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Higher organized structures facilitating metabolic and

genetic processes require energy for synthesis and
assembly (i.e. they are antientropic)

So how did it start? Physical energy sources prevailed in a „sterile“ prebiotic

earth (lightning, heat, UV radiation)

De novo synthesis of organic compounds?

Was there enough energy on the early earth?

(kilojoules x m-2 x year-1)
Solar radiation* 24000
Impact shock waves 200
Radioactivity 117 Proc. Royal Society London A, 116:197-211.
Electrical discharges 2.9
Volcanoes 5.4
Mg/ZnCO3
Chemical energy Important for the origin of life,
yet to be estimated ?

*Energy of UV radiation in wavelength range absorbed ?

by organic compounds thus able to initiate photo-
chemical reactions. All values based on estimates!
Miller AL, Urey, HC. 1959. Science 130:245-251. UV light
Chyba CF, Sagan C. 1992. Nature 355:125-131.

Paper chromatography,
Miller-Urey 2D, Ninhydrin spraying,
ID via Rf values
experiments
–
origins
of life?

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Parker, E.T et
al. 2011.
PNAS
108:5526-
5531.
Re-analyzed
Stan Millers
original
samples by
HPLC-UVFD
after
OPA/NAC
derivatization.
A. Original
OPA/NAC=
sample o-phthal-
B. Standards dialdehyde/N-
C. Blank acetyl-L-cysteine

Parker, E.T et al. 2011. PNAS 108:5526-5531. Miller-Urey Experiments – origins of life?
Re-analyzed Stan Millers original samples after derivatization, shown are the
molar ratios (relative to glycine = 1) of amino acids and amines detected

Gas phase
CO
CO2

Aqueous phase
Amino acids
Aldehydes
HCN

Demonstrated the
abiotic formation
of simple
Biomolecules!

Miller-Urey Experiments
But how to get from an “organic soup” to cellular life?
• Simulated potential prebiotic conditions
• Polymerization of monomers requires input
• Generated several organic molecules, including of energy and removal of water!
amino acids and nucleic acid bases – precursors
for polymer formation! • Catalytic (surface catalysis) function of
suspended clay/mineral particles?
• BUT
Open waters of primordial earth are not • A primitive catalytic reaction centre?
a very likely site for early development stages to
more complex organic structures because • RNA world/iron sulfur world?
water participates in hydrolytic
cleavage reactions!

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CO2 CH3-SH
CH3CO2H
FeS and similar minerals offer 3 dimensional
CH3 CH3
HO-C=O
compartments of diffusion limited hydrophobic -CO CH3S-CO-CH3
HCO2H H2S, CO
surfaces able to bind and concentrate organic Fe, Co, Ni
OC- Mineral -CO-CO-CH3
compounds! cluster

FeS and NiS catalysts lining these cavities are CO

HO2C-CO-CH3
able to form C-C bonds! Alanine
NH3

Peptide Huber C, Wächtershäuser, G. 1998. Science. 281:670-672.

library Huber C, Wächtershäuser, G. 1997. Science. 276:245-247.

A potential model for early metabolism –

FeS can react under hydrothermal conditions with H 2S primordial hydrogenase and ATPase…
to form pyrite (FeS2) and H2 in an exothermic reaction
FeS + H2S → FeS2 + H2 -38.5 kJ/mol FeS + H2S → FeS2 + H2
In the presence of UV hydrogen might be formed by
2Fe(II) + 2H+ → H2 + 2Fe(III) 2H+
Hydrogen might then serve as reducing power for biosynthetic
reactions!
In an early anaerobic world (prior to oxygenic PS!) this could
2e-
have been coupled to S0 as electron acceptor to generate energy 2H2O
ATP
via membranes (involving primordial hydrogenase and ATPase)
2H2O + S0 → H2S + 2OH-
2H2O + S0 → H2S + 2OH-! (with 2OH- + 2H+ → 2H2O)

An exothermic reaction such as

FeS + H2S → FeS2 + H2 -38.5 kJ/mol
can in principle drive the autocatalytic
assembly of complex
organic molecules!

