This document summarizes research on the psychrophilic degradation of long chain alkanes by microorganisms. Key findings include: 1) Pseudomonas frederiksbergensis and Rhodococcus erythropolis were able to degrade n-alkanes from C10-C22 at 4-20°C with optimal degradation by P. frederiksbergensis occurring at 15°C and 7.0 pH. 2) P. frederiksbergensis degraded alkanes via terminal oxidation and detection of metabolites, starting with hydroxylation of alkanes to fatty acids. 3) Genes encoding alkane hydroxylase and alcohol dehydrogenase from P. frederik

1. Technical biochemistry
TUHTechnical University Hamburg-Harburg
PSYCHROPHILIC DEGRADATIONPSYCHROPHILIC DEGRADATION
OF LONG CHAIN ALKANESOF LONG CHAIN ALKANES

2. OutOutlines of the researchlines of the research
 IntroductionIntroduction
 Objective of the researchObjective of the research
 Results and discussionResults and discussion
IntroductionIntroduction

3. Importance of the researchImportance of the research
DesertDesert
SeasSeas
SoilSoil
LakesLakes Problem of oil spillProblem of oil spill??
Petroleum
 Aromatics
 Resins
 Asphaltenes
 AlkanesAlkanes
What are alkanes?

4. Major components of crude oil (20 –Major components of crude oil (20 – 50 %)50 %)
Alkanes
CnH2n+2
 Biodegradation of alkanesBiodegradation of alkanes
 Biodegradation in natureBiodegradation in nature
 Microbial activityMicrobial activity
 LowerLower MWMW
 HigherHigher MWMW

5. 0 - 20 °C Cold polar seasPsychrophiles
Extremophiles microorganisms
and their habitats
Growth conditionsExtremophiles
Thermophiles 50 - 110 °C Hot springs
Acidophiles pH < 2 Acidic fields
Alkaliphiles pH > 9 Alkaline soils
Halophiles 3 – 20 % salt Highly saline lakes
Habitats
 90 % of marine water masses are colder than 4°C.
 Two-third of sea water covering 70 % of the earth is cold.
Psychrophiles?!

6. CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH33
Intermediary metabolismIntermediary metabolism
ßß-Oxidation-Oxidation
CHCH33-(CH-(CH22))nn-CH-CH22CHOCHO
AldehydeAldehyde
NAD+
NADH
alcDHalcDH
NAD+
NADH H2O
CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH22COOHCOOH
Fatty acidFatty acid
aldDHaldDH
alkBalkB
CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH22OHOH
Primary alcoholPrimary alcohol
Alkanes degradation pathwayAlkanes degradation pathway

7. Mode of alkanes uptakeMode of alkanes uptake
AdaptationAdaptationEmulsificationEmulsification ModulationModulation
Diffusion &
active
transport
Emulsification
Direct contact (Alkane
droplets)
Solubility
biosurfactantsalkanes
OutsideOutside MembraneMembrane InsideInside
Biosurfactants biosynthesis
CnH2n+2
Intermediary metabolism
Emulsification
Intermediary metabolism
Biosurfactants biosynthesis

8. Genes encoding those enzymes could
be under the same repressor control
or
A part of the same operon
Retledge, 1988
Mode of alkanes uptakeMode of alkanes uptake
biosurfactantsalkanes
OUTSIDEOUTSIDE MembraneMembrane INSIDEINSIDE
Biosurfactants biosynthesis
CnH2n+2
Diffusion &
active
transport
Emulsification
Intermediary metabolism
Direct contact (Alkane
droplets)
Solubility
Biosurfactants biosynthesis
Emulsification
Intermediary metabolism

9. Mode of contaminant uptakeMode of contaminant uptake
Diffusion ( cell wall)
Diffusion (cell wall) Diffusion (Cytoplasmic membrane)
Multi-Enzymes ComplexesOrganic Contamination
CnH2n+2
CnH2n+2CnH2n+2

10. Results and discussionResults and discussion

11. Source of the mixed cultureSource of the mixed culture

12. Morphological & biological characteristicsMorphological & biological characteristics
PPseudomonasseudomonas frederiksbergensisfrederiksbergensis
RRhodococcushodococcus erythropoliserythropolis
2700 x
 16S rDNA
 Fatty acids analysis
RR.. erythropoliserythropolis (+)(+)
PP.. frederiksbergensisfrederiksbergensis (-)(-)
2700 x

