1

Biodegradation of synthetic surfactants:

linear alkylbenzenesulfonate (LAS)
and related compounds
David Schleheck
SO
3
-
LAS
Prof. Alasdair McLeod Cook
University of Konstanz
2
Universitt
Konstanz
`Surfactants=surface active compounds
water soluble amphiphiles,
with hydrophobic and hydrophilic moiety
support the washing process,
enhance the removal of impurities (`soil) from surfaces
and disperse the soil in the wash liquor.
3
SO
3
-
O
SO
3
-
O SO
3
-
SO
3
-
SO
3
-
O
O
O
OH
N
+
CH
3
CH
3
SO
3
-
N
+
CH
3
CH
3
CH
3
N
SO
3
-
CH
3
SO
3
-
COO
-
SO
3
-
SO
3
-
SO
3
-
O
CH
3
anionic
cationic
zwitterionic
nonionic
(a) (b)
(c) (d)
(e)
(f)
(g)
(h)
(i)
(j)
Illustrative examples of surfactants
(a) linear alkylbenzenesulfonate (LAS)
(b) branched-chain alkylbenzenesulfonate
(ABS) [tetrapropylenebenzenesulfonate]
(c) 4-(1-n-octyl)benzenesulfonate (OBS)
(d) linear hexadecanediphenyletherdisulfonate
(LADPEDS)
(e) 1-alkanesulfonate
(f) secondary alkanesulfonate
(g) methylestersulfonate
(h) alkylmethyltaurate
(i) fatty-acid sulfate
(j) soap
alkylethoxylate
alkyltrimethylammonium
alkylsulfobetaine
History of surfactants
soap from saponification of fat with soda,
by the ancient Sumerians (2500 BC)
`Persil 1907, first detergent formulation with
soap as surfactant, for manual laundry
`Fewa 1932, first detergent with synthetic surfactant,
fatty-acid sulfate (SDS)
Machine-washing in the 1960s,
branched-chain alkylbenzenesulfonates (ABS),
and other synthetic, xenobiotic surfactants
from the petrochemical industry
SO
3
-
ABS
4
ABS surfactant is only
poorly biodegradable ...
in the 1960s in Germany
SO
3
-
ABS =branched-chain
alkylbenzenesulfonate
60 64 68 72 76 80 84 88 92
0
200
400
600
year
gMBAS / sec
... and was replaced by the
fully-biodegradable LAS surfactant
SO
3
-
LAS =linear-chain
alkylbenzenesulfonat
Anionic-surfactant load of River Rhine
(...)
(...)
5
LAS today
2.4 Million tons LAS per year,
3 g LAS per person and per day,
about 4 %of the carbon-load of
communal wastewater,
are degraded in
sewage treatment plants
by microbial communities:
SO
3
-
COO
-
SO
3
-
H
2
O, CO
2
, SO
4
2-
LAS
SPC
sulfophenyl-
carboxylate
No. 1
No. 2
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
COO
-
SO
3
-
COO
-
SO
3
-
2
8
C10
3
7
4
6
5
5
2
9
3
8
4
7
5
6
6
5
2
10
3
9
4
8
5
6
6
5
2
11
3
10
4
9
5
8
6
7
7
6
C11
C12
C13
LAS
Diagramatic representation
of commercial LAS:
subterminal 4-sulfophenyl-
substitution on C
10
- C
13
alkyl chains
(radical reaction)
20 congeners,
18 in pairs of R/S-enantiomers
38 individual compounds
are completely degraded
6
0 10 20 30
0.3
a
b
s
o
r
b
a
n
c
e

(
2
2
0

n
m
)
retention time (min)
21
LAS
138
84
63
42
3
SPC
day
sewage sludge
agricultural soil
forest soil
lake sediment
(Donget al. J AM 2004)
LAS-salts medium+air
samples for HPLC-analysis
biomass on
polyester fleece
Laboratory trickling filters to esthablish degradative communities
Enrichment and isolation of
-proteobacterium strain DS-1
(now Parvibaculum lavamentivorans gen. nov. sp. nov.)
O
SO
3
-
SO
3
-
COO
-
O
SO
3
-
SO
3
-
LADPEDS
Dowfax8390
initial
degradation
onl y !
biomass
from
sludge
`DS-0
(Schleheck et al. AEM 2000)
7
Growth of strain DS-1 in pure culture:
dependence on the addition of a solid surface,
`solid support (polyester fleece, glass wool, glass particles),
to the surfactant-salts medium
0 2 4 6 8
0
0.1
0.2
G
r
o
w
t
h

