (NH4)2SO4-N (40A) (Fig.2).

Each treatment had three replicates (3 plots). To prevent N ‘contamination’ through runoff from

fertilized plots, the experimental plots were distributed in three rows along the slope, with the

controls (without fertilization) being located in the top row. The fertilization began in January 2007

and it is still being applied. Three equal applications have been applied throughout the year:

middle autumn/winter, spring and summer.

Soil and Root sampling

The soil samples used for the study of ammonia oxidizing archaea (AOA) and ammonia oxidizing

bacteria (AOB) preferential distribution were

collected in July 2012. Three soil locations were

randomly chosen in each control plot (C, F and E)

(Fig. 3). None of the sample was taken from the

modified plots. For each sample rhizospheric soil

and roots were taken from seed germinated Cistus

ladanifer plants at a depth of 5 - 6 cm. Samples

were stored in clean plastic cups and refrigerated.

In the laboratory, individual samples were

immediately weighted and prepared for the

microbial enrichment cultures.

For the activity and community structure comparison of ammonia-oxidizing communities in the

non-fertilized and fertilized (altered) plots the sampling was performed in October 2012. Three

rhizospheric soil samples were taken from different seed germinated Cistus ladanifer at a depth

Fig.3 Experimental design scheme and the relative location of the experimental plots (Green - Control; Orange - 40A; Blue - 40AN; and Grey - 80AN)




of 5 - 6 cm in each plot (Fig.3). Samples were maintained cold in clean plastic cups. A composite

sample was obtained by homogenization of three samples from each of the three field replicate

plots. 1 gram of soil from each composite sample was used as inoculum for the sequential

enrichment cultures (AOB and AOA). A soil extract was prepared for each composite sample with

1 gram diluted in 10 ml of ultra-pure water for nitrite and ammonia quantification.

Ammonia oxidizing microorganism’s enrichments cultures

To compare the nitrifiying activity in Cistus ladanifer rhizospheric soil and roots three sequential

enrichments cultures were prepared to allow a selection and subsequent increase of the ammonia

oxidizing microorganism’s (AOM) population.

In the first enrichment 36 enrichment cultures were prepared: 18 cultures for AOB (9 for roots, 9

for rhizospheric soil) and 18 for Archaea (9 for roots, 9 for rhizospheric soil). Ammonia oxidizer

Bacteria enrichment cultures were achieved in 25 mL McCartney flasks containing 20 mL of Fresh

Synthetic Crenoarchaeota (SFC) with 1 mM of NH4Cl. 1 g of soil or roots were used to inoculate

20 ml of medium. SFC medium (Konneke et al. 2005; De La Torre et al. 2008) consistes of NaCl

(1 g L-1), MgCl2·6H2O (0.4 g L-1), CaCl2·2H2O (0.1 g L-1), KCl (0.5 g L-1), MgSO4.7H2O (5 g L-1)

and KBr (0.1 g L-1). After sterilization in autoclave (121ºC; 10min) the medium was supplemented

with: per litter of medium, 1 mL of non-chelated trace element mixture, 1 mL of vitamin solution,

10 ml of KH2PO4 solution (4 g L-1), 1 ml of tungstate-selenite solution, 1 ml of sodium bicarbonate

solution (1 M), and 1 ml of NH4Cl (1M). These solutions were prepared with distilled water and

sterilized in autoclave (121º for 10 min) or by filtration (0.45 µm) for heat-sensitive compounds.

The final pH is between 6.5 and 7. Ammonia oxidizer archaea (AOA) first enrichment cultures

were prepared with streptomycin in a concentration of 2.5 µg ml-1. Cultures were incubated at

28ºC without shaking during 30 and 60 days for AOB and AOA respectively. After the first

enrichment stage, AOM cultures producing more than 1 mg/L NO2- (positive cultures) were sub-

cultured into a second enrichment stage transferring 5% (v/v) inoculum to 20 mL SFC fresh

medium supplemented with sodium chlorate (5 mg L-1). For the second enrichment for AOA

streptomycin was replaced by ampicillin at 100 µg ml-1. AOM cultures that didn’t produce nitrites

(negative cultures) or those producing less than 1 mg /L of nitrites were discarded. The third

enrichment for AOB was obtained by transferring 5% (V/V) of inoculum into 20 ml of fresh SFC

medium. For AOA cultures ampicillin was replaced by 0.05% chloramphenicol.

For the study of ammonia oxidizing communities in the non-fertilized and in fertilized plots,

rhizospheric soil was used to inoculate 24 initial enrichment cultures: 12 initial cultures for AOB

and 12 for AOA. 1 g of soil was used to inoculate 20 ml of SFC medium with 1 mM of NH4Cl,

supplemented with Calcium Carbonate (7.5 gl-1), Pimaricine (0.04 gl-1), and sodium chlorate (5

mg l-1). The first enrichment for Archaea had ampicillin in a concentration of 100 µg/ml. For AOB

the medium was not altered in the sequential enrichments. For AOA cultures streptomycin (100

µg ml-1) was added to the 2nd and Chloranphenicol 0.05% to the 3rd enrichments.




In every enrichment 5 % (v/v) of inoculum was transferred from both positive and negative

cultures. Cultures were incubated at 28ºC without shaking during 30 and 60 days for AOB and

AOA respectively.

Ammonia susceptibility:

SFC medium was supplemented with 5mM of sodium chlorate, pimaricine (0.04 gL-1),

calcium carbonate (7.5 gL-1) and different ammonia concentrations: 1 mM, 10 mM, 50 mM, 100

mM and 500 mM. The medium was inoculated at 5 % with 2nd enrichment AOB community

cultures. Cultures were incubated at 28°C during 15 days without shaking. Growth was indirectly

estimated by assessing cultures activity by nitrite concentrations determinations at several time-

points by the Griess method.

pH susceptibility:


calcium carbonate (7.5 gL-1) and 1Mof ammonia concentrations. pH was altered with 1M and 10M

HCl and 1M NaOH to achieve pH 5.6, 6.7, 7.1 and 7.9. The medium was inoculated at 5 % V/V

with 2nd enrichment AOB community cultures. Cultures were incubated at 28°C during 30 days

without shaking. Growth was indirectly estimated by assessing cultures activity by nitrite

concentrations determinations at several time-points by the Griess method.

Effect of organic compounds in AOB nitrite production


calcium carbonate (7.5 gL-1) and different organic compounds: urea (46.8 mg/L), peptone (101

mg/L), sodium acetate (0.5 %) and glucose (0.5 %).Concentrations for urea and peptone were

calculated to have the same amount of N as the control media (1M of NH4C, corresponding to

0.78 mM of N). Media with a combination of ammonia and urea or peptone was prepared with 0.5

M of ammonia chlorate and urea (mg/L) and peptone (mg/L). Media with sodium acetate or

glucose had 1M of ammonia, but not sodium bicarbonate.The medium was inoculated at 5 % with

2nd enrichment AOB community cultures. Cultures were incubated at 28°C during 30 days without

shaking. Growth was indirectly estimated by assessing cultures activity by nitrite concentrations

determinations at several time-points by the Griess method.

AOM Indirect Growth Measurement

AOM indirect growth was estimated by measuring nitrite concentration at several time-

points by the Griess method (Standard Methods, 1999). 200 µl were removed from each culture

and the ammonia-oxidizing reaction was stopped with a 4 M KCl solution in a 1:2 proportion. 50

µl of the sample were mixed with 125 µl of a sulphanilamide solution (10 g L-1) diluted with 10%

concentrated HCl and 125 µl of N-(1-naphthyl)-ethylenediamine solution (100 mg L-1). The assays

were conducted in a 96-well microplate and the absorbance was read at 540 nm in a

spectrophotometric microplate reader (Tecan Spectra Rainbow Micrplate Reader 400-700nm,




Switzerland). Nitrite standard curve was prepared using sodium nitrite solutions with

concentrations ranging between 1–100 μM. Nitrite concentration of experimental samples were

obtained from the resulting standard curve of nitrite concentration vs. absorbance.

The nitrite concentration through time was used to establish a nitrite production profile

and typical parameters were determined. For the 1st enrichment, length of Lag phase, nitrite

production rate, maximum nitrite concentration and area under the curve were used to compare

the different cultures. For the 2nd and 3rd stages of enrichment, nitrite production rate and

maximum nitrite concentration were used to compare ammonia oxidizing community’s cultures.

Lag phase was defined as the time segment between the beginning of incubation time

and beginning of exponential nitrite production, expressed by days. Nitrite production rate was

define at exponential phase with linear regression and was expressed as mg of nitrite produced

per liter per incubation day (mg/L/d). Maximum nitrite concentration was determined as the

highest nitrite concentration value after the 1st day of incubation, expressed as mg of nitrite per

liter.

Additionally, ammonia concentration was determined in the end of each culture

enrichment stage by the Berthelot reaction. 50 µl of each culture was first mixed with 50 µl of 5 %

sodium citrate solution (pH 7). After 1 min of incubation at room temperature, 50 µl of 2-

phenylphenol nitroprusside (PPS-nitroprusside) solution was added. Then 25 µl of hypoclorite

buffer (pH 13) was added followed by 100 µl of deionized water. The microplate was incubated in

the dark at room temperature for 45 min before reading. The assays were conducted in a 96-well

microplate and the absorbance was read at 660 nm in a spectrophotometric microplate reader

(Tecan Spectra Rainbow Micrplate Reader 400-700nm, Switzerland). The PPS-nitroprusside

solution was prepared with 3.22 g of 2-hydroxybiphenyl sodium salt tetrahydrate and 0.015 g of

sodium nitroprusside dehydrate dissolved in 100 ml of deionized water. The buffer was prepared

by dissolving 1 g of tri-sodium phosphate dodecahydrate in deionized water and 10 ml of sodium

hypochlorite.

Subsequentially ammonia consumption (%) (equation 1) and nitrite production yield (%) (equation

2) were determined. The yield is calculated given in account the N weight, not the total molecule

weight.

