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Universidade de Aveiro 2011

Departamento de Biologia

José António Amaro

Correia Medeiros

Optimal sample size for assessing bacterioneuston

structural diversity

Tamanho

da

amostra

ideal

para

avaliar

a

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Universidade de Aveiro 2011

Departamento de Biologia

José António Amaro

Correia Medeiros

Optimal sample size for assessing bacterioneuston

structural diversity

Tamanho da amostra ideal para avaliar a diversidade

do bacterioneuston

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Aplicada, Ramo de Microbiologia Clínica e Ambiental realizada sob a orientação científica da Professora Doutora Ângela Cunha, Professora Auxiliar do Departamento de Biologia da Universidade de Aveiro e da co-orientação do Doutor Newton Gomes, Investigador Auxiliar do Centro de Estudos do Ambiente e do Mar (CESAM).

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Dedico este trabalho a todos aqueles que colaboraram directa e indirectamente para que fosse possível realizá-lo

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O júri

presidente Profª Doutor João Serôdio Professor Auxiliar

Departamento de Biologia da Universidade de Aveiro vogal Profª Doutora Maria Ângela Sousa Dias Alves Cunha

Professora Auxiliar

Departamento de Biologia da Universidade de Aveiro (Orientadora) vogal Doutor Newton Carlos Marcial Gomes

Investigador Auxiliar

Centro de Estudos do Ambiente e do Mar (CESAM) (Co-orientador) vogal Doutora Isabel Henriques

Investigadora em Pós-Doutoramento

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agradecimentos A conclusão desta Dissertação de Mestrado só foi possível pela intervenção directa e indirecta de algumas pessoas, a quem agradeço com imenso carinho: À Professora Doutora Ângela Cunha, por ter aceite ser minha orientadora, e que ao longo destes meses tenha estado sempre disponível, e por me ter dado imensa força e incentivo em todos os momentos.

Ao Doutor Newton Gomes, pela sua disponibilidade e simpatia. Ao Mestre Francisco Coelho e Mestre Ana Luísa que com muita simpatia e sempre com um sorriso tornaram possível a realização do trabalho laboratorial, pela disponibilidade toda que tiveram ao longo de meses, colaboração e pelas palavras de incentivo nos momentos mais desesperantes. Também não posso deixar de lembrar todos os meus colegas do LEMAM, em especial a Ana Cecília, não só pela companhia mas também por toda a simpatia que demonstraram ao longo destes meses.

A todos os meus amigos, eles foram essenciais neste meu trajecto.

Ao CESAM (Centro de Estudos do Ambiente e do Mar) pelo suporte financeiro.

E para finalizar, e não por ser o menos importante, até pelo contrário, agradeço imenso aos meus pais, que sem eles eu não teria conseguido chegar até aqui, sempre me apoiaram, deram força e carinho. Sem eles não seria o que sou hoje em dia e espero continuar a dar-lhes motivos para se orgulharem de mim.

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keywords Surface microlayer, Bacterioneuston, Diversity, DGGE

abstract The surface microlayer (SML) is located at the interface atmosphere-hydrosphere and is theoretically defined as the top millimeter of the water column. However, the SML is operationally defined according to the sampling method used and the thickness varies with weather conditions and organic matter content, among other factors. The SML is a very dynamic compartment of the water column involved in the process of transport of materials between the hydrosphere and the atmosphere. Bacterial communities inhabiting the SML (bacterioneuston) are expected to be adapted to the particular SML environment which is characterized by physical and chemical stress associated to surface tension, high exposure to solar radiation and accumulation of hydrophobic compounds, some of which pollutants. However, the small volumes of SML water obtained with the different sampling methods reported in the literature, make the sampling procedure laborious and time-consuming. Sample size becomes even more critical when microcosm experiments are designed. The objective of this work was to determine the smallest sample size that could be used to assess bacterioneuston diversity by culture independent methods without compromising representativeness and therefore ecological significance. For that, two extraction methods were tested on samples of 0,5 mL, 5 mL and 10 mL of natural SML obtained at the estuarine system Ria de Aveiro. After DNA extraction, community structure was assessed by DGGE profiling of rRNA gene sequences. The CTAB-extraction procedure was selected as the most efficient extraction method and was later used with larger samples (1 mL, 20 mL and 50 mL). The DNA obtained was once more analyzed by DGGE and the results showed that the estimated diversity of the communities does not increase proportionally with increasing sample size and that a good estimate of the structural diversity of bacterioneuston communities can be obtained with very small samples.

