Transcript Marine Microbes_Marine Ecology_2014

www.glogster.com
Marine Microbes: Who are they
and why should you care?
Marine Ecology 2014
Natalie Ortell
PhD Candidate
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.scienceray.com
www.microbewiki.kenyon.edu
www.nps.gov
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)
www.whataretheywaitingfor.com
• Evolution of life and biodiversity of the
ocean
– More abundant/diverse than any other
organism
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/begi
n/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,...)
Including:
Hydrothermal Vents
Open Ocean
Symbionts
Human Guts
Deep sea sediments
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?
Marine Viruses
“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.
Stereotypical phage
• 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
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.
2.
3.
4.
5.
6.
7.
Culture
Epifluorescence microscopy
DNA extraction
DGGE
qPCR
Sequencing
Cloning
Culture-dependent
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
www.bitesizebio.com
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 doublestranded 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?
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 photooxidation 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 Valdez
DWH
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 Nfixation
• 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
DGGE
16S Sequencing
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
Thaumarchaeota (ml-1)
Total prokaryotes (ml-1)
2x107
1.6x105
2x107
1.4x105
7
2x10
1.2x105
1x107
1x107
105
107
8.0x104
8x106
6.0x104
6x106
4.0x104
6
4x10
2.0x104
2x106
0
0
Surface
ST 1
Bottom
Surface
Bottom
ST 2
Surface
Bottom
ST 1
Surface
Bottom
ST 2
• amoA abundance follows a similar pattern as Thaumarchaeota (Wilcoxon p<0.05)
High NO3-
PC 2
High NH₄⁺
Principal components analysis (PCA) revealed
three distinct environments
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