( [𝑁𝐻4+]𝑓𝑖𝑛𝑎𝑙 − [𝑁𝐻4

+]𝑖𝑛𝑖𝑡𝑖𝑎𝑙 ) × 100 (equation 1)

(([𝑁𝑂2−]𝑚𝑎𝑥𝑖𝑚𝑢𝑚 × 0.31)/([𝑁𝐻4

+]𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 × 0.782)) × 100 (equation 2)

DNA extraction:

DNA was extracted from 10 - 15 ml of the enrichment cultures using a direct boiling method

(Sambrook et al. 2001). The samples were centrifuged at 4000 rpm for 10 minutes to remove the

medium precipitated CaCO2. The supernatant was centrifuged at 4000 rpm for 10 min. The pellet

was ressuspended in TE buffer (pH 8) and centrifuged at 13 000 rpm for 5 min to wash the cells.

1 30% of NO2- molecular weight is nitrogen 2 78% of NH4+ molecular weight is nitrogen




The supernatant was discarded and the pellet was ressuspend with TE supplemented with 0.1%

tween. The samples were incubated at 100ºC for 10 min.

PCR amplification

Each 50 μl PCR reaction contained: 0.2 µl (1 unit) BioTaq DNA Polymerase (Bioline, 500 units),

5 µl (1x) NH4+ Buffer (Bioline, 10X), 2 µl (2 mM) MgCl2 (Bioline, 50 mM), 1µl (0.2 mM each) dNTPs

(10 mM each), 1 µl (50 ρmol/µl) of each primer, and 2.5 µl of BSA (0.1 %). 1 μl of extracted DNA

was used as template.

Thermal conditions for the bacterial 16S RNA PCR were as follows: 3 min initial denaturing step

at 94 °C, followed by 35 cycles of 1 min denaturing at 94 °C, 2 min annealing at 50 °C and 1 min

extension at 72 °C, with a final extension step of 3 min at 72 °C.

All PCR products were verified on 1% agarose gels stained with ethidium bromide under UV light.

Primers used for amplification, targets and positive controls are listed in table 1.

Primer Sequence target Positive control Tested

dilutions reference

104F 5’-GGCGVAYGGGTGAGTAA-3’ Bacterial 16S

rRNA Escherichia coli

1:1; 1:10;

1:25

Sandra Chaves

907R 5’-CCGTCAATTCMTTTRAGTTT-3’ Muyzer et al. 1995

ArchF 5’-GAATTGGCGGGGGRGCA -3’ Archaeal 16S

rRNA

Halobacterium

salinarium

DSMZ

1:1

Sandra Chaves,

personal

communication ArchR 5’-TGTGTGCAAGGRGCAGGG – 3’

amoA-1F 5′-GGGGTTTCTACTGGTGGT-3′ Β-proteobacteria

amoA

Nitrosomonas

europaea 1:1; 1:10

Rotthauwe et al.

1997 amoA-2R 5′-CCCCTCKGSAAAGCCTTCTTC-3’

Arch-

amoA-rer 5´- TTCTTCTTTGTWGCCCARTA – 3’

Archaeal amoA - 1:1

Sandra Chaves,

personal

communication Arch-

amoA-for 5’ – CTGAYTGGGCYTGGACWTC – 3’

PCR- RFLP fingerprinting:

Selection of the restriction endonuclease was based on the in silico analysis of several selected

amoA sequences from the β-ammonia oxidizing bacteria group of AOB available on GenBank,

access numbers given by Purkhold et al. (2000), using the NEBCutter V2.0

(http://tools.neb.com/NEBcutter2/) from new England Biolabs. The enzyme HinfI and HaeIII was

selected given its ability to discriminate between the different strains, with 2 or 3 hydrolysis region,

and did not produced fragments with less than 20 bp length.

Amplification of amoA genes for RFLP analysis was performed as described above. The

restriction was done separately for each enzyme. Reactions contained 10 µl of PCR product from

each sample, 5 U of HinfI and 1x NEBuffer 2 (New England Biolabs GmbH, Frankfurt, Germany)


Table 1. Primers used for PCR-amplifications of archaeal and bacterial 16S rRNA and amoA genes.

http://tools.neb.com/NEBcutter2/



or 5 U of HaeIII and M buffer (TAKARA bio INC. Kyoto, Japan). Restriction reactions were

performed at 37 ºC for 24 h. Full reaction volumes were loaded and visualized on a 1.5 % agarose

gel 1:3 high resolution ultra-pure agarose - 1000, 2:3 ultra-pure agarose (Invitrogen

Carlsbad, Estados Unidos) under UV light, after staining with ethidium bromide (EtBr) for 15 min.

Statistical analysis

The statistical analysis was performed using SPSS 20 software for Windows. All data was

checked for normality with the Levine’s test. The coefficient variation (equation 3) was calculated

as a normalized measure of dispersion for all the data. Linear data was checked for multiple

comparisons of means with ANOVA at p<0,05. Kruskal-Wallis test was performed for non-linear

data at p<0,05. Rank transformation followed by a two-way ANOVA was performed for the AOB

community’s ammonia sensibility data, as described by Iman and Conover (1983).

𝐶𝑉 = 𝜎 (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑛𝑡𝑖𝑜𝑛)

µ (𝑚𝑒𝑎𝑛) (equation 3)

Results and Discussion

Where does the Ammonia-oxidizing community prosper?

Cistus ladanifer as the most abundant plant species in the experimental study, was

expected to have the highest influence on soil microbial community. So, the activity of ammonia

oxidizing communities in Cistus ladanifer root surface and rhizospheric soil was assessed in order

to understand in which of these niches presents a higher ammonia oxidation activity. As described

in methods, 3 sequential enrichments were done. Active AOB and AOA cultures were defined as

cultures with nitrite accumulation through time, where the maximum nitrite concentration is higher

than the nitrite concentration found in lag phase.

Nitrite production by Ammonia-Oxidizing Bacteria

Ammonia-oxidizing bacteria growth is difficult to measure, so most of the studies are

based on the activity, or nitrite production. With the nitrite measurement through the incubation

time it is possible to have nitrite production curves to characterize that community.

In the rizospheric soil community 1st enrichment, nitrite accumulation can be seen during

the beginning of incubation, 2 days for soil and root AOB cultures (fig.4 a and d), but decreases

after that period of time. For soil AOB cultures nitrite starts to accumulate 7 days after the first

decrease. Nitrite production recovery for root AOB cultures starts after 15 days of incubation. Both

soil and roots AOB cultures had a 2nd decrease in nitrite concentration, more evident for root AOB

cultures at 22 days of incubation (fig. 4 d) than soil AOB cultures at 13 days of incubation (fig.4

a). If in the cultures AOB where the only functional community active then the nitrite production

from ammonia should be constant. However the community’s nitrite production dynamics includes

diauxic growth and the variations in nitrite concentration can be considered as a result of the

culture dynamics, since the coefficient variation (CV) for Griess method is 0.1, lower than these

variations.


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The 1st enrichments are prepared from soil and roots, adding nutrients and organic

compounds to the medium. Since the microbial inoculum corresponds to the community present

in that soil and root surface, both AOM and other heterotrophic bacteria, capable of producing

nitrite, are present in the culture. AOM have a slow growth and most AOB species need several

days to weeks for growth, depending on the conditions such as substrate concentration, oxygen

availability, the temperature and pH values (Bock and Wagner, 2006). Since heterotrophic

bacteria tend to out-grow the AOM in selective media, using the soil inoculum nutrients, the

observed diauxic growth can be explained by the change in nutrient source from the ones in the

medium to the nutrients in soil and roots.

Concerning the studied nitrite production parameters: lag phase, time period, in days (d),

between the beginning of incubation and beginning of exponential phase; nitrite production rate,

mg of nitrite produced per day per liter of medium (mg/L/d), calculated in the exponential phase;

and maximum nitrite concentrations (mg/L), the enrichment community cultures from both soil

and roots AOB cultures presented a high heterogeneity (table. 2 in annex) for maximum nitrite

concentration (1.04 and 1.46, respectively) and nitrite production rate (1.59 and 2.3). Lag phase

for soil and root AOB culture present the lowest variation (0.26 and 0.32). Root AOB cultures had

higher CV for the studied parameters compared to soil AOB cultures.

The 3 studied plots (plot C, E and F) from soils without N addition, are considered field

replicates in the experimental design, however considering the natural heterogeneity found in

soils (Ochua-Hueso et al. 2011), it is expected to have a higher heterogeneity between

communities from the same plot and between communities between the 3 plots.

Comparing the maximum nitrite concentration produced by the AOB cultures in both

niches (Table 2), soil AOB cultures had higher maximum nitrite concentrations (3.59 mg/L) than

root surface AOB cultures (1.35 mg/L), though significant differences were not found (ANOVA

p=0.95, table 3 in annex). Particularly, communities from plot C had the highest values (9.36

mg/L, 3.06 mg/L and 11.27 mg/L) in soil AOB cultures. In root surface AOB cultures, plot C also

presents the highest values for maximum nitrite concentration (2.26 mg/L, 0.75 mg/L and 6.62

mg/L) (table 2). Soil communities also present higher nitrite production rates than root surface

communities (0.46 mg/L/d and 0.15 mg/L/d, respectively) (table.2), though significant differences

were not observed (ANOVA, p=0.95) (table. 3 in annex). The highest nitrite production rate was

also observed for plot C in soil AOB cultures and root AOB cultures (table.2).Concerning lag

phase, root AOB cultures had higher lag phases than soil AOB cultures (23.33 d and 16.67 d,

respectively) (table 2), lag phase was significant different between the soil and root AOB cultures

(sig of 0.049, ANOVA with p= 0.95) (table. 3 in annex).

Since root cultures had cultures without constant nitrite production, nitrite production rate

could not be calculated and where given a value of 0 mg/L/d. Lag phase for these cultures is 30

days, corresponding to the complete incubation time.

The soil used to prepare the 1st enrichment can have more nutrients and organic

compounds available than roots with complex organic compounds that need to be degraded


Table. 2. 1st enrichment nitrite production parameters for the Rhizospheric Soil AOB community cultures and root surface AOB community culture of Cistus ladanifer from non-fertilized soils without N addition: Plot C, E and F. Parameters include lag phase, expressed by day, nitrite production Rate expressed by mg nitrite/L/d and maximum nitrite concentrationexpressed as mg/L. Plant 1, 2, 3 are field replicates from which the samples were collected.



before being used as energy source for heterotrophic bacteria. This could explain the higher lag

phase in root community cultures, particularly in plot C communities.

To correctly compare the AOB community activity, cultures without growth of other

microorganisms are needed. Therefore a 2nd enrichment was performed.

From 9 inital rhizospheric soil community cultures, 7 were considered active and used as

inoculum for a 2nd serial enrichment. From root community cultures 3 were considered active and

used to prepare the 2nd enrichment (table. 1 in annex).

