Marine microbial ecology

Introduction | Microbial components | Microbial processes | What do we study? | How do we collect samples? | How do we study microbes? | Movement by microbes | Agents of death | Research staff | Links | View components

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Introduction

This program studies the microscopic life in the Southern Ocean: single celled plants and animals (phytoplankton and protozoa), bacteria and viruses.

Why are marine microbes important?

These organisms comprise most of the living matter in the sea. Photosynthesis by phytoplankton takes up CO₂, producing the food that supports, directly or indirectly, the wealth of marine life for which Antarctica is renowned. However, only a small proportion of the carbon taken up flows directly on to organisms such as krill, fish and whales. Most of this carbon is cycled by microorganisms in the so-called microbial loop.

Marine microbes also have major effects on the world's climate. By absorbing carbon dioxide, they contribute to the uptake of CO₂ from the atmosphere, thereby moderating the global Greenhouse Effect. The Southern Ocean is one of the world's important 'sinks' where carbon is transported to the deep ocean by sinking particles. Some microorganisms also produce chemicals which, when ventilated to the atmosphere, form aerosol particles that can trigger the formation of clouds.

The rare but beautiful Stephanoeca norrisii, a choanoflagellate comprising a central cell surrounded by a lorica of silica rods that it uses to capture food
Image: John van den Hoff

SEM of the chitinous lorica (outer shell) of a tintinid
Image: Fiona Scott

SEM of the dinoflagellate Protoperidinium latistriatum
Image: Fiona Scott

SEM of Triparma columaceae subspecies alata,
a member of the Parmales
Image: Fiona Scott

SEM of the silica skeleton of
the silicoflagellate Dictyocha speculum
Image: Fiona Scott

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Microbial components

Phytoplankton are single-celled algae. Like all plants, they use carbon dioxide and light to produce food in the process of photosynthesis. Most range in size from 1 - 100 micrometres (1 micrometer = 1/1000 millimetre, abbreviation = µm), although some needle-shaped species reach 4 mm in length. Phytoplankton are commonly subdivided into three size classes: picoplankton (<2 µm), nanoplankton (2 - 20 µm), microplankton (> 20 µm). For most of the year, nanoplankton dominate the Southern Ocean with superimposed blooms of microplankton in summer. Picoplankton are much less important in the Southern Ocean than in temperate waters. There are at least 360 different phytoplankton species identified in Antarctic waters, many of which can swim.

LM of the common sea-ice diatom Nitzschia stellata
Image: Fiona Scott

SEM of Chaetoceros bulbosus,
a common Antarctic marine diatom
Image: Fiona Scott

Protozoa are single celled animals that consume phytoplankton, bacteria and organic matter [see video images in Agents of death]. Their respiration releases much of the carbon dioxide incorporated by phytoplankton. However they also help remove CO₂ from the atmosphere by converting their microscopic food into their own cell mass, making it available for higher levels of the food web whose bodies and faecal pellets sink into the deep ocean.

A protozoan ingests a small, green diatom
Image: Fiona Scott

SEM of a heliozoan, Acanthocystis perpusilla
Image: John van den Hoff

Protists is the general term for single celled organisms, including phytoplankton and protozoa. Each litre of surface water from the Southern Ocean can contain from 0.5 million to 60 million protists.

Bacteria are abundant in the Southern Ocean. Typically there are about 600,000 cells per ml of seawater. They are vital components of the microbial community, breaking down particulate matter (cells and detritus), releasing nutrients for use by other organisms and releasing CO₂. They also take up dissolved organic matter, converting it to cell mass, and making it available to grazers.

Fluorescence microscope image of the natural marine bacterial community selectively stained to show live bacteria as yellow/green and dead bacteria as red. Large red blobs are phytoplankton.
Image: Andrew Davidson

Viruses are the most abundant biological agents in seawater. Concentrations in Antarctic waters range from 1 to 4 million particles per ml. They infect phytoplankton, protozoa and bacteria and may be responsible for up to 50% of deaths of marine bacteria. Bursting cells release their contents into the water, where they fuel bacterial growth. As each virus infects a particular species of microbe, viruses may be important in controlling the abundance and composition of microbial communities in Antarctic waters.

