1
SUPPLEMENTARY DATA
Bio ans o ma ion o o ganic mic opollu an s by anae obic sludge enzymes
Lo ena Gonzalez-Gil a*, Daniel K ah b, Ann-Ka h in Gha as b, Ma a Ca balla a, A ne
Wick b, Lissa Helmholzb, Juan M. Lema a, Thomas A. Te nes b
a Depa men o Chemical Enginee ing, School o Enginee ing, Uni e sidade de
San iago de Compos ela, Rúa Lope Gómez de Ma zoa, E-15782 San iago de
Compos ela, Spain
b Fede al Ins i u e o Hyd ology (B G), D-56068 Koblenz, Am Mainze To 1,
Ge many
* Co esponding au ho
2
Con en s
Sec ion I. Chemical s uc u e and physicochemical cha ac e is ics o OMPs ........... 3
Sec ion II. Cha ac e iza ion o lysa es ........................................................................ 6
Sec ion III. Remo al o OMPs in anae obic sludge (posi i e con ol) ...................... 8
Sec ion IV. Remo al o OMPs unde di e en lysa e condi ions .............................. 9
Sec ion V. Iden i ied TPs by LC–QToF-MS measu emen s .................................... 16
Re e ences ................................................................................................................ 17
3
Sec ion I. Chemical s uc u e and physicochemical cha ac e is ics o OMPs
Table S1. Applica ion and main physicochemical p ope ies o he selec ed OMPs.
OMP
Applica ion
MW
(g/mol)
s
(mg/L)
H
(a m m
3
/mol)
pKa log Kow
10,11- DiOH-CBZ
Me aboli e o CBZ
270.2 290 n. . 8.2 1.8
10,11-DiH-10-OH-
CBZ (10-OH-CBZ)
Me aboli e o CBZ
254.3 550 n. . 14.1 1.7
2-OH-CBZ
Me aboli e o CBZ
252.3 120 n. . 9.2 2.2
3-OH-CBZ
Me aboli e o CBZ
252.3 110 n. . 9.2 2.3
Acesul ame
A i icial swee ene
163.2
5.8·10
5
9.0·10
-6
5.7
-1.3
Ace aminophen
Analgesic
151.2 1.4·104 n. . 9.4 0.46
N-ace yl-SMX
Me aboli e o SMX
295.3 1.2·103 3.1·10-15 5.7 1.2
Acyclo i
An i i al d ug
225.2
2.5
3.2·10
-22
9.3
-1.6
A enolol
Be a blocke
266.3 1.3·104 1.4·10-18 9.6 0.16
Benzo iazole
Co osion inhibi o
133.2 9.7·103 3.1·10-16 9.6 1.1
Beza ib a e
Lipid
- egula o 361.8 0.36 6.1·10-11 3.8 4.2
Ca bendazim
Fungicide
191.2 29 7.5·10-10 4.2 1.5
Ca bamazepine (CBZ)
An icon ulsan
236.3 112 1.1·10-10 15.9 2.5
Ci alop am
An idep essan
324.4 31.1 2.7·10-11 9.8 3.7
Cla i h omycin
An ibio ic
748.0
0.34
1.7·10
-23
9.0
3.2
Climbazole
An imyco ic
292.8 8.3 2.8·10-9 7.5 3.8
Codeine
Opioid
299.4 9·103 7.6·10-14 8.2 1.2
Dia izoa e
X- ay con as medium
613.9
8.9
2.8·10
-18
2.2
1.4
Diclo enac
Analgesic
296.2 2.4 4.7·10-12 4.2 4.2
Diu on
He bicide
233.1 42 5.0·10-10 nonionic
2.7
E y h omycin
An ibio ic
733.9 1.4 5.4·10-29 8.9 3.1
Fluconazole
Fungicide
306.3 1.0 n. . 2.6 0.58
Iop omide
X
- ay con as medium
791.1 23.7 1.0·10-28 4.2 -2.1
Iopamidol
X
- ay con as medium
777.1 1.4·105 1.1·10-25 4.2 -2.4
Iomep ol
X- ay con as medium
777.1
155
n. .