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palavras-chave Microcamada Superficial (SML), Bacterioneuston, Diversidade, Eletroforese em gel de gradiente desnaturante (DGGE)

resumo A microcamada superficial marinha (SML) situa-se na interface atmosfera-hidrosfera e teoricamente é definida como o milímetro mais superficial da coluna de água. Operacionalmente, a espessura da SML depende do método de amostragem utilizado e é também variável com outros fatores, nomeadamente, as condições meteorológicas e teor de matéria orgânica, entre outros. A SML é um compartimento muito dinâmico da coluna de água que está envolvida no processo de transporte de materiais entre a hidrosfera e a atmosfera. As comunidades bacterianas que habitam na SML são designadas de bacterioneuston e existem indícios de que estão adaptadas ao ambiente particular da SML, caracterizado por stresse físico e químico associado à tensão superficial, alta exposição à radiação solar e acumulação de compostos hidrofóbicos, alguns dos quais poluentes de elevada toxicidade. No entanto, o reduzido volume de água da SML obtidos em cada colheita individual com os diferentes dispositivos de amostragem reportados na literatura, fazem com que o procedimento de amostragem seja laborioso e demorado. O tamanho da amostra torna-se ainda mais crítico em experiências de microcosmos.

O objectivo deste trabalho foi avaliar se amostras de pequeno volume podem ser usadas para avaliar a diversidade do bacterioneuston, através de métodos de cultura independente, sem comprometer a representatividade, e o significado ecológico dos resultados. Para isso, foram testados dois métodos de extracção em amostras de 0,5 mL, 5 mL e 10 mL de SML obtida no sistema estuarino da Ria de Aveiro. Após a extracção do DNA total, a estrutura da comunidade bacteriana foi avaliada através do perfil de DGGE das sequências de genes que codificam para a sub unidade 16S do rRNA. O procedimento de extracção com brometo de cetil trimetil de amônia (CTAB) foi selecionado como sendo o método de extração com melhor rendimento em termos de diversidade do DNA e mais tarde foi aplicado a amostras de maior dimensão (1 mL, 20 mL e 50 mL). O DNA obtido foi mais uma vez usado para análise dos perfis de DGGE de 16S rDNA da comunidade e os resultados mostraram que a estimativa da diversidade de microorganismos não aumentou proporcionalmente com o aumento do tamanho da amostra e que com amostras de pequeno volume podem ser obtidas boas estimativas da diversidade estrutural das comunidades de bacterioneuston.

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Table of contents

Index 8

List of figures 10

List of abbreviations 12

I. Introduction 14

1. Sea Surface Microlayer 16

2. The surface microlayer environment 18

2.1. Physical properties 18

2.2. Chemical properties 19

2.3. Biological properties 20

3. The bacterioneuston 20

3.1. Abundance and diversity 20

3.2. Activity 21

3.3. Ecological role and biotechnological applications 21

4. Sampling the surface microlayer in natural environments 22

5. Microcosms assays 23

6. Justification and objectives 24

II. Material and Methods 26

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2. Extraction of total DNA from environmental samples 29

3. PCR Amplification of 16S rDNA gene sequences 20

4. DGGE 32

5. Data analysis 32

III. Results and Discussion 34

Diversity of bacterial communities 36

IV. Conclusion 40

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List of figures and tables

Figure 1 – Conceptual model of the sea surface microlayer (modified from Hardy e Word, 1986).

Figure 2 – Ria de Aveiro, Portugal, with indication of the sampling site. Figure 3 – Glass plate sampler.

Figure 4 – SML collecting through rubber blades.

Figure 5 – DGGE profiles resulting from the separation of fragments of 16s rDNA genes amplified by PCR from DNA extracted from samples of 0.5, 5 and 10 mL of SML sample by two different extraction protocols. M – marker.

Figure 6 – DGGE profiles resulting from the separation of fragments of 16s rDNA genes amplified by PCR from DNA extracted from samples of 0.5, 5, 10, 20 and 50 mL of of SML sample with CTAB-containing extraction buffer. M – marker.

Table 1 – Mean ± SD of the values of the Shannon diversity indices calculated from denaturing-gradient gel electrophoresis (DGGE) profiles of bacterial 16S rDNA obtained from samples of 0.5, 5 and 10 mL extracted with or without CTAB.

Table 2 – Mean ± SD of the values of the Shannon diversity indices calculated from denaturing-gradient gel electrophoresis (DGGE) profiles of bacterial 16S rDNA obtained from samples of 0.5, 5 and 10 mL extracted with CTAB-containing extraction buffer.

