BioSeNPs were produced by incubating anaerobic granular
sludge (13 g L1 wet weight) in an oxygen-free growth medium
at 30 C and pH 7.3 for 14 days. The growth medium and incubation
conditions were applied as these were successfully used for
the reduction of selenate using the same inoculum [26]. The
growth medium contained (in mg L1): NH4Cl (300.1), CaCl22H2O
(14.7), KH2PO4 (245.0), Na2HPO4 (283.9) and KCl (245.9). Acid and
alkaline trace elements and vitamins were not added to growth
medium to avoid their interaction with the formed BioSeNPs.
2.24 g L1 of sodium lactate and 0.86 g L1 of sodium selenite were
used, respectively, as carbon and selenium source. This medium
was flushed with nitrogen to maintain anaerobic conditions.
Anaerobic granular sludge from a full scale UASB reactor used for
treating paper mill wastewater in Eerbeek (The Netherlands),
described in detail by Roest et al. [27], was used as inoculum.
The production of elemental selenium was confirmed by the
appearance of red colorization of the medium. The supernatant
was collected by simple decanting and concentrated by centrifuging
(Hermle Z36 HK) at 37,000 g and 4 C. The pellet was re-suspended
in Milli-Q (18 MX cm) water and purified by the protocol
from Dobias et al. [28] with minor modification. Briefly, the concentrated
BioSeNPs were sonicated in ice cold water for 1 h at
100 watt and 42 kHz. NaOH (6 N) was added to raise the pH to
12.5 and the concentrated BioSeNPs were again sonicated at
42 kHz in ice cold water for 2 h to lyse any remaining biomass
present in the supernatant. The pH was lowered back to 7.3 by
addition of 1 N HCl. An equal volume of n-hexane was added and
the resultant mixture was kept overnight in a separatory funnel.
The BioSeNPs were collected from the aqueous phase and washed
three times with Milli-Q (18 MX cm) water. After washing, the
BioSeNPs were re-suspended in Milli-Q water and the pH was
adjusted to 7.3 by the addition of a few drops of 1 N NaOH before
adding them for adsorption experiments. The BioSeNPs preparation
was carried out in duplicate to ensure that the characteristics
of the nanomaterial were reproducible.