In the 2º enrichment’s community’s cultures (fig. 4 b and e) constant nitrite production

can be observed. No nitrite decrease is observed for the root or soil AOB cultures (fig. 4 b and e).

The nitrite concentration decrease observed in the 1st enrichment indicated a growth of other

microorganisms than AOB. With a serial transfer, nutrients and organic compounds found in the

1st enrichment were diluted, decreasing the growth of heterotrophic bacteria.

As nitrite-oxidizing bacteria could be one of the possible groups of bacteria existing in the

inoculum, in the 2nd enrichment a nitratation (nitrite oxidation to nitrate) inhibitor, sodium chlorate,

was added to the medium. Chlorate appears to affect specifically the nitrite-oxidizing bacteria,

and has little to none inhibitory effect on the growth and activity of ammonium oxidizers (Belser

and Mays, 1980). With a nitratation inhibitor the rate at which nitrite accumulates will be the rate

of ammonium oxidation (Norton and Stark, 2011), allowing a more correct determination of

ammonia oxidation parameters: maximum nitrite concentration and nitrite production rate.

Soil AOB cultures had a higher nitrite production rate and maximum nitrite concentrations

(2.15 mg/L/d and 27.26 mg/L) than root AOB cultures (1.19 mg/L/d and 14.91 mg/L) in average

(table 3). Observing each culture from soil and root AOB cultures, in soil AOB cultures Plot E

presents the highest values for maximum nitrite concentration (30.22 mg/L and 29.49 mg/L) while

plot C has the lowest value (23.01 mg/L), while the the lowest and highest nitrite production rates

rates can be seen for in plot E (1.48 mg/L/d and 2.89 mg/L/d). For root AOB cultures the highest

Rhizospheric Soil Cultures Roots Cultures

Plot Plant Lag phase (d) Nitrite production rate

(mg/L/d)

Maximum [nitrite] (mg/L)

Lag phase

(d)

Nitrite production rate

(mg/L/d)

Maximum [nitrite] (mg/L)

Plot C 1 7 0.65 9.36 15 0.18 2.26

2 16 0.15 3.06 15 0.05 0.75

3 21 2.48 11.27 15 1.08 6.62

Pot E 1 15 0.13 2.06 30 0 0.4

2 15 0.21 2.82 30 0 0.2

3 15 0.05 0.84 30 0 0.17

Plot F 1 21 0.07 0.46 30 0 0.43

2 21 0.45 1.52 30 0 0.26

3 19 0 0.89 15 0.07 1.06

mean 16,67 0,46 3,59 23,33 0,15 1,35

Standard deviation

4,27 0,74 3,72 7,45 0,35 1,96


Table.3. 2nd enrichment nitrite production parameters for the Rhizospheric Soil AOB community cultures and root surface AOB community culture of Cistus ladanifer from non-fertlized soils without N addition: Plot C, E and F. Parameters include lag phase, expressed by day, nitrite production Rate expressed by mg nitrite/L/d and maximum nitrite concentrationexpressed as mg/L. Plant 1, 2, 3 are field replicates from which the samples were collected.



maximum nitrite concentration and nitrite production rate can be observed in plot C (27.98 mg/L

and 2.39 mg/L/d), and the lowest values can also be observed in plot C (0.21 mg/L and 0 mg/L/d)

(table. 3).

Lag phase was measured, but not analysed, since it depends on the activity level of the

previous cultures when they were transferred, requiring a similar activity level to be comparable.

A higher homogeneity can be observed for the soil AOB cultures (CV of 0.2 for nitrite

production rate and 0.08 for maximum nitrite concentration) in the 2nd enrichment, though root

AOB cultures did not presented an increase in homogeneity (CV of 1.86 for nitrite production rate

and 0.92 for maximum nitrite production) in this enrichment as did the soil AOB cultures.

As the serial transfer dilutes the microbial cells from previous cultures, the prevailing

community in the following enrichments corresponds to the fast growing microorganism, capable

of growing in the selective medium. So, only a portion of the AOB, with “fast” growth among other

AOB, are being transferred to the following enrichment, explaining the homogeneity between

communities from different plots in soil AOB cultures.

Root AOB cultures presented a culture without nitrite production activity, and one culture

with low activity, explaining the high heterogeneity observed (table. 3).

Comparing soil AOB community culture parameters, maximum nitrite concentration and

nitrite production rate, for the values obtained in the 1st and 2º enrichment, an increase of the

nitrite maximum concentration can be observed (table.2 and table. 3) by 7 fold, and an increase

in nitrite production rate by 5 fold was observed (table 2 and 3).

2nd enrichment Rhizospheric Soil Cultures Roots Cultures

Plot Plant

Lag

phase

(d)

Nitrite

production

rate (mg /L/d)

Maximum nitrite

Concentration

Lag

phase

(d)

Nitrite production

rate (mg/L/d)

Maximum nitrite

Concentration

(mg/L)

Plot C

1 5 1.55 23.01 2 2.39 27.98

2 7 2.09 27.52 - - -

3 7 2.16 26.81 30 0 0.21

Pot E

1 9 1.46 30.22 - - -

2 7 2.89 27.28 - - -

3 19 2.67 29.49 - - -

Plot F

1 - - - - - -

2 9 2.22 26.474 - - -

3 - - - 30 0.47 4.19

Average 9 2.15 27.26 18.50 1.19 14.09

Standard

deviation 4.28 0.49 2.16 14.52 2.21 12.91


Table. 4 3rd enrichment nitrite production parameters for the Rhizospheric Soil AOB community cultures and root surface AOB community culture of Cistus ladanifer from non-fertilized soils without N addition: Plot C, E and F. Parameters include lag phase, expressed by day, nitrite production Rate expressed by mg nitrite/L/d and maximum nitrite concentration expressed as mg/L. Plant 1, 2, 3 are field replicates from which the samples were collected.



Given the highest number of active cultures from rhizospheric soil (7 cultures) and root

suface (2 cultures) (table. 1 in annex), it is though that the AOB community is more spread in

rhizospheric soil and more disperse in root surface.

To confirmed these results, and confirmed that the nitrite is being produced by AOB and

not by other bacteria another enrichment was implemented.

From the 2nd enrichment, positive cultures with nitrite production were transferred, 7 for

soil AOB cultures and 2 for roots AOB cultures (table.1 in annex).

Observing the nitrite production curves for root and soil AOB cultures (fig. 4 c and f), both

profiles seem similar though a high heterogeneity can be observed for soil AOB cultures.

Concerning maximum nitrite concentration the values are similar for root AOB cultures

(28.28 mg/L) and soil AOB cultures (27.95 mg/L) (table. 4). Nitrite production rate is higher for

root AOB cultures (2.37 mg/L/d), compared to soil AOB cultures (2.13 mg/L/d) (table.4).

Since both parameters are similar for root and soil AOB communities, the assessment of

AOB preferential environmental niche was based on the number of active cultures from each

niche. As it was mentioned before AOB community in rhizospheric soil is thought to be more

spread than in root surface, where could be and more disperse. So, what differs between niches

is the community abundance, being AOB communities more abundant in rhizospheric soil than

root surface

Comparing 2nd and 3rd enrichment root AOB community it is possible to detect the

differences in the analysed parameters (table 2 and 3). Nitrite production rate and maximum nitrite

production was lower in the 3rd enrichment.

Soil AOB community presented similar results for nitrite production rate and maximum

nitrite concentration in both enrichments. As the nitrite production dynamics were constant

between enrichments, soil AOB cultures appeared to be free of contaminants.

3rd Rhizospheric Soil AOB Cultures Roots AOB Cultures

Plot Plant

Lag

phase

(d)

Nitrite production

rate (mg/L/d)

Maximum nitrite

Concentration

(mg/L)

Lag

phase

(d)

Nitrite

production

rate (mg/L/d)

Maximum nitrite

Concentration

(mg/L)

Plot C

1 19 1.67 24.98 7 1.86 26.38

2 7 2.19 28.18 - - -

3 23 1.87 22.84 - - -

Pot E

1 7 2.39 32.79 - - -

2 7 2.39 29.68 - - -

3 7 2.03 28.04 - - -

Plot F

1 - - - - - -

2 7 2.36 32.18 - - -

3 - - - 9 2.89 29.53

average 11 2.13 28.38 8 2.37 27.95

Standard

deviation 6.41 0.26 3.34 1 0.51 1.58




Nitrite production by Archaea

As the nitrifying community composed by AOB, the presence and activity of AOA was

also assessed.To study of archaeal ammonia oxidizing activity, growth inhibiton of AOB was

necessary, since they can out-grow AOA. This was accomplished by adding antibiotics in the 3

enrichments preventing bacterial growth. Since the cellular targets for the antibiotics were

different and the antibiotic was changed in each enrichment, occurrence of resistant strains was

prevented.

AOA communities can only be analysed after the 3rd enrichment, since the 1st and 2nd

enrichment could have bacterial growth from resistant strains, or tolerant to the used antibiotic

CV 0.58 0.12 0.12 0.13 0.22 0.06


a d

b e

Fig. 4. Nitrite production through incubation time for AOB cultures from Cistus ladanifer root surface and rhizospheric

soil. a) 1st enrichment soil AOB cultures; b) 2nd enrichment soil AOB cultures; c) 3rd enrichment soil AOB cultures; d)

1st enrichment root surface AOB cultures; e) 2nd enrichment root surface AOB cultures; f) 3rd enrichment root surface

AOB cultures. Cultures were incubated for 30 days at 28ºC. Standard deviation is represented as error bars

c f



concentration. Concerning the number of active cultures in the 2rd enrichment, 2 for root

community and 2 for soil community were transferred to the 3rd.

Observing the culture’s nitrite production through incubation time (Fig. 5), only soil AOA

community culture presented nitrite production and accumulation.

A variation of nitrite concentration, with successive decrease and recovery can be

observed for root and soil AOA cultures, mainly after 30 days of incubation.