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List of abbreviations

CTAB DGGE EDTA m mL PCR SML UW V µl µm µM Cetyltrimethylammonium Bromide Denaturing gradient gel electrophoresis Ethylenediaminetetraacetic acidMeterMetre Milliliter

Polymerase Chain Reaction Surface microlayer Underlying water Volt Microliter Micrometer Micromolar

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Introduction

1. The sea surface microlayer

The sea surface microlayer (SML) corresponds to the air-water interface and represents an important microhabitat that among other ecological roles, is involved in the exchange of particles, gaseous or liquid, of natural or anthropogenic origin, between the hydrosphere and the atmosphere (Franklin et al., 2005; Obernosterer et

al., 2005). The SML is characterized by unique biological, chemical and physical

properties, dissimilar of the underlying water (UW) (Franklin et al., 2005). The SML has common properties in most marine and freshwater environments and it is physically more stable than the UW, because of the resulting surface tension forces acting on this layer (Wurl and Obbard, 2004; Obernosterer et al., 2005). However, it is affected by mechanical disturbance associated with the ripples and wind, that influence the formation and thickness of the SML (Franklin et al., 2005).

The community of organisms associated with the SML is referred as neuston. The neuston represents as source and storage compartment for organic and inorganic matter and neuston activity can cause a significant impact on the exchange of matter in the atmosphere-hydrosphere interface (Obernosterer et al., 2005). The SML is a reservoir of various pollutants and plays an important role in the global distribution of anthropogenic contaminants (Wurl & Obbard, 2004). These contaminants include chlorinated hydrocarbons, organometallic compounds and polycyclic aromatic hydrocarbons (PAHs), and their concentration can be about 500 fold higher than in the underlying waters (Wurl & Obbard, 2004). Hydrophobic pollutants in the SML can originate from sewage discharges, agricultural waste, industrial and port activities (Walczak & Donderski, 2004). The rainfall also plays an important role in the enrichment of the SML. Different types of aerosols, gases and dust are deposited due to gravitational sedimentation or transport by rain (Donderski & Walczak, 2004; Wurl & Obbard, 2004). Neustonic organisms have been proposed as major contributors to the transformation of toxic compounds, configuring the interface atmosphere-hydrosphere as a bioreactor for detoxification of pollutants (Hardy, 1991; CIESM,

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inhabit the SML, particularly in the pharmaceutical and cosmetics industries (CIESM, 1999).

2. The surface microlayer environment

The SML has traditionally been defined as the top millimeter of the water column (Liss and Duce, 1997). However, in most studies, it is the depth of sampling that operationally defines the microlayer, which in turn, is dependent on the sampling technique used (Agogué et al., 2004). In field conditions, the thickness of the SML can also vary in time and space according to weather conditions and with the concentration and composition of the pool organic matter (Agogué et al., 2004). Several models have been proposed for the structure of this layer. Primarily based on the transport processes of particulate matter, the Hunter model describes a hydrodynamic layer with 50 to 300 µm in total thickness (Hunter, 1980). The Hardy and Word model (Figure 1) defines three distinct surface layers: the surface nanolayers (<1 µm) which contains various surfactant particles; the surface micron (<10 µm) rich in particles and microorganisms; and the surface millilayer (< 1000 µm) which provide habitat for larvae and eggs of zooplankton (Hardy and Word, 1986). The Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP) defined SML as the top millimeter of the water column where properties are more distinct from deeper waters and proposes the division of SML into three sublayers: the viscous layer is roughly the top 1000 µm of the water surface; the thermal sub-layer is approximately the top 300 µm of the water surface; the diffusion sub-sub-layer refers the top 50 µm.

Globally, the SML can be described as a micro-habitat composed by several distinct layers, differing from each other by their chemical and ecological characteristics, with a depth range from 1 to 1000 µm (Hardy, 1991). An estimate of the thickness of the SML based on readings of pH with microelectrodes reached a value of 50±10 µm (Zhang et

al., 1998). Currently, and based on the literature, Wurl and Obbard propose that an

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properties (Wurl and Obbard, 2004). Meanwhile for studies of biological properties,a thickness of about 1000 µm is necessary, although it might vary depending on the nature and purpose of the ecological study (Wurl and Obbard, 2004).

Figure 1- Schematic representation of the conceptual model of the sea surface microlayer (modified from Hardy and Word, 1986).

2.1. Physical properties

The SML is regarded as a physically stable environment (Franklin et al., 2005). This stability is caused by surface tension forces resulting from the accumulation of organic compounds, especially lipids and surfactants (Gasparovic et al., 2007; Wurl et al., 2009). However, due to its location, SML is susceptible to the alteration of the environmental conditions. The stability is affected by mechanical disturbances, which will affect the formation and the thickness of the SML (Franklin et al., 2005). In natural environments, the surface layer is exposed to wide variations of several physical and chemical factors, including radiation, temperature, salt concentration and mechanical disturbance (Liss, 1975; Henk, 2004; Santos et al., 2009). The physical forces and molecular interactions generated at the surface of the hydrosphere, even when

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is also responsible for the accumulation of high concentrations of small particles, that being heavier than the air but less dense than the water, accumulate at the interface between both environments, becoming part of the surface microlayer (Liss, 1975; Wakzak and Donderski, 2003; Henk, 2004).

