Marine Microbes: Who are they and why should you care?

Marine Ecology 2014

Natalie Ortell

PhD Candidate

www.glogster.com

www.news.nationalgeographic.com

‘Microorganisms have shaped and defined Earth’s biosphere and have created

conditions that have allowed the evolution of macroorganisms and complex biological communities, including human societies’

• D.M Karl (2007)

What do you think a microbe is?How do microbes help your every

day life?

Marine microbes are found in all three domains of cellular life: Eukarya, Bacteria and

Archaea,

• Too small to be seen with the unaided eye• Bacteria —Purple Sulfur Bacteria• Archaea —Cenarchaeum symbiosum• Eukarya —Diatom

www.microbewiki.kenyon.edu

www.nps.gov

www.scienceray.com

Why do we study marine microorganisms?

• Their activities have an impact on the climate (greenhouse gases, Oxygen, Carbon Dioxide)

• They play a crucial role in the global turnover of the elements (C, N, Fe)

• Evolution of life and biodiversity of the ocean– More abundant/diverse than any other

organism

www.whataretheywaitingfor.com

www.naturalpatriot.org

Marine Microbes vary greatly in size

• The size of bacteria range from 0.2 – 2 micrometers (µm) where one µm equals one millionth of a meter and is so small that hundreds of bacteria can fit into a space the size of the period at the end of this sentence.

• http://learn.genetics.utah.edu/content/begin/cells/scale/

How does the biomass compare?

All the bacteria on the planet

50 million blue whales

1 Bacterium weighs = quadrillionth of a gram (that’s 15 zeros)All bacteria = one billion tons

Microbial Habitats

•Archaea and Bacteria are found wherever there is:

– Water– Energy source– C, N, P, S, etc.– Within physicochemical limits (°C, pH,

salt,...)

Open Ocean

Hydrothermal Vents

Human GutsSymbionts

Deep sea sediments

Including:

Major differentiating features between Bacteria, Archaea and Eukarya

Archaea may be phylogenetically more closely related to eukaryotes than to bacteria

How do Microbes eat?• Autotrophy: Take energy from the environment in the form

of sunlight or by chemical oxidation and use it to create energy-rich molecules such as carbohydrates

• Heterotrophy: take in autotrophs (in some form) as food to carry out functions necessary for life

Other ways to make a living• Mixotrophy:

microorganism that can use a mix of different sources of energy and Carbon– They can take advantage of

different environmental conditions

– Mixotrophs can be either eukaryotic or prokaryotic

• Chemosynthesis: is the production of organic material by energy from chemical reactions rather than light

Bacteria are the green plants of hydrothermal vents. Through a process known as chemosynthesis”

Autotrophy vs. heterotrophy

What would you be and why?

• If you were a microbe living in:

– The euphotic zone in the Mobile Bay– Open ocean euphotic zone– Deep sea oil seep– Sponge symbiont

• Think about nutrients/light/competitors etc.

Traditional Food Chain• Unidirectional transfer of energy. The role of

bacteria was simply to eat what rained out (i.e. detritivores)

The missing link: The Microbial Loop

• Salvage pathway in which bacterioplankton repackage and reincorporate DOC back into the aquatic food web

How many virus particles are in one mL of seawater?

Vs.

How many prokaryote cells are in one mL of seawater?

“Viruses are the most abundant life form in the oceans...and if stretched end to

end, would span farther than the nearest 60 galaxies." Curtis Suttle,

University of British Columbia.

• Virus: non-cellular biological entities composed of nucleic acid surrounded by a protein coat– Contain either RNA or DNA

• Size: < 0.02 µm, vs. bacteria <1 µm

Stereotypical phage

Marine Viruses

Microbial mortality due to viral infection

– ~ 10-50% of heterotrophic bacterial mortality in surface waters due to viruses

– Density dependent of the host population,

– Infection greatest in blooms: Emiliania huxleyii

– 50% of cells infected in decaying phase of bloom - can boost overall bacterial production

• High host specificity - high virus diversity

Two viral infection cycles

Marine Viruses

• Everything has a virus• Release of OM, essential elements for heterotrophs to reabsorb and metabolize

• Prevents the movement of food up the food chain

• ¼ of ocean primary production flows through the viral shunt

Viral Shunt: moves nutrients from microbes (photo/heterotrophs) into POM and DOM releasing amino acids & nucleic acids back in the food web

Discussion Question

What do you think are the greatest controls on the microbial loop? The viral shunt?

What else could viruses be controlling besides nutrients?

Do you think that Climate Change will impact the microbial loop? Viruses?

Challenge: How do we study the global impact of what we can’t see?

Molecular Techniques used to characterize microbial communities

1. Culture

2. Epifluorescence microscopy

3. DNA extraction

4. DGGE

5. qPCR

6. Sequencing

7. Cloning

Culture-dependent

www.bitesizebio.com

Historically culturing has been the only way to study microbes

Traditionally use pure cultures to learn about microbes

Culture-based methods are used for isolation and identification of microbes.

Limited: Only 1% of cells are culturable

Epifluorescence microscopy used to enumerate total prokaryotes and viruses

Prokaryote cells

Virus particles

Cyanobacteria cells

DNA Extraction: A method to isolate microbial DNA from

samples for subsequent molecular analysis

Gel electrophoresis to check DNA Extractions and PCR amplification

DGGE---Denaturing gradient gel electrophoresis

• The separation of double-stranded DNA fragments that are identical in length, but differ in sequence.