Decrease in nitrite concentration should have been prevented by growth inhibition of

nitrite consuming bacteria, or by the addition of sodium chlorate, a nitrite oxidation inhibitor (Belser

and Mays, 1980). Interesting Treusch et al. (2005) found a gene encoding a homologue of a

copperdependent nitrite reductase (NirK) in AOA. So some strains of AOA, like AOB may be able

to consume nitrite in anoxic conditions. The decrease in nitrite can occur due to

chemodenitrification, a nonenzymatically catalyzed reaction where nitrite self-decomposes in NO

(nitric oxide), NO2 (nitrogen dioxide), N2O (nitrous oxide) or N2 (dinitrogen) in acidic conditions

(Bock and Wagner, 2006; Cleemput and Samater, 1996). Since ammonia oxidation acidifies the

medium, chemodenitrification can explain the decrease in nitrite concentration.

Concerning nitrite production rate, due to the variation in nitrite concentration, it was not

possible to calculate nitrite production rate for root AOA cultures and soil AOA culture from plant

3, plot C. The observed maximum nitrite concentration (table. 5) were very low, compared to the

AOB communities (table. 4).

Though nitrite with a concentration above 0.1 mg/L can be considered nitrite production

by the cultures, active cultures were defined as cultures with nitrite accumulation through time of

incubation, therefore in the 3rd enrichment, only the AOA culture from soil was considered active

(fig.5). As the active culture was found to be in soil, AOA communities could be more abundant

and active in the rhizospheric soil. However archaeal 16S gene PCR amplification did not

confirmed the presence of archaea (fig. 6) in root or soil community culture, since positive control

has an amplicon with a molecular weight of 700 bp and the amplicons observed (fig.6 A and B)

have molecular weights between 300 bp and 500 bp.


Fig. 5 Nitrite production Curves for the 3st enrichment culture of the Ammonia oxidizing archaea cultures from Cistus ladanifer: a)

rhizospheric soil; b) root surface

a b



A higher archaea population abundance in root

surface was expected, since archaea and particularly,

Thaumarchaeota have been found to be more abundant in

the root surface (Simon et al. 2005), especially in consortia

with mycorrhiza (Timonen and Bomberg, 2009).

Ammonia oxidizing archaea cultivation is highly

difficult compared to Bacterial cultivation and although

enrichment of archaeal ammonia oxidizers from mesophilic

aquatic environments appears to be relatively easy the same

did not happen for AOA from terrestrial environments

(Prosser and Nicol, 2008). Other incubations conditions and

mediums must be developed.

3rd Rhizospheric Soil AOA Cultures Roots AOA Cultures

Plot Plant

Lag

phase

(d)

Nitrite

production

rate (mg/L/d)

Maximum nitrite

Concentration

(mg/L)

Lag

phase

(d)

Nitrite

production rate

(mg/L/d)

Maximum nitrite

Concentration

(mg/L)

Plot C

1 47 0.68 6.71 - - -

2 - - - 60 0 1.03

3 60 0 0.44 - - -

Pot E

1 - - - - - -

2 - - - - - -

3 - - - - - -

Plot F

1 - - - - - -

2 - - - - - -

3 - - - 60 0 1.45

Average 53.50 0.34 3.58 60.00 0 1,24

Standard

deviation 6.50 0.34 3.14 0 0 0.21

CV 0.12 1 0.88 0 - 0.17

Table. 5 3rd enrichment nitrite production parameters for the Rhizospheric Soil AOA community cultures and root surface AOA community culture of Cistus ladanifer from non-fertilized soils without N addition: Plot C, E and F. Parameters include lag phase, expressed by day, nitrite production Rate expressed by mg nitrite/L/d and maximum nitrite concentration expressed as mg/L. Plant 1, 2, 3 are field replicates from which the samples were collected.

Fig. 6 Archaeal 16S gene amplification results. A)

Plant 1 plot C Rhizospheric Soil AOA culture; B)

Plant 3 plot C Rhizospheric Soil AOA culture; C)

Plant 3 plot F Root AOA culture; D) Plant 2 plot C

Root AOA culture; E) positive control,

Halobacterium salinarium l; F) negative control.

A B C D E F

600 bp 500 bp

300 bp




How would the AOB population respond to an increase in N input?

Once the preferential niche for AOB and AOA was defined it was possible to select the

samples to be used for the second task. Rhizospheric soil, having the highest ammonia oxidizing

activity, should be used as inoculum to decrease the possibility of having false-negative results.

Ammonia oxidizing bacteria activity from fertilized and non-fertilized soil

In the 1st task, “Where does the ammonia oxidizing community prosper?” a heterogeneity

between the plant’s rhizosphere communities from the same plot and between plots of the same

treatment was observed. So to have a higher representation of the nitrifying community 3 different

communities were combined. Although the control plots (C, E and F) have different activity

profiles, they represent the natural field heterogeneity.

In the 1st enrichment the 80A and 40A AOB cultures presented the highest maximum

nitrite concentration (30.7 and 30.1 mg/L, respectively), the highest nitrite production rate (3.32

and 2.88 mg/L/d) and ammonia consumption (91.1% and 92.6%) (fig.7 a, b and c). The 40AN

AOB cultures had the lowest values for maximum nitrite concentration (19.83 mg/L) and nitrite

production rate (1.68 mg/L), ammonia consumption is higher than for control AOB cultures with

85.7% of consumption (fig.7 a, b and c). Control AOB cultures have a maximum nitrite

concentration of 22.27 mg/L, a nitrite production rate of 1.97 mg/L/d and the lowest ammonia

consumption with 72.5% (fig.7 a, b and c). No significant differences were observed parameters

mentioned above, between the communities from the control soil and communities from altered

soils (ANOVA, CI of 0.95 or 0.9) (table. 5 in annex). Concerning lag phase, control AOB cultures

had the highest lag phase with 16.7 d. 40A, 40AN and 80AN AOB cultures had similar lag phases

with 8.3, 7.7 and 7.7 d respectively. A significant difference can be observed for lag phase with

CI of 0.9 (Kruskal-Wallis, p=0.061). Regarding nitritation yield or nitrite production yield, 80 AN

AOB culture had the highest yield (53.7 %), followed by the control AOB cultures (49.1 %), 40A

AOB cultures (46.6%) and 40 AN AOB cultures had the lowest yield (39.7 %) (fig.7 d). No

significant differences were found for nitrite production yield (ANOVA, CI of 0.95, p=0.9) (table. 5

in annex).

Observing the several studied parameters (fig.7 a, b, c, d), there appears to be a tendency

for the control and 40AN AOB cultures to have a lower ammonia oxidation activity than 80 AN

and 40A AOB cultures, though significant differences between control and altered soils AOB

cultures weren’t found. However, as explained in the 1st task, nitrite produced by AOB cultures in

the 1st enrichment may not be resultant of ammonia oxidation, as the soil inoculum can have

enough organic matter to allow the growth of nitrite-producing heterotrophic bacteria.

Confirmation of AOB presence in the 1st enrichment cultures was done by amoA gene

amplification. No amplicons were attained with amoA amplification (data not shown).

Amplification of the 16S gene was also performed, with positive results for all the cultures (data

not shown).




These results could indicate the outgrowth of heterotrophic bacteria as there is a lack of

amplicons for amoA and the positive results for the amplification of the 16S gene. However it does

not confirm the absence of AOB.

Specific functional groups like AOB appear difficult to study them even with molecular

tools because some techniques may lack the required sensibility. For example, the detection

threshold of some PCR conditions may not be enough to amplify the amoA gene in soils or culture.


Fig. 7 Nitrite production parameters for non-fertilized (control) and fertilized soils AOB

cultures (40A, 40AN, 80AN). a) maximum nitrite concentration in 1st enrichment; b) nitrite

production rate in 1st enrichment; c) ammonia concentration in 1st enrichment; d) nitritation

yield in 1st enrichment; e) lag phase in 1st enrichment; f) maximum nitrite concentration in

2nd enrichment; g) nitrite production rate in 2nd enrichment; h) ammonia concentration in 2nd

enrichment; i) nitritation yield in 2nd enrichment; j) maximum nitrite concentration in 3rd

enrichment; k) nitrite production rate in 3rd enrichment; l) ammonia concentration in 3rd

enrichment; m) nitritation yield in 3rd enrichment. Standard deviation is represented as error

bars.

d

e

a

b

c

f

g

h

i

j

k

l

m



A nested PCR approach of the amoCAB sequence can increase the sensitivity in samples with

low abundances in AOB (Junier et al. 2010).

In the 2nd enrichment the 40A AOB cultures had the highest value for ammonia

consumption (78.2 %), maximum nitrite concentration (46.74 mg/L) and nitritation yield (97.7 %)

(fig.7f, g, h, i). Nitrite production rate was identical (4.5 mg/L/d) for 40A and 40AN AOB cultures.

40AN had a maximum nitrite concentration of 42.3 mg/L and a nitritation yield of 72.3 %, higher

than 80AN AOB culture with a maximum nitrite concentration of 39.5 mg/L and nitrite production

yield of 68.6 %, though ammonia consumption was higher for 80AN AOB culture (77.9 %) than

for 40AN culture (76.5%) (fig.7 f, g, h, i). 80 AN also had a nitrite production rate lower than 40A

and 40AN (2.6 mg/L/d). The control AOB cultures had the lowest values for all of the studied

parameters (fig.13). No significant differences between treatments were found (Kruskal-wallis and

ANOVA CI of 0.95, table. 5 in annex).

The 40A AOB culture was more homogeneous, having less variance in the studied

parameters with a lower coefficient of variation (CV) than the 40AN and 80AN AOB cultures. Both

cultures present very high CV for the studied parameters (table. 4 in annex).

In the second enrichment there is a difference between the control AOB culture and the

AOB from soils with different N-addition treatments. The nitrite maximum concentration, yield,

ammonia consumption and nitrite production rate are higher in the 40A, 40AN and 80AN AOB

cultures (fig.7 f, g, h, i). However the differences are not significant, probably due to the samples

high heterogeneity.

From the 1st to the 2nd enrichment it is possible to observe a decrease of the maximum

nitrite concentration, nitrite production rate, ammonia consumption (42.47%) and nitritation yield

(18.83%) in the control culture. If in the 1st enrichment nitrite production was mostly done by

heterotrophic bacteria then it could be possible to explain the decrease in activity in the 2nd

enrichment. By transferring and diluting organic matter, nutrients and bacterial cells in the culture

the conditions are better for AOB activity than for heterotrophic bacteria, so activity decreases.

Amplification of amoA can be seen in plot A from 80AN AOB cultures (fig. 8 a), light bands

can be observed for plots M from 80AN AOB cultures, for plot G from 40A AOB cultures and for

plot I and K from 40AN AOB cultures. Amplicons have a molecular weight of around 500 bp,

identical to the positive control.

