Impact of Physicochemical Characteristics of Wastewater

With a few exceptions (see Section 7.4), studies regarding the performance of WRF (whole-cell) and extracellular enzymes used synthetic wastewater matrix containing a mixture of a few TrOCs at higher concentrations than that environmentally relevant. Moreover, batch and continuous experiments were operated at controlled environmental conditions. However, performance of

WRF or enzyme based treatment systems in sterile and controlled environmental conditions does not reflect their true ability to treat real wastewater. Real wastewater may contain organic and inorganic impurities that can affect catalytic efficiency of WRF (whole-cell) and their enzymes. The effect of bulk dissolved organic and inorganic impurities on the performance of wholecell WRF and laccase is critically reviewed in this section. This information is particularly important to evaluate the feasibility of scaling up WRF-based treatment systems.

Operating conditions such as temperature, pH, aeration rate and hydraulic retention time (HRT) are important parameters affecting the performance of WRF reactors. Inadequate temperature and pH can destabilize enzymes. The optimum temperature and pH for an enzyme depends on the source of the enzyme. For instance, laccase extracted from T. versicolor (Schlosser and Hofer, 2002) and P. ostreatus (Palmieri et al., 2003) has been reported to show optimal activity at 55 and 35°C, respectively. Bertrand et al. (2015) suggested that the use of thermally stable laccase may enhance the rate of pollutant degradation and also prevent bacterial contamination in the bioreactor at elevated temperature. In general, most fungal laccases and peroxidases achieve optimal activity at 25-30 and 35-40°C, respectively.

The optimum pH for laccase from T. (Han et al., 2005; Lorenzo et al., 2005), P. sanguineus (Hilden et al., 2007) and A. blazei (Ullrich et al., 2005) was reported to be in the range of 3.0-4.5, 4.0 and 5.5, respectively. In general, optimal pH for fungal laccase ranges from 3.5-6.0, which is in line with the fact that fungi prefer acidic environmental conditions for their growth (Dwivedi et al., 2011). Optimal pH for the removal of steroid hormones has been reported to be 6.0 (Auriol et al., 2007), while the maximum removal of some PhACs such as diclofenac and ketoprofen was achieved at the pH range of 4.0-6.0 (Arboleda et al., 2013; Kim and Nicell, 2006b; Marco-Urrea et al., 2010c; Margot et al., 2013; Nguyen et al., 2014b; Wangetal., 2012).

While evaluating the feasibility of enzyme based treatment systems, other operating conditions such as enzyme and substrate concentration as well as aeration rate require a thorough consideration. Removal of PhACs does not always increase with the increase in enzyme concentration. As noted earlier, Lloret et al. (2012a) observed that the removal and degradation rate of steroid hormones did not improve beyond a laccase activity of 500 U/L. In another study by Spina et al. (2015b), the minimum effective activity of laccase from T. pubescens for the removal of 18 micropollutants including pharmaceuticals and steroid hormones was in the range of 100-250 U/L.

Laccase uses oxygen as the final electron acceptor for the oxidation of pollutants, making its availability vital in laccase-catalyzed reactions. Lloret et al. (2012a) suggested that intermittent supply of oxygen could sustain enzymatic oxidation of steroid hormones, thereby reducing the energy requirements of an enzymatic membrane bioreactor. The HRT of the enzymatic bioreactors should be carefully selected because longer HRT than that required will increase the capital cost in terms of larger tanks constructions. For instance, phenolic PhACs such as steroid hormones can be removed significantly (70-90%) in 4-8 h, while recalcitrant PhACs such as naproxen, ketoprofen and diclofenac may require longer HRT (>8 h) (Lloret et al., 2012a; Lloret et al., 2010; Lloret et al., 2013; Nguyen et al., 2015). Therefore, wastewater characterization should be carried out to select adequate HRT for the enzymatic bioreactors.

Real wastewater contains dissolved organic and inorganic impurities such as metal ions and organic solvents that can: (i) inhibit growth of WRF and their ability to secret extracellular enzymes; and (ii) inactivate extracellular enzymes (Asif et al., 2017a). Inhibition and inactivation of WRF (whole-cell) growth and ligninolytic enzymes subsequently affects the removal of pollutants. The effect of metal ions on WRF growth and enzymatic activity as well as their impact on the removal of recalcitrant pollutants such as PhACs, synthetic dyes, pesticides and poly aromatic hydrocarbons has been reviewed comprehensively by Baldrian (2003) and Asif et al. (2017a). Briefly, metals ions such as Cd+2 and Hg+2 could completely inhibit the growth and metabolic activities of WRF at concentrations of 0.01-0.2 and 0.05-0.25 mM, respectively, while essential metal ions such as Cu+2, Co+2, Mn+2, Zn+2 and Ni+2 can inhibit fugal growth at concentrations ranging from 50-300 mg/L. Metal ions, particularly Cd+2 and Hg+2, inhibit/inactivate fungal growth by: (i) increasing the lag phase; (ii) reducing the protein content of the mycelium; and (iii) rupturing the cell membrane, often referred to as cell lysis (Asif et al., 2017a; Baldrian, 2003). Inhibition of WRF growth also affects its ability to secrete extracellular enzymes. Hence, the use of purified enzymes instead of whole-cell cultures for the treatment of recalcitrant pollutants was recommended (Baldrian, 2003). Bhattacharya et al. (2014) observed that the growth of P. ostreatus was inhibited by 28%, 25%, 71%, 65%, 75%, and 88% following the addition of Zn+2, Mn+2, Ag+1, Cd+2, Hg+2 and EDTA, respectively, separately each at 5 mM. Moreover, the removal of a recalcitrant compound namely, benzo[a]pyrene, was significantly reduced (55-60%) in the presence of Ag+1, Cd+2, Hg+2, and EDTA (Bhattacharya et al., 2014).