13. Growth and biodegradation of eicosane byGrowth and biodegradation of eicosane by P.P.
frederiksbergensis, R. erythropolisfrederiksbergensis, R. erythropolis and mixedand mixed
culture at 4°Cculture at 4°C
0.00E+00
1.50E+07
3.00E+07
4.50E+07
6.00E+07
7.50E+07
9.00E+07
0 4 8 12 16 20 24
0
200
400
600
800
1000
R. erythropolis P. frederiksbergensis Mixed culture
eicosane conc. (µM) eicosane conc. (µM) eicosane conc. (µM)
Growth(cell/ml)
Eicosaneconc.(µM)
Time (day)
4°C

14. Gas Chromatography - Mass SpectrometryGas Chromatography - Mass Spectrometry
(GC-MS)(GC-MS)

15. 0
0.1
0.2
0.3
0.4
0 10 20 30 40
Temperature (°C)
R.erythropolis P.frideriksbergensis
0
0.05
0.1
0.15
0.2
2.5 4.5 6.5 8.5 10.5
pH
R.erythropolis P.frederiksbergensis
µ =ln2·TD*
*TD = Time Doubling
Optimal conditions for the growth ofOptimal conditions for the growth of P.P.
frederiksbergensisfrederiksbergensis andand R. erythropolisR. erythropolis
20°C15°C 7.47.0
Growthrate(d)

16. Growth and biodegradation of eicosane byGrowth and biodegradation of eicosane by P.P.
frederiksbergensfrederiksbergensisis,, R. erythropolisR. erythropolis and mixedand mixed
culture at optimal conditionsculture at optimal conditionsoptimal conditions
0.00E+00
1.50E+07
3.00E+07
4.50E+07
6.00E+07
7.50E+07
9.00E+07
0 2 4 6 8 10 12
0
200
400
600
800
1000
P. frederikesbergensis R. erythropolis Mixed culture
eicosane conc. (µM) eicosane conc. (µM) eicosane conc. (µM)
Growth(cell/ml)
Eicosaneconc.(µM)
Time (day)

17. Growth ofGrowth of P. frederiksbergensisP. frederiksbergensis on eicosanon eicosan
and docosane crystalsand docosane crystals
EicosaneEicosane
crystalcrystal
DocosaneDocosane
crystalcrystal
C10H20, C16H34, C18H38, C20H42, C22H46

18. Biodegradative capabilities ofBiodegradative capabilities of P.P.
frederiksbergensisfrederiksbergensis
0
200
400
600
800
1000
0 2 4 6 8 10 12
Time (day)
Conc.(µM)
hexadecane eicosane decane
decane
eicosanehexadecane

19. Metabolic pathway of eicosaneMetabolic pathway of eicosane and decaneand decane
degraded bydegraded by P. frederP. frederiiksbergensisksbergensis
Eicosane
Decane

20. Metabolic pathway of eicosaneMetabolic pathway of eicosane and decaneand decane
degraded bydegraded by P. frederP. frederiiksbergensisksbergensis
CH3-(CH2)n-CH2-CH3CH2 -CH2OHCHO-CH2COOH

Terminal oxidationTerminal oxidation
CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH33


21. 0,00E+00
1,25E+07
2,50E+07
3,75E+07
5,00E+07
6,25E+07
7,50E+07
0 2 4 6 8 10 12
Time (days)
Growth(cell/ml)
5,5
5,75
6
6,25
6,5
6,75
7
7,25
7,5
pHvalue
P. frederiksbergensis (a) R. erythropolis (b) pH (a) pH (b)
Effect of pH on P. frederiksbergensis
growth
NAD+
NADH + H+
alkanes Alkanoic acid
H+
H+
H+
H+
H+
H+
Medium acidification

22. ConclusionConclusion
 TheThe isolates in this studyisolates in this study were able to degradewere able to degrade (C(C1010- C- C2222)) n-n-alkanesalkanes
from 4°C to 20°C.from 4°C to 20°C.
 P. fredP. fredeeriksbergensisriksbergensis waswas obligate psychrophileobligate psychrophile, while, while R.R.
erythropoliserythropolis waswas facultative psychrophilefacultative psychrophile..
 Alkane degradation byAlkane degradation by P. fredriksbergensisP. fredriksbergensis strated with terminalstrated with terminal
oxidation and detection of the metabolites was possible.oxidation and detection of the metabolites was possible.
 The alkane degradation inThe alkane degradation in P.P. frederiksbergensisfrederiksbergensis starts with thestarts with the
hydroxylation of the alkane and subsequently to fatty acidshydroxylation of the alkane and subsequently to fatty acids

