SUPPLEMENTARY MATERIAL
Environmental Science and Pollution Research
Assessing the use of nanoimmobilized laccases to remove micropollutants from wastewater
A. Arca-Ramos*1, E. M. Ammann2, C. A. Gasser2, P. Nastold2, G. Eibes1, G. Feijoo1, J.M. Lema1,
M.T. Moreira1, P. F.-X. Corvini2
1
Dept. of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, 15782
Santiago de Compostela, Spain
2
Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts
Northwestern Switzerland, Gruendenstrasse 40, Muttenz 4132, Switzerland
*Corresponding author: Tel.: +34881816773 +; fax: +34881816702
E-mail address: adriana.arca@rai.usc.es
1.
Schematic process of laccases immobilization
NH3+
OH
APTES
NH3+ Lac
Laccase
H2N
N
N
CH
Lac3
Glutaraldehyde
Figure S1 Scheme of the functionalization of the fsNP with 3-aminopropyltriethoxysilane (APTES) and
covalent immobilization of adsorbed laccase with glutaraldehyde.
The global procedure comprises the following steps:
1) Functionalization of the fsNP with APTES
2) Addition of laccase (sorption step).
3) Covalent immobilization using glutaraldehyde as cross-linking agent.
4) Washing of nanobiocatalyst after 24 h.
2.
Sample treatment and HPLC analysis of radiolabeled compounds
In order to determine the residual concentration of
14
C-Bisphenol A (BPA) and
14
C-Diclofenac (DCF)
samples, the 2 mL-samples were immediately processed with solid phase extraction (SPE) cartridges
OASIS HLB 6 cc (for BPA determination) and OASIS MCX 3cc (for DCF determination after acidification
with sulfuric acid), which had been previously conditioned with heptane-acetone-methanol and water
(at pH 7.5 for HLB cartridges and pH 2.8 for MCX cartridges). The cartridges were dried by a nitrogen
stream. Subsequently, the substances retained in the OASIS HLB cartridges were eluted four times with
2 mL methanol and the solvent was evaporated by heating at 64ºC under a moderate nitrogen stream.
The substances retained in the OASIS MCX cartridges were eluted twice with 2 mL of acetone and then
the solvent volume was reduced to approximately 500 µL by heating at 56ºC under a moderate nitrogen
stream. Subsequently, 1 mL of methanol was added and the evaporation process went on until the
complete depletion of the solvent. Residues were carefully re-dissolved in 100 µL acetonitrile and
transferred into HPLC vials for further analysis. Liquid chromatography analysis was conducted using an
HPLC 1200 Series (Agilent Technologies, Switzerland) coupled to a radioisotope detector “Ramona Star”
(Raytest, Germany). A Nucleodur C18 Pyramid column (150x4 mm, 3 µm particle size, Macherey and
Nagel) was thermostated at 40ºC and the flow rate was set to 0.8 mL/min. Aliquots of 50 µL recovered
from the SPE were injected in the HPLC and eluted with a water:acetonitrile gradient starting with a
water fraction of 55%, which was decreased to 2% in 17 min. Elution solvents were acidified with formic
acid to a final concentration of 0.1%.
Figure S2
Figure S2. Residual radioactivity (% initial) in the reaction media after 24h.
Table S1
Table S1. Total radioactivity associated to the reaction media (RM) and sodium hydroxide (CO2 traps) for
the treatment of 14C-BPA by free TvL and in control lacking enzyme (n=2).
Time (h)
Control
RM(Bq)
Control
(NaOH)(Bq)
Free TvL treatment
RM (Bq)
Free TvL treatment
(NaOH)(Bq)
0
4069 ± 56
-
3735±79
-
16
4158 ± 23
1.9±0.6
2141±261
14.0±1.1