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

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).

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

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 Centro de Estudos do Ambiente e do Mar (CESAM) (Arguente)

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.

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.

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.

8

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

1. Location and sampling 28

9

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

V. References 44

10

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.

11

12

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

13

14

I. Introduction

15

16

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,

1999). Other biotechnological applications have been suggested for the organisms that

17

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 sub-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-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

average thickness of 60 µm in SML is required in order to study physical and chemical

18

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

considered on their own, represent a considerable challenge to microbial life. Gravity

19

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).

20

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

bacterioneuston communities is still not fully demonstrated (Agogué et al., 2005). The

21

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).

22

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

collection of SML samples without contamination from other layers remains a

23

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

24

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

cycling, industrial processes and pathogenesis (Jessup et al., 2004; Benton et al., 2007).

25

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.

26

II. Material and Methods

27

28

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).

Figure 2 – Ria de Aveiro, Portugal, with indication of the sampling site.

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

30

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,

3.75 mM MgCl2, 0.1 mM of each primer, 0.25 mg of BSA (Bovine Serum Albumin,

31

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

32

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.

33

34

III. Results and Discussion

35

36

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.

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.

38

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 .

39

40

IV. Concluding remarks

41

42

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.

43

44

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