23. Mode of alkanes uptake byMode of alkanes uptake by
P. frederiksbergensisP. frederiksbergensis ?!?!

24. Detection of the P. frederiksbergensis
biosurfactants by (MBAS)
P. frederiksbergensis
colonies on blue agar
P. frederiksbergensis
colonies on eicosane MSM

25. Biosurfactants activity producedBiosurfactants activity produced byby P.P.
frederiksbergensisfrederiksbergensis
0
15
30
45
60
75
90
0 2 4 6 8 10
Time (day)
Surfacetension(mN/m)
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
O.D.580nm
surface tension (mN/m)
surface tension (mN/m) with pellet suspension
Growth on hexadecane

26. Cell surface hydrophobicity andCell surface hydrophobicity and
adhesion to hexadecaneadhesion to hexadecane
G
row
th
Emulsification stabilityAdhesion to hexadecane
0
30
60
90
120
0 40 80 120 160 200 240
Time (h)
O.D.580nm
0,0
0,5
1,0
1,5
2,0
Adhesion(%)
(73%)
(10%
)
(88%)
0
30
60
90
120
Emulsificationstability(%)
% Adhesion = (1- (OD shaken with hexadecane //OD original) x 100%

27. GC-profile of the fatty acids involved
biosurfactants of P. frederiksbergensis
?
1
2
4
3
?
? ?
Time (min)
?
1
Abundance
1. Methyl palmitate
2. Methyl decanoate
3. Methyl tetradecanoate
4. Methyl eicosanoate

28. Characterization ofCharacterization of the biosurfactantsthe biosurfactants
Rhamnolipids possess hemolytic
properties.
Blood agar (5 % sheep blood)

29.  Glycolipids produced byGlycolipids produced by P. fredriksbergensisP. fredriksbergensis had a potential effecthad a potential effect
on substrateon substrate utilizutilizationation by:by:
ConclusionConclusion
 Developing hydrophobic cell surface and reducingDeveloping hydrophobic cell surface and reducing
media surface tension tomedia surface tension to 25 mNm-125 mNm-1
 The stability of the substrate emulsion reached the maximum value
after 8 days and
 73% of hexadecane was converted to hexadecanoic acid in
water emulsion

30. Molecular BiologyMolecular Biology
How can we solve the problem of environmental pollution by
Genetics engineering?

31. Detection ofDetection of alkalkBB inin P. frederiksbergensisP. frederiksbergensis withwith
oligonucleotide primers specific foroligonucleotide primers specific for alkalkBB
PCR product M
DNA Purification
PCR products M
DNA Amplification
550
bp
DNA Isolation
P. frederiksbergensisP. frederiksbergensis

32. Amino acid and
corresponding
proteins
Sequence analysis of the alkaneSequence analysis of the alkane
hydroxylase genehydroxylase gene probeprobe

33. Detection andDetection and llocalization ofocalization of alkanealkane
hydroxylasehydroxylase ggeneene
SouthernSouthern hybridizationhybridization
EcoR I
1 2 3 4 5 6 7
1. M (1kb) 2. DNA probe 3. EcoR1 4. NdeI 5. NcoI 6. AvaI 7. BamI
AmplificationAmplification
M
PCR
DigestionDigestion RestrictionRestriction
7 6 5 4 3 2 1

34. CloningCloning ofof alkane hydroxylasealkane hydroxylase ggeneene
PCR

35. Cloning and sequencing of alkaneCloning and sequencing of alkane
hydroxylase ofhydroxylase of P. frederiksbergensisP. frederiksbergensis
pUC19
2894 bp2894 bp
ORF
B1
F
alkB primers
R
B2
F
A2
pUC19
prim
ers
A1
R

36. pUC19 primeralkB primer
1 bp 2 894 bp
Analysis of (ORFS) and genetic organization
in P. frederiksbergensis
2 188 bp85 bp
ORF1
alkB
alcDH
2 316 bp1 303 bp
ORF2
The arrows indicate the direction and translation
of the gene
ORF3
Unknown
protein
 alkT absence?? (downstream of the gene)
 P. frederikesbergensis DAN (one cluster)
 distribution of the gene (Scattered on the chromosome)

37. 1 2 3 4
1313 bp
Subcloning ofSubcloning of alcalcDH from the wild typeDH from the wild type
P. frederiksbergensisP. frederiksbergensis and the cloned fragmentand the cloned fragment
pET-15
5708
bp
1. M (kb) 2. CF 3. WT 4. R