2.2. Chemical properties

The accumulation of dissolved organic matter in the atmosphere-hydrosphere interface in a biofilm-like layer contributes to the development of a well-defined SML (Donderski & Walczak, 2004). At the sea, the main source of these compounds is primary production of phytoplankton, whose products of metabolism accumulate in the SML. In coastal areas, the products derived from anthropogenic activities represent an enormous contribution to the formation of the SML (Liss & Duce, 1997). The constituents with a greater ability to diminish the surface tension, in particular lipids and lipophilic components, are located on the surface and its accumulation forms a permanent multimolecular layer. The water-soluble constituents, including proteins and carbohydrates, are below the multimolecular layer (Momzikoff et al., 2004). These constituents accumulate in the SML through mechanisms of adsorption, diffusion, buoyancy, and rainfall (Donderski & Walczak, 2004). The SML also represents a reservoir of various hydrophobic pollutants with an important role in the global distribution of anthropogenic pollutants. These pollutants include chlorinated hydrocarbons, organometallic compounds and polycyclic aromatic hydrocarbons, which are derived from sewage discharges, agricultural waste, industrial and port activities. The concentration of these compounds can be more than 500 times higher than in UW (Wurl & Obbard, 2004; Coelho et al., 2011).

The rainfall also plays an important role in the enrichment of this layer. Different types of aerosols, gases and dust are deposited in the SML due to gravitational sedimentation or transportation by rain (Donderski & Walczak, 2004; Wurl & Obbard, 2004).

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2.3. Biological properties

The community of organisms associated with the SML is generally referred as neuston. The neuston includes the virioneuston (virus), the bacterioneuston (prokaryotes), the fitoneuston (microalgae), the zooneuston (microinvertebrates), the ictioneuston (fish eggs and larvae) whose abundances are often characterized as higher than in the UW (Zaitsev, 1971; GESAMP, 1995). The larvae and eggs of a large number of fish and invertebrates remain only temporarily in the SML during some development stages (GESAMP, 1995), but the SML is an important location for the development of many larvae of fish with high economical value (CIESM, 1999). The SML is significantly enriched in heterotrophic prokaryotes, heterotrophic protists, picoeucariotes and nanoeucariotes (Obernosterer et al., 2005). Compared to the underlying water, the abundance of bacteria, microalgae and invertebrates is increased in SML by factors of 102-104, 102 and 10 times, respectively, in relation to bulk water (Wurl and Obbard, 2004).

3. The bacterioneuston

3.1. Abundance and diversity

The bacterial community associated with the SML is composes the bacterioneuston. Information on the taxonomic diversity of bacterioneuston is still very scarce. By the use of culture-independent approaches techniques such as denaturing gradient gel electrophoresis (DGGE) some differences between the bacterial communities of SML and UW have been detected (Henk, 2004). The construction and analysis of genomic libraries reveals lower bacterial diversity in the SML, compared with the UW. The SML is dominated by 16S rDNA gene sequences closely related with two main groups: Vibrio, with a percentage of 68% of Pseudoalteromonas, with a share of 21% of the clones (Franklin et al., 2005). A study of estuarine bacterioneuston by DGGE revealed 16S rDNA gene sequences in SML samples that could not be detected in UW from the corresponding sampling sites (Cunliffe et al., 2008). Although new bacterial species have been isolated from the SML, evidences of the existence of typical

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need for further studies on the diversity of bacterioneuston, using culture-dependent and culture-independent approaches are needed to verify the existence of specific bacterial communities in the SML, and understand the relationship between community structure and the physico-chemical environment, is a consensus opinion (Franklin et al., 2005; Cunliffe et al., 2008).

3.2. Activity

The characterization of the patterns of activity of bacterioneuston communities is still at a prospective phase. The activity of heterotrophic bacterioneuston is characterized in different studies as being lower (Williams et al., 1986), higher (Carlucci et al., 1986; Obernosterer et al., 2005), or identical to that of bacterioplankton (Agogué et al., 2004). The rates of extracellular enzymatic hydrolysis are considered higher in SML than in UW (Kuznetsova and Lee, 2001).

3.3. Ecological role and biotechnological applications

Due to its unique location, bacterioneuston is assigned to perform an important role in the dynamics of freshwater and marine ecosystems (Zaitsev, 1971). It has also been proposed that bacterioneuston is involved in gas exchange and transport mechanisms between the atmosphere and the water column, with an important role of regulation of the methane metabolism and global climate change (Liss and Duce, 1997). Although the SML is an active site for the development of biological and chemical processes, its role still largely unknown (Hardy, 1982; Kuznetsova and Lee, 2001; Agogué et al., 2005, Franklin et al., 2005).