Hence, chemistry of the RNA world

probably took place in such
FeS cavities… The submarine setting – scenario for emergence of early life?

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Prebiotic energy flow…

Primary energy Photochemical energy Geochemical
source energy
• Modern enzymes still rely on FeS like moieties
UV light Electric Heat Heat Mineral
discharge reducing (mineral heritage?)
power
Primary products Neutral Atmos. Reducing • Encapsulation required (as before taking place
HCHO, HNO Atmos. H2, H2S,
HCN, RCHO CH4, CO in diffusion limited pores/cavities)
+ NH3
Hydrosphere • Conditions prevailing on the ancient seafloor
Primary Sugars, Amino acids, Acetic acid,
products NO2- →NH3 nucleobases methane-
are likely to support the formation of fatty acid
thiol based membranes to engulf biomolecules…
Secondary Amino acids,
CytP450
products thioesters, Polypeptides,
N-hetero- phospho- Acetic acid
cycles, anhydrides thioester
model
protocells

•Buildup of lipids/synthesis of
phospholipid membrane vesicles

• Enclosure of biochemical and

replication machinery

•Montmorillonite clay vesicles?

Formation of artificial vesicles from fatty acids and

RNA in the presence of Montmorillonit (“clay like mineral”)

Stage set for self-replicating systems

RNA world theory

First self-replicating systems RNA-based

RNA can bind small molecules (e.g., ATP,

other nucleotides)

RNA has catalytic activity; may have

catalyzed its own synthesis

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An alternative scenario?
Mulkidjanian, A.Y. et al. 2012. PNAS. 109:E821-E830.
Anoxic, CO2-
Early “protocells” dominated
Leaky “primitive” membranes primordial
No sophisticated membrane bound pumps atmosphere,
Hence - equilibrated ion concentration basins at
terrestrial
(inside/outside)
geothermal fields,
ponds of
but condensed/cooled
Ion Sea Primordial ocean Cytoplasm geothermal vapor
[mol/L] water [anoxic] enriched in metal
Na+ 0.4 >0.4 0.01 sulfides, K+, Zn2+,
K+ 0.01 ∼0.01 0.1 and PO43−, would
Fe 10−8 10−5 10−3 to 10−4 resemble cytosol
concentrations…
Zn2+ 10−9 <10−12 10−3 to 10−4
PO43− 10−6-10−9 <10−5 ∼10−2 (mostly bound)

Advanced microbial metabolism and

Pre-Prac ecophysiology - First Practical

Microbial activity determination

(FDA hydrolysis/TTC reduction)

Number of viable aerobic

This can help to assess the
heterotrophs and total
status of environmental
microbial metabolic activity are
materials such as soil,
useful indicators of organic
sediments, compost etc. as
matter turnover in natural
regards the overall activity
habitats as this takes place
of microbes present.
mainly via microbial
Relevant indicator for soil
decomposition processes.
fertility etc.

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Parameters that can be determined to estimate

As the metabolic activity of microbial metabolic activity in soil, compost, litter
droppings and other materials:
individual microbes in
environmental materials such Gas consumption/formation

as soil or compost is not easily Consumption of O2 due to microbial

respiration
determined, quicker and easier
methods have been developed Production of CO2 from microbial
decomposition of organic matter (refined
to determine the overall approach employs spiking with isotope
labeled substrates to produce 14CO2 or 13CO2)
microbial activity therein.
Release of CH4, N2 etc.

Parameters that can be determined to estimate

microbial metabolic activity in soil, compost, litter
and other materials: What is MAR?

Heat formation Microaudiography

Release of heat via actively metabolizing
microbes (measured by microcalorimetry) Enables in situ detection of metabolic

Other employable parameters activity using labeled substrates such as

ATP concentration 14C-glucose, 14C-actetate, 14CO etc.
2
Microbial biomass
Total and viable counts
MAR-FISH
…

What is MAR-FISH?

A combination
What is FISH? of
Microaudiography
Fluorescence in situ hybridization and
Fluorescence in situ hybridization
Enables in situ phylogenetic affiliation of
individual cells using labeled probes Enables in situ detection of metabolic
activity and phylogenetic affiliation at single
cell level!