SYBR Green 1 stained filter showing two highly fluorescent protist cells (yellow), many less fluorescent bacteria (green/yellow) and large numbers of very small viruses (pale green)
Image: Andrew Davidson

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Microbial processes

Components of the microbial community are instrumental in several processes of the marine food web and global chemical cycles.

Photosynthesis: Phytoplankton absorb CO₂ and harness the energy of sunlight to manufacture sugars and other cell components, releasing oxygen. The sunlight is absorbed by chlorophylls and carotenoids, which can be used to identify the various groups of phytoplankton in the water.

Respiration: All organisms (from microbes to whales) oxidize intracellular carbon reserves to produce the energy necessary for cell function (growth, movement, chemical metabolism). This process of respiration releases to the atmosphere much of the CO₂ taken up by phytoplankton.

Feeding: The ocean has been likened to a vast very dilute jelly, containing a continuum of matter ranging from small molecules to large aggregates [see Aggregation]. Microbes are able to consume matter throughout this size range, changing the kind and size of these compounds. This alters the availability of these food sources to other organisms. Bacteria release enzymes that convert complex matter to simple molecules that can be absorbed across their cell membrane. Protozoa have various means of consuming cells and can graze on a large range of particles from molecules to cells larger than themselves [see video image Agents of death]. All protists are grazed by crustaceans and other zooplankton.

Microbial loop: The processes discussed above operate simultaneously in a microbial community, whose collective metabolism is called the microbial loop. Most of the carbon in the marine ecosystem is cycled through this loop, strongly influencing the quality, quantity and size distribution of food available to higher organisms.

Aggregation: There are several processes by which particles can aggregate. Particulate and dissolved organic matter can spontaneously aggregate in seawater, a process aided by mixing. Grazing protozoa and higher organisms repackage matter into faecal pellets. Mucilage produced by algae provides a substrate that can be colonized by other cells. Aggregates support a rich and diverse microbial community within which the close proximity of organisms enhances recycling of matter via the microbial loop. Such aggregates are often called “marine snow”.

Sedimentation: There is a continuous 'rain' of particles from the sunlit upper waters to the ocean depths where there is insufficient light for photosynthesis and hence respiration rules. Much of the matter is recycled en route. Sedimentation to the deep ocean is the principal global process by which CO₂ is biologically removed from the atmosphere for geological time scales.

Succession: The composition and abundance of marine microbes varies greatly due to physical and environmental factors including, light, temperature, salinity, depth, nutrient concentrations, the nature, extent and persistence of sea ice, the depth and speed of vertical mixing of the water column, and grazing pressure.

Large areas of the Southern Ocean are unproductive. This is thought to be due to both strong vertical mixing that carries cells out of the sunlit portion of the water column and low concentrations of micronutrients (especially iron) that limit phytoplankton growth. Most microbial production occurs close to the Antarctic continent. Here they bloom in or on the bottom of the sea ice during spring, or occur as brief, spectacular water column blooms near the margin of the sea ice as it retreats southward during spring and summer.

In spring, as sunlight returns to Antarctic waters, phytoplankton concentrations begin to increase. Phaeocystis antarctica, a flagellate around 6 µm diameter that forms gelatinous colonies up to 2 cm in diameter, is often the first species to bloom in ice-edge waters. Subsequent blooms are often comprised of large (>20µm) diatoms, which are superimposed upon a background of nanoplanktonic (2 - 20µm) flagellates and diatoms. Towards the end of the season, phytoplankton abundance declines and protozoan and bacterial concentrations increase to consume the remainder of the summer's production. However, at many sites around the Antarctic coastline there is little interannual consistency in the timing, abundance or successional sequence of marine microbes.

Production by phytoplankton over the entire Southern Ocean can vary 25% between years and, at a single location, can vary by a factor of 5 - 10 between years. Small-scale variation in the physical and biological environment causes significant differences in the composition and abundances of protists (phytoplankton and protozoan) communities over distances of meters. The composition of and abundance viruses and bacterial communities can vary over distances of centimetres. Thus, while patterns are apparent in the overall community structure and function, the Antarctic marine microbial community constantly changes in response to an ever-changing environment.

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What are we studying?

The Australian Antarctic Division marine microbial programs focus on:

What is there?

Identifying the biodiversity of Southern Ocean protist populations.