(
O
D

5
8
0

n
m
)
Incubation time (days)
1 mg/ml GP
0.5 mg/ml
0 mg/ml
Growth with 1 mM LAS
GP DS-1
DS-1
GP
8 days incubation; GP, glass particles
1 mM LAS upon shaking
1 mM LAS, 4 days, Ruthenium-Red stain
Formation of biofilm on polyester fleece
8
DPG
PG
PE
PC
AL2
AL1
Percentage composition of the fatty acids present in strain DS-1
T
Fattyacid StrainDS-1
T
Method1
a
Method2
a
3-OH14:0 6.3 5.7
16:0 6.5 6.1
3-OH16:1 1.4 1.3
3-OH16:0
b
1.2 9.1
18:17c 50.4 47.4
18:0 9.7 9.0
unknown tr tr
cyclo19:0 13.6 11.8
2-OH18:1 2.8 2.6
cyclo2-OH19:0 8.1 7.0
a
Methods1and2releasedester-linkedandester-linkedplusamide-linkedfattyacids,
respectively.
b
Putativeamide-linkedfattyacids.
tr Traceamounts.
Phylogeny of strain DS-1 (Schleheck et al. IJ SEM 2004)
16S-rDNA sequence: sorted into -subclass of Proteobacteria,
but with >8 %sequence divergence to any
validly published species.
membrane components: typical of -subclass of Proteobacteria,
but in combination unique to -proteobacteria.
3-(4-sulfophenyl)dodecane 1-n-dodecanesulfonate
4-(4-sulfophenyl)
hexanoate
6-sulfohexanoate
uptake
oxidations
thioesterification
-oxidation
thioester cleavage
-oxygenation
excretion
extracellular
environment SO
3
-
SO
3
-
SO
3
-
OH
COO
-
SO
3
-
CO-SCoA
SO
3
-
CO-SCoA
SO
3
-
COO
-
SO
3
-
-O3S
-O3S
OH
-O3S
COO
- -O3S
CO-SCoA
-O3S
CO-SCoA
-O3S
COO
-
SO
3
-
COO
- -O3S
COO
- -O3S
Strain DS-1
primary alkylsulfonate
secondary alkylsulfonate
-olefin-sulfonate
methylestersulfonate
N-alkyl-N-methyltaurate
alkylsarcoside
alkaneethoxylate
alkaneethoxylate sulfate
alkyldiglycolether sulfate
alkylglutamic acid
polyoxyethylene monoalkanoate
alkyl polyglucoside
fatty-acid glucosamide
(and)
Parvibaculum
lavamentivorans
gen. nov. sp. nov.
9
The LAS -oxygenase is a novel
P450-ish heme containing monooxygenase ( P428 )
(Schleheck and Cook, AM 2005)
inhibitor concentration
s
p
e
c
i
f
i
c

o
x
y
g
e
n

u
p
t
a
k
e
r
a
t
e
(
m
k
a
t
/
k
g
p
r
o
t
e
in
)
56%
30%
7%
100%
0
0.2
0.4
control 2 mM 4 mM 8 mM
a
b
s
o
r
b
a
n
c
e
350 400 450 500
0
0.1
0.2
0.3 406 nm
428 nm
wavelength (nm)
Inhibited byP450-specific inhibitor
(Metyrapone)
Heme-specific CO-difference spectra
of crude cell extract






a
b
s
o
r
b
a
n
c
e

(
2
2
0

n
m
)
retention time (min)
30 0 10 20
0.4
mc-SPC
lc-SPC sc-SPC
LAS
C10
C11
C12
C13
+0.25 h
+68 h
+6 h
+2 h
SO
3
-
COO
-
SO
3
-
H
2
O, CO
2
, SO
4
2-
LAS
SPC
sulfo-
phenyl-
carboxylate
Strain DS-1
HPLC-chromatograms
from a cell suspension
after addition of LAS
10
0
0.4
0
0.2
0 10 20 30
25.6
7.9
10.3
9.2
7.4
retention time (min)
A

2
2
0

n
m

COO
-
SO
3
-
COO
-
SO
3
-
COO
-
SO
3
-
SO
3
-
The degradation of each
individual LAS-congener
yields several individual SPCs
HPLC-chromatogram
Strain DS-1
2-C10-LAS
3-C4-SPC
3-C4-SPC-2H
5-C6-SPC
1
1
2
3
2
3
5-C10
12.6
14.3
11.8
7.1
9.0
10.8
30 20 10 15 5 25
retentiontime(min)
a
b
s
o
r
b
a
n
c
e

(
A

2
2
0

n
m
)
SPdC
HPLC-chromatogram
SO3
-
COO
-
SO3
-
COO
-
SO3
-
COO
-
COO
-
COO
-
SO
3
-
-
OOC
SO
3
-
COO
-
COO
-
COO
-
-
OOC
-
OOC
-
OOC
COO
-
SO
3
-
-
OOC
COO
-
S
-
5-C10-LAS
6-C10-SPC
4-C8-SPC-2H
4-C8-SPC
5-C10-SPdC
4-C8-SPdC
3-C6-SPdC
firs
t
s
e
c
o
n
d