• Fingerprinting technique that estimates:– The composition of microbial

communities• Species richness

• Which has the greatest species richness?

Bacteria

Archaea

qPCR: quantitative polymerase chain

reaction

• Determine copy number of specific genes

• Simultaneously amplify and quantify target genes •SYBR green is a dye that binds to dsDNA

• dsDNA increases with PCR amplification

• SYBR green in 1000x more fluorescent bound to dsDNA

Now that we know about the role microbes play in the marine food web: what

happens during an oil spill?

blogs.forbes.com

What is Oil? Why do microbes care?

• From ancient photosynthetic material– Dead organisms buried

• Energy source for engines and microbes– Carbon rich—hydrocarbons

• Enzymes allow microbes to “combust” hydrocarbons at lower temps

Physical processes do not destroy oil

• Evaporation—volatile hydrocarbons evaporate quickly

• Dissolution—some components dissolve in water

• Dispersion—oil is broken up into small droplets and spread through the water column

• Photo-oxidation—sunlight breaks rings of PAHs (polycyclic aromatic hydrocarbons)

• Only living organisms can “destroy” oil

Why should we worry about oil spills? Won’t microbes always clean them up?

• Natural seeps on GoM seafloor = established community capable of utilizing all the different oil compounds

• Background bacteria proliferate in the presence of surface oil

• Lag time for population to respond to oil

• Oil may outpace the microbes ability

Limiting factors of microbial degradation

• Physical/chemical nature of the oil—complexity of the hydrocarbon chains

• Nutrient availability—N and P

• Oxygen availability—respiration

• Water temperature—higher temperature means higher metabolisms

• Other microbes—competition, predation, viruses

Let’s revisit the microbial loop

1. Large input of C rich-nutrient limited food increase in oil-loving bacteria

2. Change in prey alters predator speciation/activity in the microbial food web and the traditional food web

3. Move C and energy to higher trophic levels

Dispersants affect oil biodegradation

• Dispersants like dish soap break oil into tiny droplets mixing surface oil slicks into the water column– Should increase surface area for microbes to colonize but

the chemicals may have adverse affects

• Removes the oil from evaporative and photo-oxidation processes

• Moves the oil into a cooler, higher pressure area lowering microbial metabolisms

Ortmann et al 2012: Dispersed Oil Disrupts Microbial Pathways in Pelagic Food Webs

+Glucose

+Oil

Increase in ciliates = transfer of C to

higher trophic levels

+Dispersant

Increase in heterotrophs and

inhibition of ciliates

Do you think there would be a different response of the microbial community between the Exxon Valdez and the Deep Water Horizon Oil spills?

Exxon ValdezDWH

Some issues with our current knowledge…

• Microbial communities are dynamic, some turning over in a day

• Microbial communities are redundant, many species perform similar functions like N-fixation

• Lack of baseline data to compare or determine if a shift in community structure is permanent

My Dissertation: Archaea Are Awesome!!!

Abundance and diversity of Archaea in the northern Gulf of Mexico and interactions with hypoxia

1. A survey of temporal and spatial dynamics is necessary due to the lack of current knowledge concerning archaeal communities in the nGOM.

2. Studies examining the contribution of Archaea to the microbial community or ecosystem functionality have focused on cold seeps and the open ocean. How does the surface archaeal community (abundance and diversity) differ along an estuarine-shelf gradient?

3. Archaeal communities and metabolic requirements have been investigated in OMZs and upwelling systems, however, there is a lack of data from seasonally occurring coastal hypoxic systems.

Methods I use to answer my questions

16S SequencingDGGE

What I can learn?

How groups of marine Archaea change over time and respond to environmental variability including seasonal changes and extreme

environments. And ultimately how those changes impact nutrient

cycles.

Increasing global marine hypoxia

Eutrophic and Hypoxic Areas Areas of Concerns Documented Hypoxic Areas Systems in Recovery

Very little is known about Archaea and hypoxia

• Most studies have focused on zooplankton, ciliates and bacteria.

• One study in the East China Sea looked at distribution of archaeal community in hypoxic area (Liu et al. 2011).– Only one season sampled – No significant effect of dissolved O₂– Significant relationship between salinity and archaeal

community structure

• Members of Thaumarchaeota participate in the rate-limiting first step of nitrification

Chapter 3 Objectives

• To quantify the Thaumarchaeota community in oxic and hypoxic waters

• To determine the potential for ammonia oxidation, using amoA gene abundance as a proxy, in oxic and hypoxic waters.

Thaumarchaeota and amoA abundance was elevated offshore and with depth

Total prokaryotes (ml-1)

0

2x106

4x106

6x106

8x106

107

1x107

1x107

2x107

2x107

2x107

ST 1 ST 2 ST 1 ST 2 Surface Bottom Surface Bottom

Thaumarchaeota (ml-1)

0

2.0x104

4.0x104

6.0x104

8.0x104

105

1.2x105

1.4x105

1.6x105

Surface Bottom Surface Bottom

• amoA abundance follows a similar pattern as Thaumarchaeota (Wilcoxon p<0.05)

Principal components analysis (PCA) revealed three distinct environments

H

igh

NO

3-

PC

2H

igh

NH

₄⁺

High DO PC 1 Low DO

77% of the variability explained by PC1 and PC2.

Conclusions

• Thaumarchaeota are important members of coastal hypoxic microbial communities

• The absence of amoA genes may be due the existence of two populations of Thaumarchaeota in coastal Mississippi – Current primer set may not target the

ammonia oxidizers in our system– Alternate pathways or metabolisms