A guide to more than 450 Antarctic protists with photographs and descriptions is being compiled, as an aid to the identification of these organisms. New organisms are being found and described every year.

Where and how much?

Determining the distribution and abundance of protists in the Southern Ocean.

The distribution, abundance and types of phytoplankton present are being determined using their photosynthetic pigments, by light and electron microscopy, and using electronic particle counters. Factors that control population abundance are being studied in relation to oceanography, nutrients and grazers - information vital to models of global carbon budgets. We are also investigating associations between species and ecological processes or oceanic regimes.

What are they doing?

Determining the response of protist populations to enhanced ultraviolet radiation due to the Antarctic ozone hole.

Protists in the upper waters of the ocean are exposed to increased ultraviolet radiation (UV) at the time of the "spring bloom". Different species vary in their susceptibility to damage by UV, and changes of species composition have been observed in Antarctic protist communities. Such changes directly affect the abundance and nature of food available to larger organisms as well as the extent of CO₂ uptake in surface waters.

Determining the feeding rates and preferences of protozoa.

Measurements are being made of the rates of protozoan feeding in the Southern Ocean, as well as studies of their feeding selectivity. Protozoan preference for food of particular sizes and "flavours" affects their role in the microbial loop.

Determining the abundance, viability and activity of marine bacteria.

These programs link with external programs in which scientists measure phytoplankton production, carbon dioxide uptake, carbon sedimentation, nutrient concentrations and oceanography to build an integrated picture of the smallest, but the most important, components of the marine food web.

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How are field samples collected?

Ship based sampling

Samples are collected either using instruments deployed over the side of the ship or by collecting from a seawater supply line with an intake at 7 m depth in the ship's hull.

Samples for depth profiles are obtained using a Rosette sampler. The rosette has 24 x ten litre Niskin bottles that can be closed at required depths by electronic signal from the ship. The attached CTD (Conductivity, Temperature, and Depth) system sends continuous data profiling the water column's temperature, salinity and density. Water masses, oceanographic features and currents are identified from these data.

CTD sampling rosette being launched from the side of the RV Aurora Australis
Image: Fiona Scott

CTD sampling rosette being returned into the CTD bay on the side of the RV Aurora Australis at night
Image: Fiona Scott

Collecting water samples from the Niskin bottles on the rosette sampler
Image: Fiona Scott

Samples for phytoplankton pigment analysis (0.5 - 2 L) are filtered onto glass fibre filters and stored in liquid nitrogen (-196°C) for analysis on return to Australia.

The 12-bottle phytoplankton filtration rack used on board the RV Aurora Australis
Image: Fiona Scott

Duplicate samples are preserved for cell identification by light microscopy or electron microscopy, and cell counting by a process of sedimentation and inverted microscopy. Living cells are also examined and counted whilst on board the ship with microscopes shock-mounted to avoid vibrations from the ship's engines.

Bacteria and viruses are filtered onto black polycarbonate membrane filters, stained with dyes that bind to their DNA and RNA, and counted by fluorescence microscopy.

Large phytoplankton are collected using 20 micrometer mesh plankton nets. This method of sampling is selective but it allows comparisons of current phytoplankton communities with historic samples.

Sea ice sampling

It is difficult to sample the microbial community living within sea-ice. The protists live in brine channels that are formed between relatively fresh water ice crystals. To protect the delicate organisms, sampling and laboratory techniques must avoid large changes in salinity that occur during melting.

Ice communities are sampled using a SIPRE ice corer or Jiffy drill. The SIPRE corer provides a cylindrical section of the sea-ice from which samples are cut, melted in filtered seawater and examined for microbes.

Alternatively, a partial core hole is drilled and the microbe-containing brine is allowed to drain into it.

A Sipre-corer and part of the resultant sea-ice core
Image: Fiona Scott

Scientist operating an ice-drill
Image: Fiona Scott

Land-based (Continental station) sampling

Over-snow vehicles or small boats are used to get to collecting sites from our Antarctic stations. Sea-ice and water samples are brought back to the laboratory where there are low-temperature incubation tanks, culturing facilities and analytical equipment.

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How are microbes studied?

Various techniques have to be used for studying microbes, for no one technique can measure everything. All are selective and have particular advantages and disadvantages.