-o
x
y
g
e
n
a
tio
n
SPC SPdC
(-oxidation)
sulfophenyl-
di-carboxylates
... and even more intermediates: sulfophenyl-di-carboxylates
11
R
R
COO-
R
COO- 5-C7E
3-C5E
3-C13E
R
R
COO-
R
COO-
6-C9E
4-C7E
4-C13E
R
COO-
R
COO-
R
COO-
R
COO-
R
COO-
R
COO-
R
-OOC
-OOC
-OOC
-OOC
6-C13
4-C9E
6-C11E
4-C7
4-C9E
6-C11
6-C13E
R
COO-
R
COO-
R
COO-
R
COO-
R
COO-
R
COO-
R
COO-
R
-OOC
-OOC
-OOC
-OOC
-OOC
COO-
R
-OOC
7-C13
3-C9E
5-C11E
3-C5
3-C7E
3-C9E
5-C9
5-C11E
7-C13
R
R
COO-
R
COO-
6-C7E
4-C5E
2-C13E
R
R
COO-
R
COO-
COO-
R
COO-
R
COO-
R
COO-
R
-OOC
-OOC
-OOC
-OOC
5-C9
3-C7E
3-C5
5-C9E
3-C7E
5-C11E
5-C11E
COO-
R
5-C13E -OOC
4-C12E
3-C6E
5-C8E
R
COO-
R
R
COO-
R
COO-
R
COO-
R
3-C12E
4-C6E
6-C8E
COO-
R
R
COO-
COO-
R
COO-
R
R
COO-
-OOC
-OOC
-OOC
R
COO-
-OOC
3-C6E
4-C8E
5-C10E
5-C12E
R
6-C10E
5-C12E
4-C8E
R
COO-
R
R
COO-
COO-
R
COO-
R
R
COO-
-OOC
-OOC
-OOC
6-C12E
3-C8E
5-C10E
3-C6E
3-C8E
5-C10E
R
COO-
-OOC
6-C12E
R
COO-
R
COO-
R
2-C12E
3-C4E
5-C6E
LAS congener
major SPC
(50 - 80 %)
minor SP(d)C
SPC+2C
SPdC
substrate
products
excreted
strain DS-1T
-oxygenation
-oxidation
C
even
-LAS
C
odd
-LAS
SPdC
LAS congener
major SPC
SPC+2C
R =
SO3
-
33 SPCs and 17 SPdCs are generated from the 20 commercial-LAS congeners
(Schleheck et al., AEM submitted)
SO
3
-
COO
-
SO
3
-
H
2
O, CO
2
, SO
4
2-
LAS
SPC
Strain DS-1
Nr. 2
The degradation of SPC
new environmental isolates
Comamonas testosteroni
strains SPB-2 and KF-1, and
Delftia acidovorans SPH-1
(Schleheck et al., AEM 2004)
12
Delftia acidovorans SPH-1 degrades 4-C6-SPC
(major SPC from3-C12-LAS)
Comamonas testosteroni strains SPB-2 and KF-1
degrade 3-C4-SPC (major SPC from2-C12-LAS)
5 10 15
0
1
0
1
5 10 15
0
1
0
1
retention time (min)
retention time (min)
a
b
s
o
r
b
a
n
c
e

(
2
2
0

n
m
)
a
b
s
o
r
b
a
n
c
e

(
2
2
0

n
m
)
a
b
s
o
r
b
a
n
c
e

(
2
6
0

n
m
)
a
b
s
o
r
b
a
n
c
e

(
2
6
0

n
m
)
4-C6-SPC
10.4
12.6
11.7
9.6
9.3
8.2
3-C4-SPC
7.9
10.3
9.2
6.4
7.5
SPH-1
SPB-2
Enantioselective degradation of
R/S-4-(4-sulfophenyl)hexanoate (4-C
6
-SPC)
by D. acidovorans SPH-1
protein (g/ml)
0 50 100
0.0
0.5
1.0
1.5
2.0
19.9 min
19.1 min
(R) (S)
c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
M
)
sulfate
R
S
R/S 4-C
6
-SPC
COO
-
SO
3
-
H
-OOC
SO
3
-
H
13
Oxygenase activities detected in C. testosteroni strains SPB-2 and KF-1
COO
-
SO
3
-
SO
3
-
OH
OH
COO
-
COO
-
SO
3
-
SO
3
-
OH
3-C
4
-SPC 4-sulfophenol 4-sulfocatechol
4
-
s
u
l
f
o
c
a
t
e
c
h
o
l
-
1
,
2
-
d
i
o
x
y
g
e
n
a
s
e
4
-
s
u
l
f
o
p
h
e
n
o
l
-
2
-
m
o
n
o
o
x
y
y
g
e
n
a
s
e
3
-
C
4
-
S
P
C

o
x
y
g
e
n
a
s
e

?
?
incubation time (days)
g
r
o
w
t
h

(
O
D

5
8
0

n
m
)
c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
M
)
0.01
0.1
0 2 4 6
0.0
0.5
1.0
1.5
incubation time (days)
0.01
0.1
0 2 4 6
0.0
0.5
1.0
1.5
c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
M
)
g
r
o
w
t
h