Despite the shortage of information, it is believed that high concentrations of different compounds (organic and inorganic) in the SML affect the spectrum of metabolic processes of bacterioneuston as well as their rate (Walczak & Donderski, 2004). The bacterioneuston probably plays an important role in the degradation of natural compounds and various compounds of anthropogenic origin that accumulate in this layer (GESAMP, 1995).

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More recent studies reported high frequency surfactant resistant bacteria in the SML of the estuarine system Ria de Aveiro (Louvado et al., 2010). Another study studies revealed the importance of bacterioneuston as a potential source of new PAH-degrading bacteria with potential use in the bioremediation of hydrocarbon-polluted ecosystems (Coelho et al., 2011).

4. Sampling the surface microlayer in natural environments

One of the major limitations in the study of the SML is the method used for sample collection. This method will determine the thickness and concentration of organic and inorganic compounds at the SML in comparison with the UW (Agogué et al., 2004). Some factors must be taken into account when choose in the samples strategy. The objective of the study and the required sample volume will determine the most suitable device and, in some cases, the nature of the material from which is made. The choice of the material is particularly critical in samplers that operate through adsorption, since different materials adsorb different compounds (Franklin et al., 2005). Although in recent years several methods have been proposed, the collection of representative SML samples remains a major challenge. Mechanical stirring by winds and currents, the need for operator training in routine sampling in order to obtain reproducible results, and the fact that during the sampling period the samples can suffer alterations in their characteristics and concentration of solutes due to the selectivity of some materials used in the samplers, are some of the difficulties in collecting samples of SML (Wurl & Obbard, 2004). According Agogué et al., the most commonly used samplers are the metal grid (Garrett, 1965), the glass plate (Harvey & Burzell, 1972), the drum rotation (Harvey, 1966, Hardy et al., 1988), the Teflon plate (Larsson et al., 1974) and the platform (Hatcher & Parker, 1974). In general, the layer sampled with the glass plate corresponds to 50 ± 10 mm and it is suitable for several physical, chemical and biological studies (Wurl & Obbard, 2004, Zhang et al., 2003). Hydrophilic and hydrophobic membranes are also used, but only to collect bacterial cells and are not suitable for quantitative studies (Agogué et al., 2004). However, the

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challenge, because the SML is physical, chemical and biologically heterogeneous. For example, the thickness of the SML varies with wind speed and the movement of waves can disrupt or even destroy the SML. The chemical composition is also subject to rapid change in areas where surface tension is higher (Wurl & Obbard, 2004). The sampling techniques requires training in order to collect samples with high reproducibility. A study by Knap et al. (1986), reports a relative standard deviation (RSD) of 15% in the volume of collected SML by a group of ten researchers with the metal grid sampler (Garrett, 1965). The difference in the volume of the sample led to a significant difference in the estimated thickness of the SML. The time involved in the collection of the volume of SML necessary to analyse trace contaminants may become so long that considerable changes in the characteristics and concentrations of materials is likely to occur (Wurl, 2004).

5. Microcosms assays

Microcosms are small-scale experiments using model systems recreating natural environment on a simplified form, when the isolation of sources of variability in necessary to text hypothesis or characterize effects. Such model systems are even regarded as a useful approach to global processes (Benton et al., 2007).

Microcosms are attractive especially due to the small size of the experiments. This allows greater flexibility to add or remove variables to increase replication ensuring statistical significance, restricted movement of the organisms and a rapid temporal dynamics (Srivastava et al., 2001). In natural microcosms is also possible to assess the interactions between species within a community but it is more difficult to study interactions between communities. The experimental manipulations of communities in natural continuous habitats are complicated (Krebs, 1996). On the other hand, the physical limits of natural microcosms represent a natural constraint for biota, which facilitates the addition or removal of species, the complete redesign of a community and the manipulation of environmental factors of regulation (Srivastava and Lawton, 1998; Kneitel and Miller, 2002). However, natural microcosms are closed systems, and

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during the course of the experiments, considerable changes in the physical and chemical environment may occur. The fact that the organisms represented in microcosms tend to be small (insects, arthropods, annelids, crustaceans, metazoan, protozoa and bacteria), implies a rapid generation time which allows a study of several generations, unlike the studies with larger organisms (Bengtsson, 1989). The artificial microcosm they tell us whether the effects described may occur in the event, while the natural microcosms tell us whether such effects occur and are important. Although the extrapolation of microcosm results to field conditions is sometimes difficult, experimental microcosm are often the most convenient approach to establish mechanistic relations between biotic and biotic components of the ecosystem and are particularly useful in the detailed characterization of particular factors, such as pollutants or other forms of stress, on population and organisms. Standardized aquatic microcosms have obvious advantages in the speed of analysis, reproducibility between laboratories and operators, statistical significance and costs, when compared with field studies and a quick statistical analysis (Taub, 1997).