Though positive amplification of amoA gene in the 1st enrichment was not observed,

amoA amplification occur in the 2nd serial enrichment, confirming the presence of AOB and

indicating cell growth of AOB in the cultures.




In the 3rd enrichment different nitrite production curves profiles (fig. 9) can be observed,

suggesting different ammonia oxidation activities in the control, 40A, 40AN and 80AN AOB

cultures. Concerning variability, AOB cultures from the 40A plot seem less heterogeneous, while

the control and 40AN AOB cultures appear to have high variability. The number of “active”

cultures, with nitrite production, could explain the variability since the 40A and 80AN AOB cultures

had 3 active cultures (replicates), while the control and 40AN AOB cultures had 1 active culture

each.

Regarding the maximum nitrite concentration in each treatment, (fig.7, j) the lowest

concentration seems to be produced by the control AOB cultures (14.3 mg/L), followed by the

40AN and 80AN AOB cultures (21.2 and 42.3 mg/L, respectively). 40A treatment has the highest

maximum nitrite concentration (59.3 mg/L). Significant differences for maximum nitrite

concentrations between AOB cultures were found with a CI of 0.9 (Kruskal-Wallis, p=0.063)

(table. 5 in annex). Concerning variability in maximum nitrite concentration, the control and 40AN

AOB cultures had the highest variation (CV of 138.7 and 123.1). High variability was expected,

since both cultures had one active culture in the total of 3, high deviation is expected.

Nitrite production (nitritation) yield, nitrite production rate and ammonia consumption

were higher in 40A AOB cultures (97.5%, 3.8 mg/L/d and 100%, repectively), followed by 80AN

AOB cultures (72.1 % of yield, 3.5 mg/L/d of rate and 96.2% of ammonia consumption) (fig. 7 k,

l). The control AOB cultures had the lowest maximum nitrite concentration (14.3 mg/L), nitrite

production yield (24.1%) and ammonia consumption (94.3%), while 40AN AOB cultures had the

lowest nitrite production rate (0.9 mg/L/d) (fig. 7 j, k, l, m). No significant differences were found

for nitrite production rate, and ammonia concentration decrease (Kruskal-Wallis, CI of 0.95)

(table. 5 in annex). Significant differences were found for nitrite production yield with a CI of 0.9

(Kruskal-Wallis, p=0.09) (Table. 5 in annex).


Fig. 8 amoA gene amplification for the a) 2nd enrichment of AOB cultures; b) 3rd enrichment AOB

community cultures. Control AOB communities: F, E, C; 40A treatment: H, L, G; 40AN treatment: K, I, B;

80AN treatment: N, M, A. Samples outlined by green had positive amplification for amoA.

500

F E C H L G K I B N M A + -

Control 40A 40AN 80AN

500

a)

b)



Concerning variability, the control and 40AN AOB cultures had the highest CV for the

studied parameters (table. 4 in annex), though for ammonia consumption, variability was low.

As the 3rd enrichment should have growth of AOB without the interference of heterotrophic

bacteria growth, AOB culture activity for the treatments should present the effect of N-addition in

the AOB. However the presence of heterotrophic growth should not be disregarded. The decrease

in ammonia concentration in control and 40AN AOB cultures was similar to the decrease in

ammonia concentration for 40A and 80AN AOB cultures, though nitrite production (rate and

maximum concentration) is different. This observation can be explained by growth of

microorganism other than AOB or by ammonia volatilization, though AOB growth was attained in

a closed system with the possible minimal gas exchange.

Presence of AOB was confirmed by amplification of the amoA gene (fig.8 b). Amplicons

with the same molecular weight as the positive control were seen for plot C from control AOB

cultures, for plots H, L and G from 40A AOB cultures, for plot I from 40A AOB cultures and for

plots M and A from 80AN AOB cultures.

Corresponding amoA amplification and AOB ammonia oxidation activity, it is possible to

observe that cultures with nitrite production and accumulation through time also had a positive

amoA amplification, e.g., in control AOB cultures, plot C had active AOB (fig.9 a) and AOB

presence was confirmed (fig.8 b), while control AOB cultures from plot F and E did not had nitrite

production (fig.9 a) and AOB was not detected by amoA amplification (fig.8 b). This

correspondence between ammonia oxidation activity and presence of amoA was also observed

for 40A, 40AN and 80AN AOB cultures.

Considering all the parameters, and confirmation of AOB presence by amoA amplification

(fig.8 b), it is possible to hypnotize an alteration in the AOB community’s ammonia oxidation

activity in response to the N-addition, though no significant differences between treatments were

found, given the high heterogeneity. The highest ammonia oxidation activity was found in the

40AN AOB cultures and use of ammonia (decrease in ammonia) also appears to be induced in

these cultures. 80AN AOB cultures also had a high ammonia oxidation activity, similar, but lower

than the activity found for 40A AOB cultures. The control and 40AN AOB cultures had similar

activities, as 40A AOB cultures had a high maximum nitrite concentration and yield and the control

AOB cultures have a higher nitrite production rate. Considering just the nitrite production rate,

control AOB cultures can be considered more active than 40A treatment AOB cultures.

Since the same amount of ammonia, but not the same form, is being added to the soils

from 40A and 80AN treatments it could explain the similar ammonia oxidation activity for 40A and

80AN AOB cultures. 40A and 80AN treatments do differ regarding the variability, which could be

an effect of the type of ammonia compound, though more studies are needed to study the effects

of different ammonia compounds in AOB activity. Concerning the effects of N-addition in different

forms and quantities of ammonia on AOB community structure, TGGE studies performed in scope

of the SERN (Spheres of Ecosystem Response to Nitrogen (SERN): A case study in a

Mediterranean-type ecosystem in southern Portugal) project (PTDC / BIA-BEC / 099323 / 2008),

suggested that the community structure changes more according with the quantity of N and not




with the form of N (Cristina Cruz personal communication), and regarding ammonia oxidation

activity, a similar activity is seen with the same quantity and different forms of ammonia.

As soils fertilized with the 40AN treatment have half of the amount of ammonia quantity

comparing to 80AN, it was expected a lower ammonia oxidation activity for 40AN AOB cultures

than for the 80AN AOB cultures but a higher activity for the 40AN AOB cultures than for control

AOB cultures, however 40AN and control AOB cultures had a similar ammonia oxidation activity.

Both 40AN and control AOB cultures had similar CV, indicating that addition of 40 kg of ammonia

nitrate may not alter the activity of AOB.


Fig. 9. 3rd enrichment nitrite production curves through incubation time. AOB community cultures for: a) control soils without

N addition; b) soils with N addition – 40A; c) soils with N addition – 40AN; d) soils with N addition – 80AN. Replicates for

each treatment are represented as grey dotted lines and the mean curve is represented as the full colour line. standard

deviation is represented as error bars.

a b

c d



AOB community structure assessed by PCR-RFLP

Ammonia oxidation activity among AOB cultures from non-fertilized and fertilized soils

were different, as well as the susceptibility to ammonia. Therefore is was of interest to understand

if these differences would also correspond to different community structures.

To assess the community structure possible changes, PCR-RFLP was performed. The

amoA gene was amplified from DNA extracted from the 3rd enrichment AOB cultures, with primers

amoA-1F and amoA-2R. The PCR products were used for restriction with 2 different enzymes,

HaeIII and HinfI. The results for the PCR-RFLP are shown in fig.10. Both HaeIII and HinfI

restriction enzymes originated 2 distinct patterns.

HaeIII hydrolysis of amoA amplified fragment presented 2 bands with a molecular weight

of 167, 124 or 102 bp, while HinfI restriction had bands with a molecular weight of 537 and 494

bp. Four different patterns for HAE III and 4 patterns for HinfI were obtained (table.6).

C H L G K N A C H L G K N A

Hae III Hinf I

Fig. 10 amoA PCR-RFLP profiles for the 3rd enrichment AOB community cultures. Control AOB communities: C; 40A

treatment: H, L, G; 40AN treatment: I; 80AN treatment: N, A. HaeIII and HinfI were used as restriction enzymes.




Profile types were coded to allow an easier interpretation (table. 6). Cultures from the

control plot C AOB enrichment showed an A1 profile. No other culture presented the same profile.

40 A treatment showed 3 different profiles (B2 in plot H, C3 in plot L and C2 in plot G), plot I from

40AN presented a C3 profile, identical to plot L from 40A treatment. 80AN presented a C4 and

D2 of profile in plot N and A respectively. So with the exception of plot L (40A) and I (40AN) the

enrichments present unique profiles and, consequently, community structures.

In addition, a possible identification of AOB species was performed. Each profile was

compared to Nitrosomonas sp. and Nitrosospira sp. profiles done in-silico with the sequences

available in the GenBank database for β-Proteobacteria (fig. 1 and 2 in annex).

A correspondence with the AOB in-silico HaeIII profiles was found for Nitrosomonas

nitrosa (150; 140; 120; 35 bp), Nitrosomonas aesturi (150; 175; 10; 45) and Nitrosospira tenuis

(25; 146; 127; 116). Concerning HinfI restriction profiles, most PCR products were not hydrolysed

band (537 bp). Patterns with a 537 bp and other molecular weight fragments could indicate the

presence of more than 1 type of strain in the culture. A strain with a restriction site in the amplified

amoA fragment and another without that region. This was expected since the culture correspond

to a population and not pure strains.

A correspondence with the AOB in-silico profiles was found for Nitrosospira tenuis in the

PCR-RFLP profiles from plot H and plot L (40A), plot K (40AN) and plot A (80AN). The presence

of this species of Nitrosospira is also in concordance with pyrosequecing data. Nitrosospira sp.

But not Nitrosomonas sp. was found in rhizospheric soil of Cistus ladanifer (data from the project

In-Nitro: conceptualizing the effects of increased nitrogen availability in a Mediterranean

ecosystem - PTDC/BIA-ECS/122214/2010).

