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.