Microscopy

Light microscopy

Both conventional compound microscopes and inverted microscopes are used to examine samples. These are used in transmitted light, Normaski differential interference contrast, phase contrast and epifluorescent modes to distinguish cellular organelles, appendages, and stained or autofluorescent cells (red autofluorescence typically denotes cellular chlorophyll).

Images are obtained using fine grained photographic film in microscope-mounted cameras or broadcast-quality video.

Living organisms are routinely examined at sea using microscopes that are isolated from the ship's vibrations. Live material can also be examined using microscopes in a cold (0ºC) room.

Light micrographs (LM) of various organisms are shown below.

LM of the Antarctic marine diatom Corethron criophilum
Image: Harvey Marchant

LM of the diatom Asteromphalus sp.
Image: Fiona Scott

The faint rings around Thaumatomastix cells are the surface body scales, easily seen using an electron microscope (see TEM image)
Image: Fiona Scott

Two euglenoid cells with ingested diatoms
Image: Fiona Scott

Light microscopy allows direct identification of microplankton but does not have sufficient resolution to identify most nanoplankton (especially those smaller than 10 µm). It allows cell behaviour (swimming, feeding etc.) to be studied as well as direct counts of phytoplankton. However, plankton counting is extremely time consuming, and "a task which cannot be completed without ruin of mind and body" (Haeckel, 1890).

Fluorescent stains are used to distinguish live and dead bacteria ( BacLite) and highlight DNA and RNA of protists, bacteria and viruses ( SYBR Green 1).

Electron microscopy

Electron microscopy is vital for the identification of small cells as well as examining details of larger cells. Much of the taxonomy of protists is based on fine structural details such as scales, flagella, surface patterning etc.

Scanning electron microscopy (SEM) shows the surface detail of cells.

The diatom Thalassiosira gravida has intricate surface ornamentation
Image: Fiona Scott

Hooks at one end of the common Antarctic diatom, Corethron criophilum
Image: Fiona Scott

SEM of the flagellated prasinophyte Pyramimonas gelidicola
Image: Sandy Melloy

SEM of the dinoflagellate Protoperidinium defectum
Image: Fiona Scott

Kakoeca antarctica, an endemic Antarctic choanoflagellate
Image: Fiona Scott

Emiliania huxleyi a Southern Ocean coccoithophorid
Image: Fiona Scott

Transmission electron microscopy (TEM) is used to examine either thin slices of material to show internal details of cells, or shadow-cast material to reveal fine surface structures of scales, flagella and other external cell components.

Thaumatomastix is covered by intricate body scales
Image: Fiona Scott

Organic body scales of Chrysochromulina vexillifera
Image: Fiona Scott

Details of choanoflagellate lorica as seen with TEM
Image: Fiona Scott

Scales from the flagellum and body of Pyramimonas sp.
Image: Rick van den Enden

Flow cytometry

A Flow Cytometer analyses particles by passing them in single file through a laser beam. It can count up to 1000 cells per second, measuring for each one the light scattering properties (indicating size and complexity), as well as yellow-green, orange, and red fluorescence. Autofluorescence (from chlorophylls and other pigments) as well as the use of fluorescent stains help distinguish cell types.

The capacity to rapidly count large numbers of particles greatly increases the reliability and precision of cell concentration estimates, however the cells are not directly identified and must be examined by microscopy.

Flow cytometer output of BacLight® stained marine bacteria. BacLight® is a dual nucleic acid stain that causes live bacteria to fluoresce green and dead bacteria to fluoresce red when excited with the 488 nm argon laser of the flow cytometer. The upper coloured plot shows the dead bacterial population in red and the live in green. These populations are also shown as a contour plot (upper right). The lower plots show these populations as counts of green (FL1) and red (FL3) fluorescent particles.

Pigment analysis

Phytoplankton, like all photosynthetic plants, use chlorophylls and carotenoids to absorb light for photosynthesis. Chlorophyll a, or a derivative, is present in all types of phytoplankton and is commonly used as an indicator of the phytoplankton biomass. It enables living phytoplankton to be distinguished from zooplankton, detritus and dead phytoplankton. It is highly fluorescent and can be measured in unconcentrated seawater samples. Other chlorophylls and carotenoids are present in phytoplankton, many of which have restricted taxonomic distribution. These pigments can be used as quantitative markers for particular taxonomic groups.

