2. Immobilised-laccase bioreactors for wastewater treatment
There are several bioreactor configurations that can be used with
immobilised laccase enzymes for the removal of pollutants from
wastewater (e.g., stirred tank, fixed bed, fluidised bed and membrane).
The selection of a determined configuration and the operation strategy
will depend on the reaction kinetics and the properties of the
immobilisation carrier.[14] In Figure 2 different
reactor configurations usually utilised for immobilised-laccase
bioprocesses are schematically depicted. Likewise, in Table 2 the
advantages and drawbacks of each reactor configuration are presented.
Despite the numerous publications about laccase immobilisation and its
applications in the removal of pollutants, there are few papers
reporting the removal of pollutants by immobilised laccases in
bioreactors. In Table 3 recent published research on immobilised-laccase
bioreactors for wastewater treatment and removal of pollutants from
aqueous solutions is gathered.
Ahmad et al. (2020) studied the degradation of tetracycline (20 mg/L) by
laccase from T. versicolor immobilised by covalent grafting on
silica monoliths in a plug tubular reactor operating at a flow rate of 1
mL/min with continuous recycling.[15] They found
that tetracycline was degraded by 40-50% in 5 h. Additionally, the
silica monolith immobilised laccase presented high operational stability
during 75 h which, according to the authors, indicated the applicability
of the developed reactors on a large scale. However, the scalability of
such minireactors is dubious.
Ladole et al. (2020) immobilised laccase enzymes in peroxidase mimicking
magnetic metal organic frameworks (MMOFs).[16] The
immobilised biocatalysts (laccase@MMOFs), with a particle size below 100
nm, were placed in a fixed-bed reactor (working volume 50 mL) and tested
for the degradation of the industrial dyes Methylene Blue (MB) and
Crystal Violet (CV) in continuous mode. The former was degraded by 96%
and the later by 98% in 15 min and from there onwards degradation was
kept steady. However, a considerable amount of dye removal (47% for MB
and 56% for CV, in 15 min) was due to MMFOs. Therefore, laccase was
only responsible for the additional dye removal (about a half) which
make the developed bioprocess questionable. In addition, the authors
indicated neither the dye concentrations nor the hydraulic retention
time used.
López-Barbosa et al. (2020) immobilised crude laccase fromPycnoporus sanguineus on silanised silica nanoparticles,
synthesised in the presence of either water or acetone, by covalent
binding with glutaraldehyde.[17] The immobilised
laccase was accommodated in a flow reactor configuration and tested for
the continuous decolouration of the dye Congo Red (CR). They found that
CR (7 g/L) was removed by 39% with the nanoparticles synthesised in the
presence of acetone. However, it was not indicated whether some amount
of dye was adsorbed on the nanoparticles. Likewise, neither the working
volume nor the hydraulic residence time (HRT) of the reactors were
provided which makes difficult to assess the scale-up feasibility of the
developed system.
Yuan et al. (2020) compared the efficiency of a horizontal rotating
reactor (HRR) with laccase immobilised on strips of bacterial
nanocellulose and a vertical mixing reactor (VMR) with laccase
immobilised on wafers of bacterial nanocellulose for the decolouration
of the textile dye Reactive Blue 19 (RB19).[18]The total volume of both reactors was 600 mL. The HRR showed a much
better performance for the RB19 decolouration than the VMR. Thus, it led
to about a 2-fold higher RB19 decolouration over a wider temperature
range together with higher reusability and detoxification than those
obtained by the VMR. This was likely due to the HRR provided higher
oxygen availability and larger contact area than the VMR. However, in
both cases the addition of the synthetic mediator 1-hydroxibenzotriazole
was required which is neither economic nor ecological.
Zdarta et al. (2020) used a packed-bed reactor with laccase immobilised
by adsorption on 3D chitin scaffolds for the continuous removal of the
antibiotic tetracycline (1 mg/L). However, neither the volume of the
reactor nor the HRT were indicated. Thus, it is difficult to assess the
real potential of the developed approach.[19]
Girelli et al. (2021) utilised a packed-bed reactor with laccase
immobilised by covalent binding on silica-chitosan carriers for the
removal of phenol and a mixture of phenolics (4-methylcatechol,
catechol, caffeic acid, syringic acid, vanillic acid, p-coumaric acid,
and tyrosol) in continuous mode at a flow rate of 0.7
mL/min.[20] They found that phenol (525 mg/L) was
degraded by 33% in 8 h and by 49% in 14 h and the phenolic mixture
(525 mg/L) by 90% in 21 h. However, the volume of the reactor used was
very small (about 16 mL) which makes the feasibility of the developed
approach for large scale applications questionable.
Masjoudi et al. (2021) investigated mini-membrane reactors (working
volume 50 mL) with laccase immobilised by covalent binding on
polyvinylidene fluoride (PVDF) membranes modified with multi-walled
carbon nanotubes (MWCNTs) for the removal of carbamazepine (5 mg/L) and
diclofenac (5 mg/L).[21] They observed degradation
efficiencies of 27% in 48 h for the former and 95% in 4 h for the
latter and suggested that the developed system had potential for large
scale water treatment. However, without scaling up and economic studies,
such assertion is debatable.