38. Alignment of P. fredriksbergensis alcohol
dehydrogenase amino acids sequences with
different organisms
 TGXXXGXG (cofactors binding site)
 TXXXL (active centre)

39.  Member of SCR family
 Highly conserved regions
Phylogenetic tree of P. frederiksbergensis
alcDH based on amino acids sequences
Accession number of AAR134804

40. Alignment of P. fredriksbergensis alkane
hydroxylase amino acids sequences with
different organisms
 Residues of eight histidine box
 hydrophobic amino acids

41.  Member of alkB family
 Highly conserved regions
Phylogenetic tree of P. fredriksbergensis
alkB based on amino acids sequences
sp.
Accession number ofnumber of AY452488AY452488

42. 20
Optimal temperature of native alkane
hydroxylase of P. fredriksbergensis
0
100
200
300
400
500
0 5 10 15 20 25 30 35 40
Temperature (°C)
Lossofdecaneconc.(µM)
Measurements were carried out at different temperatures (after 2 h of
incubation)
20

43. Periplasm spacePeriplasm space
Genetics of alkane degradationGenetics of alkane degradation
CH3
H
uptake??
Cytoplasmic membraneCytoplasmic membrane
Outer membraneOuter membrane
alkSp2
O
alkB alkF alkG alkH alkK alkL alkJ
ß-Oxidation
RegulationRegulation
Chemotaxis?Chemotaxis?
CytoplasmCytoplasm
palkB
alkSalkS
alkFalkF
alkTalkT
alkH
NAD
H
alkH
NAD
H
alkKalkK
alkG
Fe++
alkG
Fe++
BB
O ATP
CoA
NADH
FAD
TCA cycle
alkSTalkSp1
alkS
Catabolic
repression
alkanesalkanes
absenceabsence
alkanes
presence
ơS
R SCOA
alcDH
FAD
alcDH
FAD
CH3
HO
AA alkLalkL
alkNalkN
alkB
Fe++
alkB
Fe++
alkSalkS
alkFalkF
alkTalkT
alkG
Fe++
alkG
Fe++
alkB
Fe++
alkB
Fe++
alkS
alkanes
presence RegulationRegulation

44. 0
0,2
0,4
0,6
0,8
1
0 8 16 24 32 40
Temperature (°C)
0
0,2
0,4
0,6
0,8
1
2 4 6 8 10 12
pH
Activity(U/ml)Optimal reaction conditions of recombinant
alcohol dehydrogenase (alcDH)
U = (ε [cm2
µmol-1
] of NADH = 6.22)
pH = 9 T= 30°C

45. 0
25
50
75
0 50 100 150 200 250 300
Time (min)
Activity[µM/min]
10°C 30°C
40°C 50°C
0
20
40
60
80
0 2 4 6 8 10
Time (day)
RT
4°C
Thermostability of recombinant
alcohol dehydrogenase (alcDH)
U = (ε [cm2
µmol-1
] of NADH = 6.22)

46. methanol
ethanol
propanol
1-butanol
isobutanol
amylalcohol
isoamylalcohol
1-octanol
tetradecanol
10 mM
0
0,2
0,4
0,6
0,8
Specific
activity
[U/mg]
Substrate
10 mM 50 mM
Substrate specrtum recombinant alcDH
activity
Measurements were carried out at pH = 9.0 and T = 30°C
Substrate specrtum of recombinant
alcohol dehydrogenase (alcDH)
 A broad substrate
spectrum
 Tendency towards
primary alcohols
 100 mM inhibited the
enzyme activity

47. ConclusionConclusion
 The gene cluster of alkane hydroxylase ofThe gene cluster of alkane hydroxylase of P. fredriksbergensisP. fredriksbergensis waswas
different from known alkane hydroxylases found in otherdifferent from known alkane hydroxylases found in other alkanesalkanes
degrading bacteria.degrading bacteria.
 The gene encodingThe gene encoding forfor alcalcDH belonged the groups of NADDH belonged the groups of NAD++
dependent, shortdependent, short chain alcohol dehydrogenasechain alcohol dehydrogenase..
 The enzyme of alcDH had a wide range of substratesThe enzyme of alcDH had a wide range of substrates spectrumspectrum withwith
TT == 3030°°CC
 pHpH == 9.0.9.0.
 Alcohol dehydrogenase ofAlcohol dehydrogenase of P. fredriksbergensisP. fredriksbergensis has the tendency
towards primary alcohols

50. Technical biochemistry
TUHTechnical University Hamburg-Harburg