5.6
-1.8
Isop o u on
He bicide
206.3 65 1.1·10-10 nonionic
2.9
Mecop op
He bicide
214.6 620 1.8·10-8 3.1 3.1
Me op olol
Be a blocke
267.4
1.7·10
4
1.4·10
-13
9.6
1.9
Oxazepam
An i
-anxie y 286.7 20 5.5·10-10 10.9 2.2
P imidone
An icon ulsan
218.3 500 1.9·10-10 11.5 0.91
So alol
β
-blocke 257.3 1.4·105 1.0·10-10 10.1 0.24
Te bu yn
He bicide
241.4 25 2.1·10-8 4.3 3.7
T amadol
Opioid
263.4 1.2·103 1.5·10-11 9.4 3.0
T ime hop im
An ibio ic
290.3 400 2.4·10-14 7.1 0.9
Venla axine
An idep essan
277.4
267
2.0·10
-11
10.1
3.3
Molecula weigh (MW), Hen y’s law cons an (H), solubili y a 25 °C (s), acid dissocia ion cons an (pKa), oc anol-
wa e coe icien (Kow). n. . e e s o no ound.
Da a ob ained om D ugBank. PhysP op, PubChem and The Human Me aboli e (hmdb) da abases.
4
Table S2. Chemical s uc u es o he selec ed OMPs.
Compound
Chemical s uc u e
Compound
Chemical s uc u e
10,11-DiOH-CBZ
10-OH-CBZ
2-OH-CBZ
3-OH-CBZ
Acesul ame
Ace aminophen
Ace yl-SMX
Acyclo i
A enolol
Benzo iazole
Beza ib a e
Ca bendazim
Ca bamazepine
(CBZ)
Ci alop am
Cla i h omycin
Climbazole
Codeine
Dia izoa e
Diclo enac
Diu on
5
E y h omycin
Fluconazole
Iomep ol
Iopamidol
Iop omide
Isop o u on
Mecop op
Me op olol
Oxazepam
P imidone
So alol
Te bu yn
T amadol
T ime hop im
Venla axine
6
Sec ion II. Cha ac e iza ion o lysa es
The measu emen o p o ein concen a ion (Figu e S1) and β-galac osidase (Figu e S2),
phospha ase (Figu e S2) and ace a e kinase ac i i ies (Figu e S3) allow o selec ing he
mos in e es ing lysa es o pe o m he OMP ans o ma ion assays. P o ein
concen a ion and he enzyma ic ac i i ies inc eased wi h bead bea ing ime, al hough
his imp o emen is less p onounced om 80 s onwa ds. In compa ison wi h sonica ion,
he maximum p o ein eleased h ough bead bea ing (160 s) was signi ican ly lowe , as
well as β-galac osidase and ace a e kinase ac i i ies, while phospha ase ac i i y o bo h
lysa es was compa able.
The use o phospha e bu e inc eased p o ein concen a ion by app oxima ely 55% and
80% compa ed o he use o HN-bu e wi h sonica ion and bead bea ing, espec i ely.
Ace a e kinase ac i i y also inc eased in phospha e bu e , especially upon lysis by bead
bea ing. Ne e heless, β-galac osidase and phospha ase ac i i ies emained almos
equal. Finally, he addi ion o de e gen s p io bead bea ing (80 s) clea ly inc eased
p o ein concen a ion, phospha ase and ace a e kinase ac i i ies, bu β-galac osidase
ac i i y diminished.
Figu e S1. P o ein concen a ion in lysa es ob ained by di e en ex ac ion p ocedu es.
Cell lysis by ul asonica ion wi h HN-bu e (i.e., basic lysa e, AD) o wi h phospha e
bu e (AD PO4); cell lysis wi h HN-bu e by bead bea ing o 40 s (AD40), 80 s
(AD80), 120 s (AD120), 160 s (AD160), wi h phospha e bu e by bead bea ing o 160
s (AD160PO4), wi h HN-bu e and de e gen s oc yl hioglucoside (AD80T), β-
dodecylmal oside (AD80M) o CHAPS (AD80C) by bead bea ing o 80 s. E o ba s
depic he s anda d de ia ion o iplica ed measu emen s (n=3).