Xia et al. (2021) tested a fixed-bed reactor with laccase immobilised on
polyethylenimine functionalised magnetic nanoparticles for the removal
of phenol in continuous mode.[22] They found that
the degradation rate was kept over 70.3% in 48 h when operated under
optimal conditions (15 mg laccase nanoparticles, 50 µg/mL phenol and 25
µL/min flow rate). Nonetheless, the volume of the solution treated was
very small (27 mL) making difficult to evaluate the viability of the
system for large scale applications.
Yamaguchi and Miyazaki (2021) studied the removal of the endocrine
disruptor BPA (100 µM) by laccase immobilised by cross-linking on
polyethylene glycol acrylamide (PEGA) resin in batch and flow
reactors.[23] They found a BPA removal of 144 µM/h
at 30ºC in the former and of 2880 µM/h at 50ºC in the latter. However,
the volume of the batch reactor was not mentioned and that of the flow
reactor was very tiny (a polytetrafluoroethylene tube of 39.25 µL).
Therefore, the feasibility of the developed system for large scale
applications is very uncertain.
George et al. (2022) investigated the removal of trace organic
contaminants (TrOCs) from the secondary effluent of municipal wastewater
by cross-linked laccase aggregates (CLEAs) in a 2-L perfusion reactor
that operated in continuous mode for 500 h.[24]They reported that 60 min of operation was required to obtain the steady
state for the maximum degradation (93%). The obtained results were
promising but issues related to the decrease of laccase activity along
time need to be solved.
Jankowska et al. (2022) built a novel enzymatic membrane reactor
consisting of a nanofiltration or ultrafiltration membrane combined with
laccase encapsulated in sodium alginate beads and entrapped between
polystyrene electrospun fibers.[25] The developed
approaches were tested for the decolouration of the azo dyes Acid Yellow
23, Direct Blue 71 and Reactive Black 5 at a concentration of 5 mg/L
each and a working volume of 20 mL. They found a decolouration of almost
100% of all tested dyes during 3 successive cycles which was due to a
synergistic action between the membrane and the biocatalyst. In
addition, a toxicity (Artemia salina test) decrease of about 70%
was shown. However, ABTS (0.5 mM) was used as a redox mediator which is
toxic and expensive. Also, the treated volume (20 mL) was very small
which makes difficult to assess the practicability of the developed
biosystem to treat industrial wastewater volumes.
Lassouane et al. (2022) applied cross-linked crude laccase fromTrametes pubescens entrapped in calcium alginate beads to remove
BPA in a 2-L fluidised-bed reactor during 3 successive batches of 10 h
each with increasing BPA concentrations (60, 80 and 100
mg/L).[26] A BPA degradation higher than 75%
after the third successive cycle was attained, indicating the
operational stability of the developed biocatalyst. Also, the addition
of redox mediators was not necessary. Moreover, BPA removal was only due
to laccase action since BPA adsorption onto the carrier was negligible.
Therefore, the developed approach seems very promising for the removal
of xenobiotics in continuous mode on a large scale.
Mehandia et al. (2022) co-immobilised a partially purified bacterial
laccase and the natural mediator acetosyringone by entrapment in
chitosan-clay composite beads.[27] The immobilised
laccase mediator system was placed in a packed-bed reactor and applied
to treat a real textile effluent operating in continuous mode. However,
the volume of the reactor used was very small (about 35 mL), so more
studies on a larger scale are required to test the real potential of the
developed approach.
Shen et al. (2022) developed a directional microreactor with laccase
immobilised by covalent binding in internal channels made of delignified
wood treated with dimethylacetamide/ lithium chloride (DMAc/LiCl) which
showed its feasibility for the removal of 4-nitrophenol (0.1 mM;
4-NP).[28] Thus, they found a removal rate of
94.4% of 4-NP in only 30 min and an efficiency of 86.9% was kept after
25 cycles with no evidence of laccase inactivation. The authors stated
that the developed reactor was simple to prepare and easy to scale up
showing great commercial application. Nevertheless, they only used 5 mL
of 4-NP solution which makes their asserted suitability for industrial
volumes difficult to envisage.
Sotelo et al. (2022) immobilised laccase from Pycnoporus
sanguineus by encapsulation in alginate microbeads and by covalent
binding on alumina pellets.[29] The immobilised
laccases were accommodated in flow microreactors (160 µL) and assessed
for the continuous removal of acetaminophen from an artificial
wastewater. They found that acetaminophen (18 mg/L) was removed by 72%
and 15% by the alginate and the alumina immobilised laccase,
respectively, for an HRT of 30 min and a flow rate of 2 mL/h. The
authors attributed the better catalytic performance of laccase
immobilised into alginate microbeads to their higher porosity. However,
the feasibility of scaling up the developed system is open to question.
Trivedi and Chhaya (2022) prepared a laccase nanoemulsion with a
commercial laccase from T. versicolor , the surfactant sodium
bis(2-ethylhexyl) sulfosuccinate and the organic solvent
2,2,4-trimethylpentane (isooctane).[30] Then, this
laccase nanoemulsion was encapsulated in alginate beads and the produced
biocatalysts were tested for the removal of the endocrine disruptor BPA.
They found that the immobilised laccase was able to remove 94% of BPA
in a packed-bed reactor operating a flow rate of 15 mL/h and a HRT of 2
h. In addition, the developed biocatalysts were able to remove 60% and
67% of BPA (200 mg/L) from a real industrial effluent in a packed-bed
reactor (150 mg of beads and 50 mL of BPA solution) operating in batch
and continuous mode, respectively, in 4 h of reaction time.