The changes seen in AOB activity and community structure suggests a great effect of N

based fertilizers, and the response of the AOB community was dependent of the quantity and

form of ammonia, as ammonia - nitrate seem to be less available (40AN and 80AN) for AOB than

only ammonia (40A), resulting in a greater ammonia oxidation activity in 40A. It is possible that

Treatment Plot Restriction

enzyme

Bands

Molecular

weight (bp)

Type of

profile

Restriction

enzyme

Bands

Molecular

weight (bp)

Type of

profile

Control C

Hae III

124 A

Hinf I

494 1

40A

H 167; 124 (+) B 537 2

L 167 (+); 124 C 537 (+); 494 3

G 167 (+); 124 C 537 2

40AN K 167 (+); 124 C 537 (+); 494 3

80AN N 167 (+); 124 C 537; 494 (+) 4

A 167 (+); 102 D 537 2

Table. 6 Restriction band’s molecular weight from PCR-RFLP pattern results with HaeIII and HinfI. The dense bands are signalled with a (+). Each profile was given a code. A-D for HaeIII; 1-4 to HinfI.




the apparent lower activity of 40AN AOB community can be due to the quick Ammonia-Nitrate

use by plants. Because the fertilization causes an increase in plant growth (Dias et al. 2011) AOB

communities in this treatment can have less substrate available than in control without N addition

since plants may have a higher demand for nutrients, and AOB, being bad competitors for

ammonium, may have less substrate available than in control without N addition (Verhagen et al.

1994). As, since 2007 N addition has been done AOB communities in 40AN could have been

altered to have less activity, less nutrient demand, and with a higher ammonia affinity.

Contrary to that happens in 40AN treatments, 40A treatments shows less plant coverage,

having more bare soil (Dias et al. 2011), therefore the AOB communities have more available

ammonia than communities in soils with a higher plant coverage. However AOB may not be

abundant in bare soil. Delgado-Baquerizo et al. (2013) have shown that, in Mediterranean

ecosystems, with limiting nutrients, AOB amoA genes are more abundant in rhizospheric soil

under the influence of Stipa tenacissima, while AOA amoA genes are more abundant in bare soil.

However in Mediterranean environments with N addition an increase in plant growth can lead to

a stronger competition for ammonia, inhibiting an ammonia oxidation increase.

Many studies show that AOB growth and activity normally benefits from an increase in

nitrogen addition to the soil (He et al. 2012; Allison et al. 2008; Mendum et al. 1999).

Ammonia oxidizing archaea activity in fertilized and non-fertilized soils

As it was mentioned in the 1st task, AOA activity analysis can only be done in the 3rd

enrichment to ensure the absence of AOB presence and activity. Since confirmation of presence

and activity of AOA was not possible, the cultures are referred to “AOA” as possible AOA culture.

Concerning the nitrite production curves, a similar profiles could be observed (Fig. 11).

The “AOA” cultures presented 2 exponential phases with a decrease in the nitrite concentration

between the phases. Since there is not a constant nitrite production through the incubation time,

nitrite production rate was not possible to calculate. Control and 40A “AOA” cultures appear to

have a nitrite concentration peak at 30 days and 50 days of incubation, while 40A and 80AN

“AOA” cultures have a nitrite concentration peak at 35 and 52 days of incubation.

Regarding maximum nitrite production and nitritation (nitrite production) yield (fig.12)

40AN “AOA” cultures had higher values (0.96 mg/L and 1.7%, respectively), followed by control

“AOA” cultures (0.73 mg/L and 1.2%), 80AN “AOA” cultures (0.32 mg/L and 0.6%), 40A “AOA”

cultures had the lowest maximum nitrite production and nitritation yield (0.18 mg/L and 0.3%)

(fig.19). No significant differences were found between treatments for maximum nitrite

concentration, nitrite production yield and ammonia decrease (Kruskal-Wallis, CI=0.95) (table. 5

in annex).




Concerning the previous enrichments for these

cultures, in the 1st enrichment, maximum nitrite

concentration of “AOA” cultures was similar to the 1st

enrichment “AOB” cultures maximum nitrite

concentration, though nitrite production rate was lower in

“AOA” cultures than “AOB” cultures (data not shown). So

the 1st antibiotic used had an effect on nitrite production

rate. The activity of the “AOA” cultures decreased with

each serial enrichment, being lower in the 3rd and last

enrichment. So “AOA” ammonia oxidation activity

analysis should and were performed at the 3rd

enrichment.

These results are reverse to those found for

AOB. For “AOA” a higher activity is found in 40AN

treatment followed by control, 80AN and finally 40A with

the lowest “AOA” activity.

The fact that the nitrite production activity for

“AOA” is higher in the treatments where AOB activity is

lower can be explained by the AOA higher ammonia

affinity. So under limiting ammonium concentrations

AOA activity is favoured therefore having a more

important role in nitrification than AOB (Martens-

Habbena et al. 2009). Although in some cases AOA can

outgrow AOB, this appears to not be applicable in this


Fig. 12 3rd enrichment nitrite production

parameters for non-fertilized and fertilized

soils “AOA” cultures. a) maximum nitrite

concentration; b) nitritation yield or nitrite

production yield; c) ammonia concentration.

Standard deviation is represented as error

bars.

a

b

c

Fig.11 3rd enrichment nitrite production curves through incubation time. AOA community cultures for: a) control soils without

N addition; b) soils with N addition – 40A; c) soils with N addition – 40AN; d) soils with N addition – 80AN. Replicates for

each treatment are represented as grey dotted lines and the mean curve is represented as the full colour line. standard

deviation is represented as error bars.

a b

c d



case study. Although nitrite production is higher in control and 40AN treatment, the AOB activity

in these treatment is still higher than “AOA”.

Although AOB nitrite production is higher in 40A and 80AN treatments and “AOA” nitrite

production is higher in control and 40AN “AOA” treatments, the ammonia oxidation and nitrite

production attributed to AOB is higher than the nitrite production attributed to AOA. Thus, in soil

and under these incubation conditions, both AOM coexist in the environment, being the observed

differences due to AOA slower growth rate (Prosser and Nicol, 2008). The lower ammonia

oxidation, and consequently, nitrite production in the enrichment cultures can, then be explained

by the lower number of cells in the soil population.

Additionally, considering that AOA uses, preferentially, mineralized ammonia (urea) and

organic nitrogen source (amino acids) (He et al. 2012; Tourna et al. 2011; Hatzenpichler, 2012),

then growth and activity in a medium with ammonia as nitrogen and energy source may not be

the best to ensure a high nitrite production by AOA. So optimal conditions for these cultures

should be studied to understand which ammonia oxidizing group of organisms has the greatest

importance in the control and 40AN treatment.

The presence of Archaea was monitored in the 3 enrichments, amoA archaeal gene

presence was confirmed for cultures with confirmed archaea presence.

In the 1st enrichment, amplification of the 16S gene was negative (data not shown). In the

2nd enrichment bands of the same molecular weight as the positive control were observed in the

M, B, and K plots (80AN and 40AN treatments) (data not shown), amplicons of other molecular

weights were also observed.

In the amplification results of the archaeal 16S gene for the 3rd enrichments (fig 13),

amplicons with several molecular weights can be observed. For the control “AOA” cultures, plots

F and E, bands with around 200 bp can be observed. For 40A “AOA” cultures, bands with around

600 bp can be observed in plots L and G. The plots K and I from 40AN “AOA” cultures present

bands with around 600 bp, 500 bp and 300 bp. 80AN “AOA” cultures had bands with around 800

bp, 600 bp and 1000 bp for plots N and M.

However most of those appears to be unspecific amplifications, since the molecular

weight of the amplicon doesn’t correspond to the positive control. The presence of amoA gene

was studied in the cultures with bands with a correct molecular weight (600 bp). No positive

amplification occur for the amoA gene (data not shown).


Control 40A 40AN 80AN

Fig. 13 16S Archaeal gene

amplification for 3rd enrichment AOA

community cultures. Control AOB

communities: F, E, C; 40A treatment:

H, L, G; 40AN treatment: K, I, B; 80AN

treatment: N, M, A. Samples outlined

by green had positive amplification for

16S rRNA, red outline indicates non-

specific amplification

F

3rd enrichment

E C HC

LC

GC

KC

I B N M A +

600 bp

600 bp



Although it was not possible to amplify archaeal amoA gene, nitrite production can be detected,

thus the lack of amplification of amoA gene can be due to low cell density. More enrichments or

time in each enrichment could increase AOA cell density and enable amplification.

AOA community are more sensible to N adittion and normally prosper in oligotrophic

environments with low nutrient concentration (Hatzenpichler et al. 2006).

How does the AOB population responds to abiotic factors?

AOB susceptibility to ammonia

AOB species can be characterized by their ammonia tolerance. Nitrosomonas europaea

can growth with an ammonia concentration up to 500 mM. However other strains can grow with

concentrations below 50mM. In community the response to high ammonia concentrations can be

different. Some communities can grow with concentrations as high as 1000mM (Spieck and Bock,

2005; Hatzenpichler, 2012). So depending on the community susceptibility to ammonia it will more

susceptible to change activity when confronted by a change in substrate. However in soils with

N-addition, when ammonia is frequently added to soil, it is expected for the AOB populations to

be altered and have a higher ammonia tolerance.

Concerning the three nitrite production parameters, maximum nitrite concentration and

nitrite production rate for the control, 40A, 40AN and 80AN AOB cultures incubated with different

concentrations of ammonia (fig.14) a similar pattern of response can be observed with the

increase of the ammonia concentration. With 1 mM of ammonia, the control ammonia

concentration, 40A AOB cultures had the highest maximum nitrite concentration (36.7 mg/L) and

nitrite production rate (4.3 mg/L/d), followed by 40AN and 80AN AOB cultures that had similar

results, while the control had the lowest values for the parameters (fig.14). The added10 mM of

ammonia to the medium increased the nitrite production rate and maximum nitrite concentration

of the AOB cultures (fig.14). 40A AOB cultures had the highest parameter values (42.1 mg/L and

6.02 mg/L/d) between treatments with this ammonia concentration, followed by 40AN, 80AN and

control (12.8 mg/L and 2.3 mg/L/d) AOB cultures (fig.14). With 50 mM of ammonia, an increase

of maximum nitrite concentration can be observed for 40A, with the highest value followed by

40AN and 80AN (51.1 mg/L, 42.4 mg/L and 23.1 mg/L, respectively), control AOB cultures had a

decrease in maximum nitrite concentration (1.7 mg/L). A decrease in nitrite production rate can

be observed for the AOB cultures. The 40A AOB cultures with the highest rate (5.4 mg/L/d)

followed by 40AN (3.4 mg/L/d), 80AN (2.5 mg/L/d) and control (0.17 mg/L/d) (fig.14). Concerning

the effect of adding 100 mM of ammonia in the medium a decrease in maximum nitrite

concentration and nitrite production rate can be observed, with the exception of an increase in

nitrite production rate 40AN AOB cultures (3.8 mg/L/d). The 40AN AOB cultures presented the

highest maximum nitrite concentration (32.8 mg/L) and rate, followed by 40A (20.6 mg/L and 2.1

mg/L/d, respectively), 80AN AOB cultures (5.8 mg/L and 0.5 mg/L/d). Control AOB cultures had

the lowest vales for the studied parameters (0.3 mg/L and 0 mg/L/d) (fig. 14). With 500 mM of

ammonia, nitrite production rate of AOB cultures was not possible to calculate, since nitrite




accumulation through time did not occur. No significant difference was found between treatments

and ammonia concentration (2-way ANOVA, CI=0.95) (see table. 6 in annex).