Treatment of Real Wastewater by WRF and Ligninolytic Enzymes

Performance of enzymatic bioreactors for the removal of PhACs from real wastewater has been investigated in only a few studies (Auriol et al., 2008; Cruz-Morato et al., 2013; Spina et al., 2015a). For example, 80-95% removal of micropollutants such as 17a-ethinylestradiol, nonylphenol and bisphenol A from groundwater samples was reported by Garcia-Morales et al. (2015) following 12 h treatment with a crude laccases from Pycnoporus sanguineus. In another study by Spina et al. (2015a), removal of micropollutants including PhACs such as naproxen ketoprofen and salicylic acid by crude laccase from T. pubescens was observed to range between 70-99%. Tran et al. (2013) studied the performance of purified laccase from T. versicolor for the removal of an insect repellent (i.e., /V,A/-diethyl-meta-toluamide, DEET) from municipal wastewater, and achieved 55% removal. Because the available studies did not compare the performance with suitable "control,” the impact of specific wastewater constituents on removal performance could not be clarified. This prevents a uniform comparison of the results reported. However, Auriol et al. (2007) reported that removal of steroid hormones in municipal wastewater was reduced by 30-45% compared to their removal form solution in ultrapure water.

As noted in Section 7.2.4, addition of redox-mediators can improve the extent of PhAC removal by enzymes. However, redox-mediators generate highly reactive radicals that can interact with the active sites of enzymes and can inactivate enzymes

(Ashe et al., 2016; Tran et al., 2013). Similar to that in studies employing synthetic wastewater (Khlifi et al., 2010; Nguyen et al., 2016b), impact of redox-mediators on enzymatic activity was reported by Garcia et al. (2011) during the treatment of municipal wastewater. Although complete removal of oxybenzone from municipal wastewater was achieved after 6 h by a laccase— mediator (ABTS) system, the initial laccase activity was reduced by 64% at the end of the experiment. It is worth mentioning that the extent of laccase inactivation observed in laccase-mediator system treating oxybenzone in municipal wastewater was similar to that obtained during the removal of oxybenzone from synthetic wastewater (Garcia et al., 2011).

In addition to wastewater-derived interfering organic and inorganic compounds, performance of fungal species for the removal of PhACs is also affected by microbial contamination. Several studies have shed light on the effect of microbial contamination on the performance of fungal bioreactor by operating bioreactors under non-sterile environment using either synthetic (Nguyen et al., 2013; Yang et al., 2013a) or real wastewater (Badia-Fabregat et al., 2017; Cruz-Morato et al., 2013; Cruz-Morato et al., 2014; Ferrando-Climent et al., 2015; Jelic et al., 2012; Zhang and GeiRen,

2012). In a study by Yang et al. (2013a), removal of bisphenol A and diclofenac by whole-cell T. versicolor under non-sterile conditions was observed to be reduced significantly (40-50%) as compared to sterile batch experiments. They attributed the reduction in the removal of bisphenol A and diclofenac to microbial contamination that was confirmed via microbial analysis. Recently, the performance of fungal bioreactors containing T. versicolor or P. chrysosporium has been reported for the removal of PhACs from municipal and hospital wastewater (Badia- Fabregat et al., 2017; Cruz-Morato et al., 2013; Cruz-Morato et al., 2014; Ferrando-Climent et al., 2015; Jelic et al., 2012; Mir-Tutusaus et al., 2016; Zhang and GeiRen, 2012). However, microbial contamination did not allow long-term operation of the fungal bioreactors in the aforementioned studies. Notably, removal of PhACs achieved by fungal bioreactors operated under non-sterile conditions was lower than that observed in fungal bioreactors operated under sterile conditions.

Microbial contamination can affect whole-cell WRF bioreactors and enzymatic bioreactors by: (a) reducing the enzyme secretion capacity of WRF due to disruption in its growth, and (ii) consuming the enzymes produced by WRF (Espinosa-Ortiz et al., 2016; Libra et al., 2003; Yang et al., 2013a). Because bacteria are fastgrowing prokaryotes, they can easily outgrow WRF and prevail in fungal bioreactors (Hai et al., 2009; Libra et al., 2003). A number of strategies have been reviewed by Asif et al. (2017c) for the prevention of microbial contamination. These strategies include immobilized fungal growth (Hai et al., 2013), biomass renovation (Blanquez et al., 2006) and influent pretreatment (Mir-Tutusaus et al., 2016) as well as the use of micro-screen (Van Leeuwen et al., 2003) to allow bacterial washout. However, these strategies could only extend the operation of fungal bioreactors without bacterial contamination for a few days.

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