Experiments in microbial laboratory microcosms are very useful to answer ecological questions that experiments in natural microcosms are not able to respond (Jessup et

al., 2004; Benton et al., 2007). The increasing information on the physiology and

genetics of many microorganisms allows us to understand the ecological processes at all scales of biological organization (Jessup et al., 2004). However, experiments in model microbial systems have limitations: the small scale required by microbial microcosms makes it more difficult handling of heterogeneity compared with microcosms of plants and animals. The evolution of many organisms in microbial microcosms can occur within few days, which can lead to a changing dynamic in the interaction before the end of the experiment (Jessup et al., 2004; Benton et al., 2007). Microbial microcosms offer a complementary approach to field studies and laboratory studies of microorganisms. Some of the advantages are related to the simplicity of the systems that easily allows the testing of theoretical predictions. Also, they provide the opportunity to explore more practical problems (e.g.: toxicology and environmental microbiology) that can reveal much about the role of microorganisms in nutrient

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6. Justification and objectives

Considering that the SML corresponds to the first millimetre of the water column, laboratory microcosms impose serious limitations on sample size when analysis of the SML properties is intended. Using very small samples for the analysis of bacterioneuston communities may be a poor approach to the community structure and actually underestimate bacterial diversity. The objective of this work was to verify the representativeness of small samples of SML in the analysis of the structural diversity of bacterioneuston communities by 16S rDNA DGGE profiling with the aim of obtaining a methodological approach suitable for application in microcosm experiments. As a preliminary step, two methods of DNA extraction (with and without CTAB ) were compared.

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1. Location and sampling

The SML samples were collected at S. Roque Channel, a small ramification of Espinheiro Channel (Figure 2). This site was chosen because it is located in sheltered area adjacent to the city of Aveiro. It is an eutrophicated area, suffering from anthropogenic impacts. Samples were collected in slack high tide in November 2010. The SML sample was collected using a glass plate (Figure 3) and an acrylic plate. Just before sampling, both plate were cleaned with ethyl alcohol and distilled water and rinsed with water from the sampling site. The protocol followed was adapted by Agogué et al. (2005).

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29 Figure 3 – Glass plate sampler Figure 4 – SML collecting through rubber blades

The plates were immersed in vertical position, gently removed from the water in the same position, and allowed to drip for 5 seconds. The water was collected in a sterilized glass bottle by forcing the plates through the collection grid (Figure 4). The procedure was repeated, alternating plates, to achieve the volume needed for the experiment. Samples were transported to the laboratory in an isothermal box and processed within 2 hours after collection.

2. Extraction of total DNA from environmental samples

For the extraction total community DNA, a protocol by Hurt et al. (2001) optimized by Costa et al. (2004) was followed. All materials used in the extraction procedure were sterilized in order to prevent contaminations. Six different sample volumes were tested and, for some volumes, the extraction was repeated with some changes in the protocol in order to assess if different extraction procedures would affect the results of the analysis. The tested volumes were 0.5, 1, 5, 10, 20 and 50 mL of SML water. For the extraction of the smallest volumes (0.5 and 1 mL) triplicates of SML water were directly transferred to microtubes. For sample volumes of equal or larger than 5 mL, cells were concentrated by filtration of 3 replicates triplicate through 0.2 µm polycarbonate membranes (GE Osmotics). The membranes were washed with 2 mL of

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TE buffer (10 mM Tris-HCl - Fluka, 1 mM de EDTA - Fluka, pH 8.0), and the cell suspension resulting from the wash was collected in sterile microtubes. Each microtube received 0.4 g of glass beads and 0.5 mL ice-cold ethanol. The microtubes were agitated twice using the FastPrep FP120 bead beating system (Qbiogene, USA) at 5.5 m/s for 30 sec. Samples were kept on ice during between agitation periods. Suspensions were centrifuged at 16.000 x g for 5 min. (Haereus Pico17, Thermo scientific) and the supernatant was discarded. To each pellet, 1.2 mL of extraction buffer containing 100 mM sodium phosphate, 100 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl, 1% hexadecyltrimethylammonium bromide (CTAB) and 2% SDS (pH 7.0) (Hurt et

al., 2001). After mixing, the extraction mixtures were incubated for 30 min. at 65ºC

with gentle mixing by invention of the microtubes every 10 min. A parallel series of triplicates of 0.5, 5 and 10 mL was incubated for 30 min. at 65ºC immediately after agitation in the FastPrep system with the extraction buffer without CTAB. The extracts were centrifuged at 16.000 x g for 5 min. The supernatant was transferred to new sterilized microtubes and 1 mL of a solution of chloroform-isoamyl alcohol (24:1 v/v) was added. The mixture was incubated on ice for 5 minutes. The tubes were carefully agitated and centrifuged at 16.000 x g for 5 min. The aqueous phase was transferred to sterilized microtubes and nucleic acids were precipitated by incubation at room temperature with 0.6 volumes of isopropanol for at least 30 min. Pellets were obtained through centrifugation at 16.000 x g for 20 min, washed twice with 0.5 ml 70% ice cold ethanol and air dried before resuspension in 0.2 ml of RNase-free water.