7
Figu e S2. β-galac osidase and phospha ase ac i i ies in lysa es ob ained by di e en
ex ac ion p ocedu es. Cell lysis by ul asonica ion wi h HN-bu e (i.e., basic lysa e,
AD) o wi h phospha e bu e (AD PO4); cell lysis wi h HN-bu e by bead bea ing o
40 s (AD40), 80 s (AD80), 120 s (AD120), 160 s (AD160), wi h phospha e bu e by
bead bea ing o 160 s (AD160 PO4), wi h HN-bu e and de e gen s oc yl hio-
glucoside (AD80T), β-dodecylmal oside (AD80M) o CHAPS (AD80C) by bead
bea ing o 80 s. E o ba s depic he s anda d de ia ion o quad uplica ed
measu emen s (n=4).
Figu e S3. Ace a e kinase (AK) ac i i y in lysa es ob ained by di e en ex ac ion
p ocedu es. Cell lysis by ul asonica ion wi h HN-bu e (i.e., basic lysa e, AD) o wi h
phospha e bu e (AD PO4); cell lysis wi h HN-bu e by bead bea ing o 80 s (AD80),
160 s (AD160), wi h phospha e bu e by bead bea ing o 160 s (AD160 PO4), wi h
HN-bu e and de e gen s oc yl hioglucoside (AD80T), β-dodecylmal oside (AD80M)
o CHAPS (AD80C) by bead bea ing o 80 s. E o ba s depic he s anda d de ia ion
o iplica ed measu emen s (n=3).
8
Sec ion III. Remo al o OMPs in anae obic sludge (posi i e con ol)
Figu e S4. Remo al o ace yl-SMX (a), cla i h omycin (b), amadol (c), ime hop im
(d), a enolol (e) and enla axine ( ) and o ma ion o SMX (a), O-desme hyl- amadol
(c, seconda y y-axis), N,O-didesme hyl- amadol (c, seconda y y-axis) and 4-
desme hyl- ime hop im (d, seconda y y-axis) in posi i e con ol expe imen s
pe o med wice in iplica e wi h anae obic sludge. E o ba s ep esen maximum and
minimum alues o he wo expe imen s.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80
C/C0
Time (h)
Ace yl-SMX
Ace yl_SMX
SMX
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80
C/C0
Time (h)
T ime hop im
T ime hop im
4-DM- ime hop im
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80
C/C0
Time (h)
Cla i h omycin
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80
C/C0
Time (h)
A enolol
a)
b)
d)
e)
0.00
0.05
0.10
0.15
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80
C/C0
Time (h)
T amadol
T amadol
O-DM- amadol
N,O-DDM- amadol
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80
C/C0
Time (h)
Venla axine
c)
)
9
Sec ion IV. Remo al o OMPs unde di e en lysa e condi ions
Figu es S5-S7 show signi ican e ec s wi h espec o he basic lysa e when di e en
ex ac ion condi ions, co ac o s and inhibi o s we e used. Tables S3-S5 summa ize he
maximum emo al e iciencies achie ed in he OMPs ans o ma ion assays. The
ex ac ion condi ions and he addi ion o co ac o s and inhibi o s we e es ed in h ee
independen assays, wi h lysa es ob ained om anae obic sludge samples a di e en
days. Hence, o a oid a iabili y be ween assays no caused by he speci ic condi ions
es ed, he ∆Remo al alues wi h espec he basic lysa e (Tables 1-3, manusc ip ) we e
calcula ed conside ing he co esponding emo al o each basic lysa e, which is also
speci ied in Tables S3-S5.