Control AOB cultures had the highest maximum nitrite concentration and nitrite production

rate with 10 mM of ammonia,

indicating an optimal ammonia

concentration for ammonia

oxidation activity of 10 mM, having

an activity decrease with 50 mM.

The 40A, 40AN and 80AN

AOB cultures had the highest

maximum nitrite concentration with

50mM of ammonia and highest

nitrite production rate with 10 mM of

ammonia with 1 mM of ammonia.

40A and 80AN AOB cultures start to

have an ammonia oxidation activity

decrease with 100 mM of ammonia,

while 40AN AOB cultures had a

similar rate and maximum nitrite

concentration with 10 mM, 50 mM

and 100 mM, with and activity

decrease with 500 mM of ammonia.

So AOB cultures from soils

with different N-addition treatments

appear to have a distinct ammonia

tolerance. Control AOB cultures are

the most susceptible to ammonia,

being inhibit with 50mM of ammonia. 40A and 80AN treatments have higher tolerance, being

inhibit with 100mM of ammonia. 40AN treatment, though it has a lower activity at standard

conditions (1mM) than 40A and 80AN, it shows a higher ammonia tolerance, still having a high

activity at 100mM. More ammonia concentrations should be tested to fill the gap between 100mM

and 500mM and to know the inhibitory concentration of ammonia for the 40AN treatment.

AOB susceptibility to pH changes

Ammonia oxidation is considered to be optimal at neutral to slightly alkaline soil pH values

and the optimum pH for AOB cell growth being between 7.6–7.8 (Norton and Stark, 2011; Bock

and Wagner, 2006). However several nitrifiers have been found in environments with suboptimal

pH, either acidic or alkaline (Bock and Wagner, 2006), and are able to be active (are capable of

autotrophic nitrification) in soils with pH values from 3.0 to 10.0 (Norton and Stark, 2011). Still,


Fig.14 Nitrite production parameters of the control, 40A, 40AN and 80AN

AOB cultures in relation with different ammonia concentration in the

medium(1 mM, 10 mM, 50 mM, 100 mM and 500 mM): a) nitrite production

rate; b) maximum nitrite concentration. Standard deviation is represented

as error bars.

a

b



the response of AOB populations to pH changes depends on their capacity to adapt to the change,

and while some species are more tolerant, others are more susceptible and have a limited pH

range in which they are active.

Concerning the effect of pH on AOB

activity it is possible to observe that AOB

cultures incubated in media with pH 6.6 had

the highest activity (fig.15). With pH 5.6 only

the AOB cultures from the 40A soil

treatment present activity, with a nitrite

production rate of 0.362 mg nitrite /L /d (fig.

15 b), and the highest maximum nitrite

concentration of 2.603 mg/L. Though a

maximum nitrite concentration can be

determined for the other AOB cultures,

nitrite concentration through time shows

high variation, not being possible to

determine nitrite production rate since nitrite

accumulation is not observed.

With pH 6.6, as the control pH, it is

possible to observe the same pattern of

activity with AOB soils from 40A and 80AN

treatment having the highest rate and

maximum nitrite concentration as it is

observed before. With pH 7.1 AOB cultures

from 80AN has the highest nitrite production

rate (1.243 mg nitrite /L /d), followed by AOB cultures from the control (0.146 mg nitrite /L /d) and

AOB cultures from 40AN (0.042 mg nitrite /L /d). With pH 7.9 none of the AOB cultures are

considered active, though maximum nitrite concentration was determined.

So pH changes have different impact on the AOB cultures, depending on their adaptation

to increased N input in the ecosystem. AOB cultures adapted to N input of just ammonia (40A)

appear to be more active at slightly acidic pH (>5.6, < 7.1), having the activity inhibit at neutral to

basic pH. While AOB cultures adapted to N input of ammonia – nitrate tend to be active at pH >

6.6 and pH < 7.9. AOB cultures from soils without N have the same response pattern to pH as

AOB cultures from 40AN treatment, with a total activity inhibition at pH < 6.6, and lower activity at

pH 7.1. So AOB cultures from control and 40AN appear to more susceptible to pH change both

with the decrease or increase of pH, while AOB cultures from 80AN is more susceptible to pH

decrease and AOB cultures from 40A are more susceptible to pH increase.

Though neutral to basic pH are described to be optimal for AOB activity and growth, these

are described for species, while AOB populations seem more adapted to coping with low pH

(Hatzenpichler, 2012). It is thought that the effect of acidity on ammonia oxidation can be related

a

b

Fig. 15 Nitrite production parameters of the control,

40A, 40AN and 80AN AOB cultures in relation with

different pH in the medium (5.6, 6.6, 7.1 and 7.9): a)

maximum nitrite; b) nitrite production rate

concentration. Standard deviation is represented as

error bars.




to the exponential decrease in NH3 availability. However ammonia oxidation activity under acidic

conditions might be possible through ureolytic activity, aggregate formation, or by biofilm

formation (Bock and Wagner, 2006). Some AOB species, encoding ureases enzymes, are

capable of using urea and catalyse the formation of NH3 and CO2 intracellularly, having a source

of substrate for ammonia oxidation activity (Norton and Stark, 2011; Bock and Wagner, 2006),

and possible regulating the pH in the surrounding area of the cell (Hatzenpichler, 2012).

Biofilms and aggregate formation allow the population to create a more beneficial

environment and regulate pH, substrate concentration and other factors than may alter AOB

activity, giving the population a higher resistance to change.

Effect of organic compounds in AOB nitrite production

To assess the response of the AOB community from soils without N addition to the

presence of organic compounds, urea, peptone, glucose and acetate were used to supplement

the media. Urea and peptone can be used as energy sources in litotrophic growth (with nitrite

production), in mixotrophic or organotrophic growth (lower to none nitrite production). Glucose

and acetate can be used as carbon sources in mixotrophic growth (with nitrite production) with

ammonia as energy source (electron donor). Control media had ammonia as energy source and

sodium bicarbonate as carbon source. In media supplemented with acetate and glucose (carbon

source), sodium bicarbonate was not added. While media supplemented with urea or peptone

had sodium bicarbonate as carbon source.

Concerning the AOB population activity, nitrite production was observed in cultures

supplemented with just urea and with a combination of urea and ammonia, though AOB cultures

with a combination of N compounds tend to have lower nitrite production (fig.16 a). However in

AOB cultures incubated in a media supplemented with peptone, a combination of peptone and

ammonia, acetate or glucose, nitrite production was not observed (fig. 16 b, c).

Fig. 16 Nitrite production through incubation time

by AOB cultures from soils without N addition, in

media with: a) urea; b) peptone; c) organic C

compounds. Media with ammonia was used as

control media. Standard deviation is represented

as error bars.

a b

c




Regarding nitrite production rate, AOB cultures incubated with media supplemented with

just ammonia had similar rates as the AOB cultures incubated in control medium (1.68 and 1.69

mg nitrite /L /d), while in a media supplemented with both ammonia and urea the AOB nitrite

production rate was lower (1.17 mg nitrite /L /d) (Fig. 17 a). For AOB cultures incubated with

peptone, peptone with ammonia, acetate or glucose, nitrite production rate was low (0.02, 0.03,

0 and 0.01 mg nitrite /L /d) (fig. 17 b), therefore the cultures were considered non-active. Maximum

nitrite concentration was higher in the control AOB cultures (14.32 mg/L), followed by AOB

cultures in media supplemented with urea (9.2 mg/L), and AOB cultures in media with urea and

ammonia (5.95 mg/L). Though activities tend to be different according to the nitrogen compound,

no significant differences were found (Kruskal-Wallis, p of 0.95 table. 7 in annex). Amplification

of amoA was performed to confirm the presence of AOB, as addition of organic compounds will

increase the growth of heterotrophic bacteria than might still be in the inoculum. Positive

amplification for amoA was observed in the AOB cultures in media supplemented with just urea

and urea with ammonia (fig.18).

Considering the activity observed for AOB cultures, it is possible to say that the AOB

populations from soils without N addition are capable of using urea for litotrophic growth if

ammonia is not present, as it is described for some species of Nitrosomonas, as Nitrosomonas

ureae (Bock and Wagner, 2006). The use of urea is described as a source of ammonia, but not

as an energy source. Glutamine as source of ammonia for lithotrophic growth was also described

(Bock and Wagner, 2006), and though isolated aminoacids were not tested for their effect on AOB

Fig. 17 Effect of organic compounds in nitrite production parameters for AOB cultures from soils without N addition,

incubated in media with: a) effect of N organic compounds in maximum nitrite concentration; b) effect of N nitrite in

production rate; c) effect of C organic compounds in maximum nitrite concentration; d) effect of C organic

compounds in nitrite production rate Standard deviation is represented as error bars.

a b

c d




activity, supplementing peptone in the incubation medium seem to inhibit nitrite production.

Mixotrophic growth and activity can occur in Nitrosomonas and Nitrobacter, with ammonia as

energy source and organic compound as carbon source, chemoorganotrophic growth with

acetate or pyruvate as electron donor was also described for AOB (Bock and Wagner, 2006).

However none of the cases were observed, since the cultures were not active and amoA

amplification had negative results (no amplification or non-specific amplification) (fig.18).

Thus, AOB populations from soils without N addition appear to be able to use urea, but

are susceptible to the presence of peptones, glucose and acetate. The susceptibility of AOB

populations from soils with N addition should be studied, as more organic compounds should be

tested.