3. PCR Amplification of 16S rDNA Gene Sequences

The partial sequence of 16S rDNA gene was amplified by PCR following a nested-PCR approach, using primers U27F (5´AGAGTTTGATCCTGGCTCAG- 3´) e 1492R (5´-GGTTACCTTGTTACGACTT-3´) for the Bacteria domain (Weisburg et al., 1991), synthesized by IBA (IBA GmbH) and DNA extracted from environmental samples as template. The PCR reactions were performed in 25 mL of reactional mixtures containing 1 mL of sample, 1 U of Taq DNA polymerase, 1x KCl buffer, 0.2mm dNTPs,

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Sigma Co.) and deionized water. The polymerase chain reaction used repeated cycles, each of which consists of three steps:

Step 1 - The reaction solution containing DNA molecules, polymerases, primers and nucleotides is heated to 95°C for 5 min followed by 25 cycles of denaturation at 94 ° C for 45 s.

Step 2: Lowering the temperature to 55°C by 45 s causes the primers to bind to the DNA, a process known as hybridisation or annealing. The polymerases then begin to attach additional complementary nucleotides at these sites, thus strengthening the bonding between the primers and the DNA.

Step 3: The temperature is again increased, this time to 72°C by 90 s. This is the ideal working temperature for the polymerases used, which add further nucleotides to the developing DNA strand. The PCR reaction was completed a final extension step at 72 ° C for 10 min.

The amplification products were used as templates for a second amplification with primers 968F -GC (5´-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAA CCT TAC-3´) and 1401R (5´-CGG TGT GTA CAA GAC CC-3´) (Nubel et

al., 1996) synthesized by IBA (IBA GmbH). The PCR reactions were performed in 25 mL

of reactional mixtures containing 1 µL of the first-round PCR product, 1 U of Taq DNA polymerase, 1x KCl buffer, 0.2mm of DDNP's, 3.75 mM MgCl2, 0.1 mM of primer, 4% acetamide (Fluka) and deionized water. The PCR conditions were as follows: a step of initial denaturation at 94 ° C for 4 min. followed by 25 cycles of denaturation at 94 ° C for 1 min, annealing at 53 ° C for 1min and extension at 72 ° C for 2 min. The PCR reaction was completed a final extension step at 72 ° C for 7 min.

All PCR reactions were performed in a thermocycler Multigen TC 9600 - G (Labnet International, Inc) with reagents from MBI Fermentas (Vilinius, Lithuania), except when indicated otherwise. The presence of amplification products was confirmed by electrophoresis on 1.5% agarose gel (Fluka) with ethidium bromide (VWR) to 100V for approximately 25 min in TAE buffer (0.04M Tris-Acetate, Sigma Co.; 0.001M EDTA, Sigma Co.). A positive control of DNA extracted from Pseudomonas putida KT2442 and

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the products of the amplification with primers NAPH-1F and NAPH-1R were used as positive controls (Gomes et al., 2005). To assess the size of the fragments resulting from amplification, a molecular weight marker was used (Gene Ruler TM DNA Ladder Mix, MBI Fermentas). The gels were visualized with Gel Doc (Bio Rad).

4. DGGE

DGGE of the amplified sequences was performed in a DCode System (Universal Mutation Detection System; Bio-Rad). The GC-clamped amplicons were applied to a double-gradient polyacrylamide gel containing 6 to 9 % acrylamide (Rotiphorese) with a gradient of 40 to 60 % of denaturant. The run was conducted in Tris-acetate-EDTA buffer (0.5M Tris-Base, Sigma, 0.05M EDTA, Sigma; 0.1M CH3CO2Na, Sigma , pH 8.0) at 60 °C at a constant voltage of 220 V for 16 h. The DGGE gels were silver stained according to the method of Heuer et al. (2001). The image was acquired with a scanner (Epson).

5. Data analysis

The Shannon index of diversity (H) was used to compare the complexity of the DGGE profiles. The band position and relative intensity (abundance) of each lane (community) were used as parameters in the PRIMER 5, to indicate categories (Costa

et al., 2006).