(
O
D

5
8
0

n
m
)
B
A
OD
580
LAS
SPC
sulfate
SPC
Mineralization of individual LAS
congeners by defined pairs of
heterotrophic bacteria:
Strain DS-1
Strain DS-1
Strain SPH-1
+
14
The three-member community
and commercial LAS
0 10 20 30
5 10 15 7.5 17.5 12.5
0
0.2
retention time (min)
strain DS-1
alone
with SPB-2
with SPH-1
with strains
SPB-2 and
SPH-1
commercial LAS
a d b c
A
B
C
D
E
*
4 5 6 7 8 9
a
b
s
o
r
b
a
n
c
e

(
2
2
0

n
m
)
DS-1
SPB-2
DS-1
SPH-1
DS-1
SPH-1
SPB-2
DS-1
Only 4 major SPCs
are completely degraded
COOH
SO3H
COOH
SO3H
COOH
SO3H
SO3H
COOH
Strain
DS-1
Strain
SPB-2
Strain
SPH-1
H
2
O
sulfate
CO
2
H
2
O
sulfate
CO
2
3-C
4
3-C
5
4-C
6
4-C
5
2-C
10
3-C
11
2-C
10
3-C
11
SO3H
SO3H
SO3H
SO3H
The defined three-member bacterial community
as a model system for LAS-degradation
15
R
OH R
COO
-
R
CO-SCoA R CO-SCoA R
COO
-
SO
3
-
COO
-
SO
3
-
COO
-
SO
3
-
SO
3
-
CO-SCoA
O
R
COO
-
SO
3
-
SO
3
-
OH
OH COO
-
COO
-
SO
3
-
COO
-
SO
3
-
O
O
COO
-
COO
-
O
Ac-SCoA
SO
3
-
CO-SCoA
OH
R
CO-SCoA R
SO
3
-
Strain DS-1
Strain SPB-2
extracellular space
CO2
H2O
HSO3
-
=R
(I)
(II)
(III)
(IVa)
(IVb)
(IVc)
(IVd)
(V)
(VI) (VII)
(VIII)
HSCoA
HSCoA
HSCoA
2-C10-LAS
2-C12-LAS
2-C14-LAS
3-C4-SPC
3-C4-SPC-2H
5-C6-SPC
RO-SCoA
(...)
Reactions catalyzed by P. lavamentivorans
T
DS-1 after uptake of LAS: (I), -oxygenation; (II), -oxidations; (III),
thioesterification; (IVa-IVd), spirals of -oxidation contributed by ,-desaturation, addition of water, (true) -
oxidation, and -ketothiolysis; (V) thioester cleavage and excretion.
Reactions catalyzed by C. testosteroni SPB-2 after uptake of 3-C4-SPC: (VI) initial oxygenation(s) of 3-C4-SPC
and side-chain removal; (VII), 4-sulfocatechol ortho-cleavage, (VIII), modified ortho-degradation pathway and
central metabolism.
Mineralization of LAS by two-member communities
Conclusions
- The range of products generated during degradation of commercial LAS
is more complex than initiall y realized (the SPCs, SPC-2Hs, SPC+2Cs, and SPdCs)
- The community able to completel y degrade LAS
is more complex than initiall y realized (the narrow substrate range for SPCs)
- This is the first community which allows access to all aspects of a
heterotrophic pathway to degrade complex mixtures of surfactant molecules
(the three pure cultures which can be grown as biofilm-community)
16
toxicity and biofilm:
- the role of the solid support ?
- the nature of the inducible biofilm(s) ?
- howto protect the cell with biofilm ?
novel pathways and enzymes:
Strain DS-1 - the novel P428-heme monooxygenase
- `standard -oxidation enzymes ?
- the transport systems ?
the novel organism: P. lavamentivorans
T
DS-1
- initially degrades a wide range of surfactants
- is strain DS-1 widespread in nature ?
the SPC-degraders - enantioselective degradation ?
- oxygenolytic side-chain removal ?
- 4-phenolsulfonate-2-monooxygenase ?
- the transport systems ?