Fluorometry

The concentration of chlorophyll a in surface waters is continuously monitored on cruises of the Aurora Australis by measuring the fluorescence of water pumped from an intake at 7m depth. The temperature and salinity of this water is measured simultaneously.

Vertical profiles of chlorophyll a are measured with a fluorometer attached to the CTD. This shows the vertical distribution of phytoplankton much better than discrete samples from the rosette sampler (typically collected at 15m intervals).

Note however that fluorescence is reduced by sunlight and the response per unit chlorophyll varies during the day.

HPLC

HPLC (high performance liquid chromatography) is used to separate, identify and quantify the various chlorophylls and carotenoids in phytoplankton. Many of these are markers for particular taxa and can be used to estimate their contributions to the phytoplankton community.

HPLC is an excellent technique for mapping populations, since it is feasible to analyse more than 1000 samples per cruise. However it does not identify taxa directly and must be combined with microscopy to determine the key species present.

Special software (CHEMTAX) was developed collaboratively with CSIRO Marine Research to calculate the relative contributions of different groups of phytoplankton from the pigment content of field samples.

Feeding experiments

Studies of feeding by protists that are heterotrophic (dependent on external food) or mixotrophic (can use photosynthesis or external food) use two approaches:

First approach

Uptake of fluorescently labelled particles (dextrans, bacteria, latex microspheres, algae) is detected using epifluorescent microscopy or flow cytometry. Results show the size spectrum and uptake rate of food available to protists. Cultures of the choanoflagellate Acanthocorbis unguiculata have been incubated with fluorescent microspheres as a surrogate food source. Smaller microspheres (0.25 µm diameter) are ingested faster, and by a larger percentage of cells, than larger FM (0.5 and 1.0 µm diameter).

SEM of the choanoflagellate Acanthocorbis unguiculata
Image: John van den Hoff

Left: Eight cells of Acanthocorbis unguiculata under transmitted light illumination
Right: The same eight cells under blue light fluorescence illumination. Each cell has attracted fluorescent microspheres (the minute yellow-green dots) which have become attached to the lorica and ingested into the cell body
Images: Fiona Scott

Time course graph of the percentage of cells of Acanthocorbis unguiculata ingesting Fluorescent Microspheres (FM) of various diameters

Second approach

The growth rate of phytoplankton and the grazing rate of protozoa and zooplankton upon them can be measured simultaneously by incubating a series of water samples with various dilutions with filtered seawater. In brief, the phytoplankton grow at the same rate irrespective of dilution, but the grazing rate declines at higher dilutions because the food particles are further apart and harder to find. Extrapolation to infinite dilution estimates the phytoplankton growth rate in the absence of grazing. Knowing the growth rate, the grazing rate can be determined.

Response to UV irradiation

Field and laboratory studies have been undertaken to determine the tolerance of microbial species and communities to UV exposure. Ozone depletion over Antarctica enhances the UV-B (280 - 320 nm wavelength) irradiation, which penetrates near-surface waters to 50 m depth. UV radiation has been shown to reduce growth, production and survival in the top 10 -15 m of the water column.

UV light can impact all levels of the microbial community. Microbes differ greatly in their susceptibility to UV-induced damage and significant changes to community structure and function have been observed in natural assemblages exposed to antarctic sunlight.

Cultures

Controlled laboratory studies of the physiology of key protist species are performed using cultures maintained at the Australian Antarctic Division. Cultures are isolated by selecting single cells from a field sample and maintained in various nutrient-enriched culture media based on seawater supplemented with various nutrients.

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Movement by microbes

Movement by microorganisms in water is very different from that of humans and other large animals due to the overwhelming effects of viscosity at small size scales. Microbial swimming (equivalent to humans swimming in molasses) requires special techniques. Favoured techniques are the "flexible oar" (cilium), the "corkscrew" (forward facing flagellum) and cellular deformation.