Parametric analysis of variance (ANOVA) was used to assess significant differences between samples, providing that data were normally distributed. SPSS Statistics 17 has been made the test of data normality and homogeneity of variance.

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Diversity of bacterial communities

The abundance and distribution of microorganisms in marine or estuarine water is influence by complex biotic and abiotic interactions. Substrate availability and predation are two of the main factors that regulate bacteria distribution (Shiah & Ducklow, 1995). The methods of independent culture are fundamental to the characterization of the structure of microbial communities in the environment (Amann

et al., 1995). DGGE profiling is a widely used tool to evaluate the structural diversity of

natural bacterial assemblages. rDNA gene fragments amplified by PCR are separated by sequence, rather than by size, in a gel electrophoresis conducted in a gradient of chemical denaturants.

The DGGE profiles of 16S rDNA gene fragments amplified by PCR from DNA extracted from different volumes of SML water are presented in Figures 5 and 6.

Figure 5 – DGGE profiles resulting from the separation of fragments of 16s rDNA genes amplified by PCR

from DNA extracted from samples of 0.5, 5 and 10 mL of SML sample by two different extraction protocols. M – marker.

The DGGE profiles presented in Figure 5 show a large number of equally abundant bands in the communities of bacterioneuston of both DNA extraction methods.

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37 Table 1 – Mean ± SD of the values of the Shannon diversity indices calculated from denaturing-gradient

gel electrophoresis (DGGE) profiles of bacterial 16S rDNA obtained from samples of 0.5, 5 and 10 mL extracted with or without CTAB.

DNA Extraction

Method

Without CTAB With CTAB

Sample volume 0,5 5 10 0,5 5 10 Shannon diversity indices 2,45±0,01 2,41±0,30 2,47±0,24 2,57±0,17 2,47±0,12 2,36±0,21

The average values of the Shannon diversity index,one of the indices used to measure the diversity of a community, calculated for each sample size presented in (Table 1) did not reveal significant differences (ANOVA p > 0.05) when the two extraction methods or the different sample volumes were compared. This analysis suggests that the DNA extraction method used was not relevant in the outcome of the analysis of diversity of the bacterioneuston community and that identical results could be obtained with samples between 0.5 and 10 mL.

Figure 6 – DGGE profiles resulting from the separation of fragments of 16s rDNA genes amplified by PCR

from DNA extracted from samples of 0.5, 5, 10, 20 and 50 mL of of SML sample with CTAB-containing extraction buffer. M – marker.

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The DGGE profiles of samples with sizes varying from 0.5 to 50 mL are presented in figure 6. All profiles are characterized by a large number of bands equally represented in all the samples used for DNA extraction.

Table 2 - Mean ± SD of the values of the Shannon diversity indices calculated from denaturing-gradient

gel electrophoresis (DGGE) profiles of bacterial 16S rDNA obtained from samples of 0.5, 5 and 10 mL extracted with CTAB-containing extraction buffer.

Sample volume 0,5 1 5 10 20 50 Shannon diversity indices 2,76±0,10 2,71±0,27 2,23±0,14 2,38±0,10 2,31±0,10 2,40±0,35

The Shannon diversity index did not reveal significant differences (ANOVA p > 0.05) between the different samples volumes tested (Table 2). This analysis suggests that identical insights into the structural diversity of the bacterioneuston community could be achieved with samples with sizes varying from 0.5 to 50 mL .

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The dynamics of the SML and the associated processes are still largely unknown. For a better understanding of the SML, several studies have been conducted over the last years revealing unique and important biological significance. However, the validation of some of the hypotheses sustained by field observations as to the ecological role of bacterioneuston and the factors involved in the regulation of bacterioneuston abundance, diversity and activity often requires the use of laboratory microcosms for controlled experiments. One of the problems of microcosm experiments with bacterioneuston is the small size of the samples can be collected in each sampling moment or experimental condition. In this study we tested the effect of two DNA extraction methods on assessment the structural diversity of bacterioneustons by 16S rDNA profiles, as well as the effect of sample size on the estimates of community diversity. The Shannon diversity index estimates did not significantly vary with increasing sample sizes, from 0.5 mL to a maximum tested volume of 50 mL Also, the protocol used for total community DNA extraction did not affect the results of DGGE analysis. The results show that good approaches to the structure of bacterioneuston communities can be achieved with samples as small as 0.5 mL and that very small sample sizes can be used for the analysis of the structural diversity of bacterioneuston communities by DGGE. Although promising for the designs of microcosm experiments with bacterioneuston, these results were obtained with SML samples from an eutrophicated site where elevated cell abundances are expected. The extrapolation to samples from more oligotrophic site should be considered with precaution because with lower cell abundances, a drastic reduction in sample size can reduce representativeness and ecological significance of the results.

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Referências

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