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Scheme for MAR-FISH Ideally, a technique suitable for

easily assessing total microbial activity
should be
•Nonspecific
•Sensitive
•Fairly quick (avoiding long incubation times)
•Fairly simple (simple lab equipment)
•Not too expensive
•…

3-Oxo-3H-spiro[isobenzofuran-1,9'-
xanthene]-3',6'-diyldiacetate (FDA)
First experiment:
FDA (fluorescein diacetate) is hydrolyzed by
a number of different hydrolytic enzymes, 1 mol FDA + 2 mol H2O
such as proteases, lipases, and esterases. → 1 mol F + 2 mol Acetate

2 CH3CO2H
2 H 2O
The hydrolysis of fluorescein diacetate:

3',6'-dihydroxy-3H-spiro[isobenzofuran-
1,9'-xanthen]-3-one

Procedure:
1 Mol fluorescein and 2 mol acetate are formed via
hydrolytic activity from 1 mol FDA •Groups weigh 1 g of sample A and B into each of
three 100 ml Erlenmeyer flasks (i.e. 3 flasks
Acetate can be quantified via an appropriate containing sample A and three flasks containing
analytical method (HPLC, coupled enzyme assay, sample B).
GC or similar) •Add 500 µl of a stock solution of FDA (2 mg/1 ml
acetone) to two flasks containing sample A and to
but two flasks containing sample B.
•Add 500 µl of acetone to one flask containing
the resulting fluorescein can be more easily sample A and one flask containing sample B.
quantified by fluorometry or spectrophotometry. These two Erlenmeyer flasks will serve as controls
to verify the formation of color not derived from the
hydrolysis of FDA!

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Procedure: Procedure:
Flasks required per group for FDA hydrolysis assay
•Add 500 µl of a stock solution of FDA (2 mg/1 ml
Flask Sample Sample FDA Ace- Phosphate Incu-
acetone) to one additional empty flask containing no
A B stock tone buffer bation
soil. This will serve as the blank. (2 mg
•Add 50 ml of 60 mM phosphate buffer (pH 7.6) to per ml)
each of the above flasks (7 per group) and swirl to 1 1g - 500 µl - 50 ml Shaker
mix the contents. 2 1g - 500 µl - 50 ml Static
•Incubate two of the flasks, one containing buffer,
3 1g - - 500 µl 50 ml Shaker
FDA and sample A and one containing buffer, FDA
and sample B statically at ambient temperature in 4 - 1g 500 µl - 50 ml Shaker
the dark for 1.5 h. All the other flasks are incubated 5 - 1g 500 µl - 50 ml Static
at 30oC for 1.5 h in the dark in a water bath shaker
6 - 1g - 500 µl 50 ml Shaker
set at 100 rpm.
7 - - 500 µl - 50 ml Shaker

Procedure: Procedure:
•Add 2 ml of acetone to all flasks to stop reaction.
•Filter the contents through filter paper. •Calculate the group and the class mean values and
•Take 1 ml of the filtrate from each of the samples the corresponding standard deviation and standard
and dilute to 2- and 5 fold respectively with error for the fluorescein standards using the data
phosphate buffer. from all groups in the practical.
•Take absorbance readings at 490 nm and Zero •Establish graphically a standard curve based on the
spectrophotometer using only phosphate buffer class mean values (add error bars) obtained for the
before measuring samples! fluorescein standards.
•Construct a standard curve with 0.00, 0.5, 1.00, •Calculate the concentration of fluorescein released
1.25, 1.75, 2.5, 3.75, and 5.0 mg/L fluorescein in 50 in your 7 flasks by reference to the standard curve
ml of phosphate buffer plus 2.5 ml acetone to obtained after reading the sample absorbance
account for sample matrix. Zero spectrophotometer values. Take into account the dilution factor!
using the 0 mg/L fluorescein standard before
measuring these fluorescein standard samples!