Movement using flagella

The prasinophyte Pyramimonas sp. showing four flagella. Image: Harvey Marchant

A gamete of Pyramimonas sp. moving with a forward facing flagellum. Image: Harvey Marchant

A small flagellate moving with a long trailing flagellum. Image: Harvey Marchant

A small flagellate moving with a corkscrew motion. Image: Harvey Marchant

A dinoflagellate showing its transverse flagellum. Image: Harvey Marchant

Movement using cilia

Movement by cilia 1. Image: Harvey Marchant

Movement by cilia 2. The ciliate Strombidium sp. Image: Harvey Marchant

Movement by cilia 3. Image: Harvey Marchant

Happy ciliate. Image: Harvey Marchant

Movement by cellular deformation

Movement by deformation: amoeboid. Image: Harvey Marchant

Movement by deformation: Euglenoid. Image: Harvey Marchant

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Marine microbes - Agents of death

Marine microbes are subject to attack by a range of organisms with voracious appetites and a variety of feeding strategies, but many are also consumers.

Viruses - inject their DNA or RNA and take over the cell metabolism of the host resulting in viral multiplication and eventual cell rupture.

Protozoa -

1. Puncturers - Many dinoflagellates and ciliates pierce the prey cell with a peduncle, digesting and absorbing its contents.
2. Engulfers - cover prey cells with their cytoplasm and digest them. This is the prime strategy for amoeboid cells but some other cells, notably dinoflagellates, can project a veil of cytoplasm (pallium) externally through their cell wall to enclose prey. They can sometimes successfully engulf cells larger than themselves.
3. Ingesters - feeding currents created by cilia or flagella draw prey cells to the predator where they are captured and ingested e.g. ciliates and choanoflagellates.

Mesoplankton

1. Biters - copepods
2. Swallowers - salps
3. Crushers - krill

Microbes have evolved various strategies for avoiding or resisting these modes of attack, giving them a competitive advantage.

[based on a talk by V. Smetacek]

Ciliates feeding

Click on the images below to view the QuickTime movie

A ciliate draws prey cells to it. Image: Harvey Marchant

Ciliate attempts to catch particles. Image: Harvey Marchant

Ciliate draws particles toward it. Image: Harvey Marchant

Ciliate feeding inside a dead diatom frustule. Image: Harvey Marchant

A ciliate feeding on the surface of mucilage from Phaeocystis antarctica. Image: Harvey Marchant

Ciliate gullet. Image: Harvey Marchant

A ciliate beats its cilia to draw in food particles. Image: Harvey Marchant

A tintinnid ciliate beats its cilia to draw in food particles. Image: Harvey Marchant

A choanoflagellate draws particles onto its feeding basket by beating its flagellum. Image: Harvey Marchant

Dinoflagellate containing ingested diatoms. Image: Harvey Marchant

Two protoazoa, Anisonema sp. containing ingested diatoms. Image: Harvey Marchant

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Research Staff

Prof Harvey Marchant

Biology Program Leader

Harvey driving his desk

Dr Simon Wright

Senior Research Scientist

Simon driving the HPLC

Dr Andrew Davidson

Research Scientist

Andrew driving the microscope

Ms Fiona Scott

Fiona about to drive a Hagglunds oversnow vehicle

Mr Rick van den Enden

Rick culturing

Dr Paul Thomson

Paul driving the flow cytometer

Ms Karen Westwood

Karen isolating cells for culturing in a coldroom

Mr Andrew McEldowney

Andrew driving the scanning electron microscope

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Links

Biological

The Tree of Life
The Plankton Net
Protist Image Database, University of Montreal
Checklist of phytoplankton in the Skagerrak-Kattegat (including heterotrophic protists)
Protistan links
Bacteria and protists links
Bacteria
Ciliates
Invertebrates
Other marine links
Photosynthesis
Phytoplankton image gallery
Kingdom Protista web links
Emiliania huxleyi homepage

Laboratory

Barcelona Workshop 2001 (Pigments as a tool to estimate the biomass of different phytoplankton groups).
Flow Cytometry Links Page
Flow Cytometry publications
Chromatography Links
LC-GC Magazine
HPLC troubleshooting
HPLC resources
Chromatography Related Web Sites
Links for Chemists

Related organisations

SCOR (Scientific Council for Oceanic Research)

SCAR (Scientific Council for Antarctic Research)

NIPR (National Institute of Polar Research, Japan)

BAS (British Antarctic Survey)

AMSA (Australian Marine Sciences Association)

BPS (British Phycological Society)

Acronyms

Antarctic Division

SCAR

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For more information, email: bio@aad.gov.au

See more information on the Australian Antarctic Division's Biology program

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