Conclusions

Considering the influence of factors such as niche, N concentration in soils, ammonia

concentration, pH and presence of organic matter in AOB population activity, it is possible to say

that this population are not resistant to change and responsive to changes. Therefore changes in

the environmental conditions can alter the nitrification rate and possibly the N flux in the

ecosystem. The susceptibility of AOB population to environmental factors may represent a fine

tuned mechanism of control necessary to guaranty the balance of N in the ecosystem.

When N in the ecosystem is not in excess (40AN treatment), the structure of the AOB

community may change in relation to the control (no N addition), without changes in the

nitrification rate. However with excess N, the AOB activity will increase, decreasing the inorganic

N content of the soil. This increase in ammonia oxidation rate can contribute to a decrease in soil

pH and consequently to an inhibition of the ammonia oxidation rate in negative feedback

regulation. This negative feedback regulation through by soil pH changes may be one of the

possible ways of nitrification control, though through time an altered AOB population may adapt


1 2 4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19 20

Fig. 18 amoA amplification for AOB cultures in media suplemente with: 1) F urea and ammonia; 2) E urea and ammonia;

3) C urea and ammonia; 4) F urea; 5) E urea; 6) C urea; 7) F peptone and ammonia; 8) E peptone and ammonia; 9) C

peptone and ammonia; 10) F peptone 11) E peptone; 12) C peptone; 13) F acetate; 14) E acetate; 15) C acetate; 16)

F glucose; 17) positive control, Nitrosomonas europaea ; 18) negative control; 19) E glucose; 20) C glucose. E, F and

C correspond to the plots from which soil AOB cultures were collected. Samples outlined by green had positive

amplification for amoA, red outline indicates non-specific amplification. Positive control has a blue outline.

500 bp

3



to the acid pH, as it is seen for the AOB culture from the 40A treatment. Consequently changes

in pH may represent a transient mechanism to regulate the AOB activity. Nitrogen addition to the

soil is known to increase the ecosystem productivity, leading to an increase in the concentration

of organic compounds, which can inhibit the ammonia oxidation rate. Therefore another possibility

of nitrification control is through negative feedback by the increase of organic compounds.

Though nitrification appears to be a highly controlled process, the AOB population can

be altered in a way that the activity may not be easily regulated.

Acknowledgements

Organization of both sampling expedition to Arrábida’s experimental field was

accomplished thanks to Cristina Cruz (CBA – Center of Environmental Biology, Faculty of

Science, University of Lisbon, Lisbon, Portugal). Assay reagents, growth mediums were provided

by Cristina Cruz as part of a collaboration in the framework of the project In-Nitro: conceptualizing

the effects of increased nitrogen availability in a Mediterranean ecosystem (PTDC/BIA-

ECS/122214/2010). Molecular analysis, PCR and RFLP analysis were performed thanks to prof.

Dr. Rogério Tenreiro and Dr. Sandra Chaves (BioFig.)

I thank Dr. Stephen Unger for helping with the field work and suggestions regarding the sampling

sites. I also thank Teresa Dias for the help with the nitrite and ammonia quantification protocol

and experimental field description and characterization.

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Annex

Table 1. Number of active AOB cultures from rhizospheric soil and root surface of Cistus ladanifer in each

enrichment after 30 days of incubation.

Initial culture

number

Number of active cultures

1st enrichment 2nd enrichment 3rd enrichment

Rhizospheric soil AOB culture 9 8 7 7

Rhizospheric soil AOA culture 9 4 2 1

Root surface AOB culture 9 4 3 2

Root surface AOA culture 9 4 2 0




Table. 2. Coefficient of variation calculated for the studied parameters: lag phase, maximum nitrite

concentration, nitrite production rate, attained for the 1st AOB enrichment cultures from rhizospheric soil and

root surface of Cistus ladanifer.

Table. 3 Statiscical analysis of studied parameters for: AOB 1st enrichment cultures from rhizospheric soil

and root surface of Cistus ladanifer

Sample

parameter Levene's test (CI = 0.95/

α = 0.05) Variance analysis

Result

p Result Transforma

tion Applied

test Confidence level (CI)

Α p Signifcant

1st

enrichment

AOB cultures

maximum nitrite concentration

0.1 linear - ANOVA 0.95 0.05

0.152

no

nitrite production rate 0.22 linear - ANOVA 0.95 0.05

0.292

no

Lag phase 0.18 linear - ANOVA 0.95 0.05

0.049

yes

Table.4 Coefficient of variation calculated for the studied parameters in the 2nd task: maximum nitrite

concentration, ammonia concentration decrease, nitrite production rate, lag phase and nitrite production

yield, attained for the 3 AOB enrichment cultures and 3rd AOA enrichment culture.

AOB cultures from:

CV

lag phase nitrite production

rate maximum nitrite concentration

Soil 0.26 1.59 1.03

Roots 0.32 2.3 1.46

Coefficient of variation (CV)

Sample (October)

treatment

Maximum nitrite

concentration (mg/L)

Ammonia Comsuption

(%)

Nitrite prodution rate

(mg/L/d) lag phase (d)

Nitrite production yield (%)

1st

enrichment AOB

Control 34.9 18.9 23.34 22.63 19.1

40A 46.9 5.8 63.3 11.31 44.6

40AN 42.03 8.57 37.1 12.3 46.34

80AN 49.8 9.52 78.71 12.3 42.77

2nd

enrichment AOB

Control 90.11 10.13 91.69 - 88.69

40A 27.27 19.7 32.54 - 7.5

40AN 68.63 40.6 74.7 - 64.54

80AN 69.1 34.38 67.7 - 65.53

3rd

enrichment AOB

Control 138.74 1.67 141.42 - 138.65

40A 0.85 0 30.1 - 1.18

40AN 123.07 3.33 141.42 - 122

80AN 21.04 2.77 37.59 - 18.8

3rd enrichment

AOA

Control 58.47 2.82 - - 58.91

40A 12.85 0.14 - - 12.88

40AN 64.66 0.78 - - 64.31

80AN 16.87 0.58 - - 17.30




Table. 5 Statiscical analysis of studied parameters for: soil extract samples, AOB enrichment cultures and

AOA 3rd enrichment culture.

Sample parameter

Levene's test (CI = 0.95/ α = 0.05)

Variance analysis Result

p Result Transformation Applied

test Confidence level (CI)

α p Signifcant

Soil

ammonia 0.003 non-linear - Kruskal-

wallis 0.95

0.05

0.347 no

nitrite 0.006 non-linear - Kruskal-

wallis

0.95 0.05 0.058

no

- 0.9 0.1 yes

nitrate 0.182 linear - ANOVA 0.95 0.05

0.266 no

1st enrichment

cultures AOB

lag phase non-linear - Kruskal-

wallis

0.95 0.05 0.061

no

- 0.90 0.1 yes


0.51 linear - ANOVA 0.95 0.05

0.734 no


0.732 no

ammonia consumption 0.251 linear - ANOVA 0.95 0.05

0.202 no

Nitrite production yield 0.420 linear - ANOVA 0.95 0.05

0.897 no

2nd enrichment

cultures AOB


0.037 non-linear - Kruskal-

wallis 0.95

0.05

0.516 no


0.220 no

ammonia consumption 0.036 non-linear - Kruskal-

wallis 0.95

0.05

0.453 no

Nitrite production yield 0.025 non-linear - Kruskal-

wallis 0.95

0.05

0.459 no

3rd enrichment

cultures AOB



wallis

0.95 0.05 0.063

no

- 0.90 0.1 yes


0.414 no


wallis 0.95

0.05

0.157 no


wallis 0.95

0.05 0.09

no

- 0.9 0.1 yes

3rd enrichment

cultures AOA



wallis 0.95

0.05

0.217 no


wallis 0.95

0.05

0.579 no


wallis 0.95

0.05

0.218 no




Table. 6 Statiscical analysis of studied parameters for: AOB cultures with different ammonia concentrations in the media and different pH.

Sample parameter Levene's test (CI = 0.95/ α = 0.05)


p Result Transformation Applied test Confidence level (CI)

α p Signifcant

Ammonia susceptibility

maximum nitrite

concentration

treatments 0.000 non-linear rank 2-way ANOVA 0.95 0.05 0.00 yes

[ammonia] 0.95 0.05 0.137 no

treatments*[ammonia] 0.95 0.05 0.991 no

nitrite production

rate


[ammonia] 0.95 0.05 0.000 yes


Nitrite production

yield


[ammonia] 0.95 0.05 0.000 yes


pH maximum nitrite

concentration


pH 0.95 0.05 0.233 no

treatments*pH 0.95 0.05 0.982 no

nitrite production

rate

treatments 0.000 non-linear rank 2-way ANOVA 0.95 0.05 0.491 no

pH 0.95 0.05 0.003 yes

treatments*pH 0.95 0.05 0.424 no

Table. 7 Statiscical analysis of studied parameters for: AOB cultures incubated in media supplemented with different organic compounds.

Sample parameter Levene's test (CI = 0.95/ α = 0.05)


p Result Transformation Applied test Confidence level (CI)

α p Signifcant

Organic carbon

compounds

maximum nitrite concentration 0.004 non-linear - Kruskal-Wallis 0.95 0.05 0.350 no

nitrite production rate 0.004 non-linear - Kruskal-Wallis 0.95 0.05 0.390 no

Nitrogen compounds

maximum nitrite concentration 0.002 non-linear - Kruskal-Wallis 0.95 0.05 0.572 no

nitrite production rate 0.003 non-linear - Kruskal-Wallis 0.95 0.05 0.847 no




In-silico RFLP patterns for species of Nitrosomonas and Nitrosospira genus:

Fig. 1 RFLP patterns for Nitrosomas know species, In-silico hydrolysisof amoA gene with: a) HaeIII; b) HinfI.


N. europaea

N. nitrosa

N. oligotropha

N. ureae

N. aestuari

N. Communi

N. Cryotolerans

N. halophila

N. marina

500

100

600

200

300

400

700

500

100

600

200

300

400

700

N. europaea

N. nitrosa

N. oligotropha

N. ureae

N. aestuari

N. Communi

N. Cryotolerans

N. halophila

N. marina

a

b



Fig. 2 RFLP patterns for Nitrosospira know species, In-silico hydrolysisof amoA gene with A) HaeIII

hydrolase; B) HinfI


500

100

600

200

300

400

700

N. multiformis

N. tenuis

N. Briensis

800

500

100

600

200

300

400

700

N. multiformis

N. tenuis

N. Briensis

800 900

900

a b

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