Procedure: Procedure:

•Report values for your group as µg of fluorescein •IMPORTANT!

released per gram of sample (dry weight) per hour Establish for each sample material the dry weight by
incubation (i.e. µg x g-1 x h-1). drying 1 g of sample A and B at 105°C for 48 hours
•In addition, calculate the mean value and standard on tin foil (determine the weight of the tin foil prior to
error and standard deviation for samples A and B adding the sample material!). Reweigh the sample
using the data generated by all groups in the class material after the drying process.
and compare to your group results.

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Useful reference!

why controls are important!

First prac (the FDA experiment) - full prac report!

1. Abstract (max 250 words)

2. Introduction (not more than 2 pages)
3. Material and methods (you can make reference
to the prac manual but must state all steps were
you differed from the procedures specified in the
manual)
4. Results (make sure you include the class data!!!)
Hydrolysis of FDA by media components. Tris-HCl and
sodium phosphate concentrations are expressed in moles 5. Discussion
per litre. Tryptone, peptone and yeast extract are 6. References (original publications, not web
expressed in percent concentrations (w/v). Fluorescence sources!)
intensity is the proportion of FDA that has been converted Reference Style as for
to fluorescein. Maximum fluorescence is based on an Applied and Environmental Microbiology
equal volume of fluorescein in water. Error bars represent (http://aem.asm.org/site/misc/2015DecemberAEMITA.pdf)
1 S.D. above and below the average of the replicates.

Second experiment:
2,3,5-Triphenyltetrazolium chloride is
reduced by a range of microbial
dehydrogenases/reductases (and by direct
Generally:
reaction with NAD(P)H+H+).
Provide raw data and calculations in an appendix!
The enzymic reduction of TTC:

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2,3,5-Triphenyl-2H-tetrazol-3- Procedure:
ium chloride • Take nine vials and to each of them add the
following:
• 6 g (wet weight) sample A
• 6 g (wet weight) sample B
• 6 g (wet weight) sample A + 0.2 g sorbic acid
(2Z,4E)-1,3,5-Triphenylformazan • 6 g (wet weight) sample B + 0.2 g sorbic acid
• 6 g (wet weight) sample A + 0.2 g Glucose
• 6 g (wet weight) sample B + 0.2 g Glucose
• 6 g (wet weight) sample A + 0.2 g cresol
• 6 g (wet weight) sample B + 0.2 g cresol
• No sample (blank)
Red, insoluble!

Procedure:

•To each of the nine vials add 1 ml of 3% TTC stock

solution + 2.5 ml deionised water. Prac data analysis
•Stir and seal with caps – a small amount of water
Sample mean
must appear on the soil surface. Standard deviation
Standard error of the mean
•Incubate vials for one week in the dark until next
prac.

Prac data analysis

Sample mean
𝑛𝑥
𝑥= 𝑛

x1 + x2 + x3 +…+ xn
=
𝑛

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Prac data analysis Prac data analysis

Sample mean Sample mean
FDA hydrolysis What does this mean?
Assume
Class mean = 500 µg x h-1 x g-1

Laboratory A. Laboratory B.
Hardly any scattering! Same mean but severe scattering!

Prac data analysis

Standard deviation
Level of data scatter or
deviation 𝑛 (𝑥−𝑥 ) 2
from the mean 𝜎 = 𝑛−1
indicates reliability
of data!
𝑥1−𝑥 2 + 𝑥2−𝑥 2 + … + (𝑥n−𝑥) 2
= n−1

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Laboratory A.
Prac data analysis 5 groups, values established = 520, 480, 475, 530, 495
Standard error of the mean Mean = 500

Range = 55 (Min 475, Max 530)

Median (50%, Q2) = 495

𝜎
𝜎𝑚 = 25% (Q1) = 479 75% (Q3) = 523

𝑛 Std. Dev. = 24.24 Std. Error = 10.84

(Kolmogorov-Smirnov = 0.195)

Laboratory B.
5 groups, values established = 850, 350, 150, 900, 250

Mean = 500

Range = 750 (Min 250, Max 900)

Median (50%, Q2) = 350

25% (Q1) = 225 75% (Q3) = 863

Std. Dev. = 350 Std. Error = 156.53

(Kolmogorov-Smirnov = 0.266)

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