Microbial Oceanography

Lecture 6: 6/5/2014

Many thanks to Drs. Carlson, Chadhain, and Ortmann for many of the slides

What is Microbial Oceanography (Ecology)

• Study of organisms too small to be seen with the unaided eye

• Use a variety of different technologies to see what microbes are doing

Members of the Microbial World

• Prokaryotic cells: lack a true membrane-bound nucleus – Bacteria and Archaea

• Eukaryotic cells: have a membrane-enclosed nucleus– More complex morphologically and generally

larger than prokaryotes• Viruses

How do we determine the identity of prokaryotes if they look so similar?

• Culturing studies in ‘traditional’ microbiology– Look at cell shape and

morphology– Look at colony shape and

morphology– Determine metabolic potential

• Molecular biology tools applied to microbiology– Determine the relatedness of

gene sequences – Measure change in genes and

gene order in genomes

http://www.sci.port.ac.uk/ec/_images/CLSMpartner5a.jpg

Culturing has been a major tool for microbiologists for years

Benefits of culturing• Isolate of a single

species/strain in lab• Determine metabolic

potential (test different substrates)

• Can carry out range of experiments

• Can isolate viruses which infect isolate

• More easily manipulate or determine genetics

Issues with culturing• May not represent

dominant/common species in environment

• May grow/behave differently under lab conditions compared to environment

• Experiments may be biased due to adaptations to the lab

• Viruses may not be ecologically relevant to the isolate

• Genetics may not be representative of those in environment

Why can culturing be so unsuccessful?

• Lab conditions do not reflect environmental conditions– High nutrient media– High/low temperatures– Different light conditions– Monocultures– Specific trace nutrients

missing

• Microbial ‘weeds’ are a major problem– Fast growing– dominate/overtake on

plates or in liquid media– Consistently end up with

these in culture– May not be dominant in

the field, or significant

SAR 11, dominated clone libraries but not in any cultures (Rappé et al 2002)

• SAR11 (Sargasso Sea) sequences ~26% of total 16S rRNA gene clones

• Not seen in any cultures• Isolated (eventually) using

a low nutrient dilution to extinction approach

• VERY slow growing– Weeks to reach high

density

What do living organisms need?• Define a group by how it “solves” some basic

problems:– Source of energy (for making ATP)– Source of electrons (NADP(H), reducing power)– Obtaining carbon

Heterotrophic Prokaryotes are not created equal

• By definition, use organic C for energy source• Particulate Organic Carbon (POC), by definition organic C > 0.7 µm

– Can be ‘worked’ on by microbes– Major source of export (detrital)

• Dissolved Organic Carbon (DOC) (< 0.7 µm) can be:– Labile, available to microbes. Lasts hours to days, simple or complex

molecules– Semilabile, mostly exported, lasts months to years, composition not

known– Refractory, not available. Can last centuries (e.g. CDOM), unknown

composition– Microbes convert DOC from mostly labile to refractory– Photodegradation and other abiotic processes also involved

Structure of Marine Ecosystems, Steele 1974

•Large phytoplankton at the base of the food chain•Lost energy dissipates as heat•Lost organic matter recycled by groups of ‘decomposer’ organisms

Effect of the size of primary producer on the biomass at higher trophic levels

Changing Paradigms, Classical view

• Classical view The Microbial Loop

• Pomeroy 1974– Observations made prior

to advancement of methods

The Microbial Loop (Azam et al. 1983)

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

• 60-75% of 1º prod is consumed by organisms <200 µm

DOM production and removal mechanisms

• In the open ocean, DOM production is ultimately limited by the level of primary production within a system

• Stressed phytoplankton release LOTS of DOM– Nutrient stressed

Where does the PP go?

• Phytoplankton release 10-50% of PP as DOM• Almost all is returned to atmosphere by

heterotrophic microbes

Bacteria in the Surface Ocean (≤ 200 m)Autotrophs

Heterotrophs

2.9 x 1027 cells

3.6 x 1028 cells(0.36 Pg C)

Annual Production

~10 Pg C

~7.5 Pg C3.6 x 1028 cells

Annual Production

Annual Net MarinePrimary Production

~50 Pg C

Autotrophic BP(~20% of PP)

Heterotrophic BP(~15% of PP)

Bacteria are major producers and consumers of organic matter, thereby shaping the composition and concentration of DOM in the ocean.

Microbial Loop: Link or Sink for Carbon?

• Link = C passed to higher trophic levels– High growth efficiencies

• Sink = C not passed to higher trophic levels, C is respired – Low bacterial growth efficiencies

BCD and BGE: del Giorgio and Cole 1998

• BCD = “bacterial” carbon demand– Total amount of carbon used by the “bacterial”

fraction– BCD = Bacterial Prod+Bacterial Respiration (C used

for production plus C released by respiration)• BGE = “bacterial” growth efficiency

– Efficiency by which bacteria convert organic substrate into biomass

– BGE=BP/BCD (fraction of total C used for production)

Why are microbes a sink for Carbon?