Figu e S5. Signi ican e ec o ex ac ion condi ions on he ans o ma ion o acyclo i
in o ca boxy-acyclo i (TP). Names in legends e e o basic lysa e (ob ained by
sonica ion) and bead bea ing o 160 s (BB160). Each ime poin ep esen s he mola
concen a ion a io (C/C0) (a e age o iplica es o he i s and las ime poin and
single alues o composi e samples o he second, hi d and ou h ime poin ). E o
ba s depic he s anda d de ia ion (n=3) conside ing e o p opaga ion.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
020 40 60 80
C/C0
Time (h)
Acyclo i
0.0
0.2
0.4
0.6
0.8
1.0
1.2
020 40 60 80
C/C0
Time (h)
Ca boxy-acyclo i
Basic lysa e BB160
a)
b)
16
Sec ion V. Iden i ied TPs by LC–QToF-MS measu emen s
Table S6. Mass accu acy, iso ope a io and e en ion ime o he iden i ied TPs
e y h omycin TP 576 and cla i h omycin TP 590 (bo h esul ing om he clea age o
cladinose, Te zic e al., 2018) as well as a enolol acid ( esul ing om he hyd olysis o
he p ima y amide, Radjeno ić e al., 2008).
T ans-
o ma ion
p oduc (TP)
Sum
o mula
Calcula ed
exac mass
o [M+H]+
(m/z)
Measu ed
exac mass
o [M+H]+
(m/z)
Mass
accu acy
(ppm)
Calcula ed
iso ope
a io (%)
Measu ed
iso ope
a io (%)
RT1
(min)
E y h omycin
TP 576
C29H53NO10 576.3748 576.3740 -1.5 34 30 6,7
Cla i h omycin
TP 590
C30H55NO10 590.3904 590.3898 -0.95 35 32 7,4
A enolol acid C14H21NO4 269.1581 268.1542 -2.8 16 17 5.0
1 Re en ion ime
Table S7. Compa ison o measu ed MS/MS agmen s p esen in bo h he MS2 spec um
o he pa en compound (e y h omycin and cla i h omycin) as well as in he MS2 spec um
o he de ec ed TP (e y h omycin TP 576 and cla i h omycin TP 590). The MS/MS
agmen s o he TP a enolol acid we e compa ed wi h hose o an au hen ic e e ence
s anda d.
Compound o
compa ison
(pa en /au hen ic
s anda d)
Measu ed
masses o
MS/MS
agmen s (m/z)
T ans o ma ion
p oduc (TP)
Measu ed masses
o MS/MS
agmen s (m/z)
Compliance
o MS/MS
agmen s
(ppm)
E y h omycin
(pa en )
576.3749
E y h omycin
TP 576
576.3710 ([M+H]+)
-6.8
158.1179
158.1177
-1.3
116.1066
116.1042
-21
Cla i h omycin
(pa en )
590.3852
Cla i h omycin
TP 590
590.3818 ([M+H]+)
-5.8
558.3593
558.3537
-10
158.1172
158.1166
-3.8
98.0960
98.0963
3.1
A enolol acid
(au hen ic
s anda d)
191.0698
A enolol acid
191.0701
1.5
165.0542
165.0525
-10
145.0649
145.0649
0
91.0539
91.0550
12
56.0496
56.0494
-3.6
17
Re e ences
Radjeno ić, J., Pé ez, S., Pe o ić, M., Ba celó, D., 2008. Iden i ica ion and s uc u al
cha ac e iza ion o biodeg ada ion p oduc s o a enolol and glibenclamide by liquid
ch oma og aphy coupled o hyb id quad upole ime-o - ligh and quad upole ion ap
mass spec ome y. J. Ch oma og . A 1210, 142–153.
Te zic, S., Udiko ic-Kolic, N., Ju ina, T., K izman-Ma asic, I., Sen a, I., Mihalje ic, I.,
Lonca , J., Smi al, T., Ahel, M., 2018. Bio ans o ma ion o mac olide an ibio ics
using en iched ac i a ed sludge cul u e: Kine ics, ans o ma ion ou es and
eco oxicological e alua ion. J. Haza d. Ma e . 349, 143–152.