• Low BGE– 15% in oceans– 35% in estuaries

• Lower than lab rat bacteria– Why?– Amount and quality of

organic C

Grazer Impact on Bacteria

• Most bacteria eaten by small flagellates (<5µm)

• Protists most important, especially 2-5 µm heterotrophic nanoflagellates (HNF)

• Bacterial production and protistan grazing loss in balance– Protists can grow as fast as bacteria

Viruses• “Agents of microbial mortality” thus play a role in

cycling of organic matter in the ocean• Can increase BP…increase remineralization• Can reduce the amount of C to higher trophic levels, “The

Viral Shunt”• Abundant: ~108 per ml in productive coastal waters;

numbers correlate with system productivity, bacterial numbers and chl a; 107 per ml in surface ocean

Diatom

Bacteria

Virus

Viruses are not pure evil

From Breitbart 2012

Bottom Line• May contribute to microbial mortality on scales similar to

grazing by zooplankton; but interactions between virus/host, virus/host/grazing are complicated.

• Conversion of POC (cells) to DOC may influence removal of C from surface ocean

• Quantifying microbial mortality due to viruses is difficult

• The real bottom line:– Just because they’re small, we can’t ignore them!

Oceanic Carbon Cycle

• Why is C an important element?– Cellular level: essential for macromolecular

synthesis– Trophodynamics: important in energy flow

between trophic levels– Biogeochemistry: stoichiometry demands ties C to

other important nutrient cycles (N,P,Si)– Greenhouse properties

The ability of the ocean to take up atmospheric CO2 is controlled by 2 major pumps

• Solubility pump - solubility of CO2

• Biological Pump – photosynthesis and respiration

DOC contours along meridonial transects in the N. Atlantic

% OC as biochemicals

What is refractory DOC?

Major DOC classes (Hansell 2013)

Hansell 2013

Why is the Nitrogen cycle important?

• N often limits production in many oceanic regimes• N can be used to follow C fluxes. Important even

when N is not limiting• N20, a greenhouse gas, is produced during

nitrification and denitrification• Uses of N

– Biosynthesis (proteins and nucleic acids)– Respiration (electron acceptor)– Energy source (chemolithotrophy)

N-cycle difficult to study• N has numerous oxidation

states– Gases, inorganic and

organic forms• Results in various forms of N

species• Many reactions which alter its

form• No convenient radioactive

isotope– 13N: radioactive, but half-

life of minutes – 14N: stable, most abundant – 15N: stable, 0.366 % of total

N

Nitrogen Fixation (N2(gas) to Organic N)

• N-fixers (diazotrophs)– Only prokaryotes do it in the

ocean• In aquatic systems, mainly

cyanobacteria• Marine diazotrophs

– Filamentous nonheterocystous cyanos, Trichodesmium

– Symbiotic cyanos (Richelia, Calothrix) in diatoms (Rhizosolenia)

– Single/unicellular cyanos, Crocosphaera & Cyanthecae relatives

N-fixation cont.

• Catalyzed by nitrogenase– Found only in a few species of prokaryotes– High Fe quota

• Energetically expensive– Lots of ATP expenditure

• Controlled by turbulence, grazing, light, nutrient and trace element availability

Adaptations to low ambient [Fe]

• Saito et al. 2010– Organism of study:

Crocosphaera watsonii• Photosynthesize during

the day, fix N at night– Temporal separation

• ‘Hot bunking’ technique– Fe used during day then

switched over for N-fixation at night

– ‘Recycle’ available Fe

Assimilatory and Dissimilatory Nitrate Reduction

• N Assimilation– Uptake of NO3

- and/or NH4 incorporation into biomass

• N Dissimilation– Release/excretion of NH4 by microbes and other

organisms

Nitrification

• Two steps– Ammonium to nitrite, ammonium is oxidized, i.e.

it is the e- donor– Nitrite to nitrate, nitrite oxidized further

• Carried out by two types of bacteria– Nitrosomonas and Nitrosococcus (step 1)– Nitrobacter and Nitrococcus (step 2)

Denitrification

• Two steps– Nitrate nitrite– Nitrite N2 or N2O

• Why is it important?– Loss of N from environments– Source of N2O (greenhouse gas)

Anammox

• Anaerobic Ammonium Oxidation• Anaerobic oxidation of NH4

+ using NO2- as the

electron acceptor• Produces N-gas• Anammox bacteria only recently discovered

– Planctomyces, a genus in the phylum Planctomycetes– 1st found in sewage treatment plant in Holland

• Accounts for “missing” NH4+ in N budgets

Kuypers et al. 2005

• Study site: Oxygen minimum zone (OMZ) in Benguela upwelling system

• Found that anammox bacteria are responsible for huge losses of fixed N to N2 gas– 1st time identified and directly linked anammox

bacteria to removal of fixed inorganic N in open ocean setting!

– Stimulated research to find anammox in other OMZ systems