3.2 Effect of hybrid (MW-H2O2) pre-treatment in anaerobic digestibility of FVW in co-digestion:
3.2.1 Biogas production
The efficiency of hybrid (MW-H2O2) feed sludge pre-treatment in enhancing anaerobic digestibility of co-digestion (MWAS+FVW) was studied in two-stage anaerobic digesters with the co-digestion ratio 3:1 (MWAS: FW). Microwave enhanced advanced oxidation process is an effective method in improving sludge solubilisation and consequently promotes biomethanation in waste activated sludge 20. In the current study, hybrid (MW- H2O2) pretreatment significantly improved the biogas production and biogas quality in co-digestion (3:1). The pretreated co-digestion (3:1) yielded 2.17-fold higher cumulative methane yield compared to untreated co-digestion (3:1) at the end of 20 days (Figure 1a). 2.91-fold higher methane production was achieved in thermophilic phase I of treated digester compared to untreated co-digestion (3:1). Besides, the biogas quality achieved in pre-treated sludge fed thermophilic digester was also significantly improved. On an average, treated phase I digester maintained 0.73 CH4/CO2 ratios, whereas untreated digester suffered a low 0.21 CH4/CO2ratio (Figure 1b). On average, the phase-II biogas quality was improved with CH4/CO2 ratio of 1.55 in the treated digester, whereas untreated digester maintained 1.05.
Literature reports suggest that pre-treatments enhance anaerobic digestibility of FVW by accelerated hydrolyzation and breakdown of macromolecules 3. Zhou et al, reported 115% increased anaerobic digestion performance and a 2-fold increase in digestion rate in thermally pre-treated co-digestion involving FVW compared to untreated digestion 17. Quite recently, Shanthi et al, reported a 31% increase in biomethane yield in anaerobic digestion of FVW by combined (Dimethyl sulfoxide and ultrasonication) pretreatment16. Moreover, combined (MW-H2O2) pretreatment achieved an 8.72-fold increase in biogas production in anaerobic digestion of dairy wastewater 20. The study also reported that combined (MW-H2O2-acid) pretreatment enhanced biogas production by 9.78-fold, emphasizing the efficiency of combined treatment under acidic conditions. In the current study, the hybrid (MW-H2O2) pre-treated FVW co-digestion outperformed untreated digestion in both phases, making it be an appropriate pre-treatment technique for organic content-rich substrate. The untreated digestion showed a lag phase in biogas production during the thermophilic condition, whereas the treated digestion exhibited an exponential increase due to the soluble substrate available for the methanogenic population (Figure 1a). Most of the studies on pretreated anaerobic digestion, suggests that enhancement in biogas production is mostly due to the initial hydrolyzation achieved through pretreatment. Besides, phase separation in anaerobic digestion of FVW has been highly efficient due to the process stability and enhanced biomethanation31,39. Moreover, the improved methane production in both phases in treated digestion could be due to hydrogenotrophic and syntrophic acetate oxidizing microbes, as the latter has a significant effect at high VFA (>1000 mg/l) concentration34.
3.2.2 Sludge solubilisation and digester performance
In the current study, the FVW was initially blended and homogenized to facilitate anaerobic digestion. Consequently, the mixed co-digested sludge achieved an initial COD solubilisation of 22% which was 2.2 folds higher than pure MWAS anaerobic digestion reported in our previous study 22. Application of hybrid (MW-H2O2) pre-treatment on mixed sludge further enhanced COD solubilisation by 33% facilitating easily available soluble organic content for subsequent utilization by acidogenic microbial population (Figure 3 and 4). The microwave enhanced oxidation process exhibits oxidative stress on anaerobic digestion by the generation of reactive oxygen species (ROS), that oxidize organic microbial fractions and causes cell wall breakdown 23. The efficiency of hybrid (MW-H2O2) pre-treatment on optimum sludge solubilisation of organic compounds without the formation of recalcitrant products is very much dependent on the dosage of hydrogen peroxide 24,40. An optimum sludge solubilisation involves the disintegration of EPS matrix and dissolution of proteins and carbohydrates fraction of activated sludge, without the formation of melanoidins through Maillard reaction23. In the current research, the dissolution of proteins and carbohydrates was consistent with the COD solubilisation as per the equivalent relationships: 1.5 gCOD/ gProtein and 1.06 gCOD/ gCarbohydrate 41. Accordingly, the soluble protein was observed to be 18.35% and 39.40% of sCOD in the untreated and treated digester respectively; soluble carbohydrate constituted 40% and 42% of sCOD in the untreated and treated digester, respectively. As the feedstock constitutes 25% FVW, the solubilized fraction of carbohydrates was observed to be significantly higher than the proteins (Figure 4). Application of pretreatment enhanced protein solubilisation by 177% and carbohydrate solubilisation by 18% (Figure 4). This indicates that the application of (MW-H2O2) pre-treatment affects EPS disintegration more than the organic constituents of FVW, as EPS constitutes more than 43% protein and 10-18% of carbohydrate in its composition 40. The higher COD solubilisation and EPS disintegration would accelerate the subsequent anaerobic digestion in the two-stage digesters.
The digester performance of the two-stage anaerobic digestion with and without hybrid treatment was compared with VFA accumulation and FOS/TAC ratio (Figure 2). VFAs are common inhibitors of anaerobic digestion and their accumulation within the digesters lead to process instability. In the current research, no significant difference in the VFA accumulation was observed in the initial thermophilic phase of both two-stage systems (Figure 2a). However, the FOS/TAC ratio was 77% higher in the untreated digester compared to the treated digester, during this phase (Figure 2b). The improved digester performance in the thermophilic phase of treated digester could be due to the buffering capability of microbial intracellular compounds released during the hybrid (MW-H2O2) pretreatment. Zhou et al, reported a similar phenomenon, wherein despite an increase in VFA accumulation by thermal hydrolysis pre-treatment, the digester maintained stable pH and VFA/alkalinity levels compared to the untreated digester, indicating the buffering ability achieved during the pretreatment 17. Besides, the increased nitrogen mineralization with co-digestion (3:1) along with the pre-treatment could also increase the buffering capability of the system42. The mesophilic phase II anaerobic digestion significantly improved FOS/TAC ratio in both the systems with the treated digester achieving an average of 0.26, which is well within the acceptable range of stable anaerobic digestion. Although, Di Maria et al, reported stable anaerobic digestion with FOS/TAC value <0.1 in a co-digestion of FVW with sludge under mesophilic conditions 8.
3.2.3 Oxidative status and sludge activity
Microwave enhanced advanced oxidation process is known for the destruction of microbial cell walls and oxidation of numerous recalcitrant organic pollutants present in wastewater through the formation of hydroxyl radicals [OH]43. An optimum concentration of H2O2 pretreatment leads to the formation of [OH] radicals, the highly reactive and strongest oxidant among H2O2 derivatives; higher concentration of H2O2 might lead to the formation of perhydroxyl radicals [\(\text{HO}_{2\ }]\), which is less reactive and decreases the release of soluble organics20 according to equation 4.
\(H_{2}O_{2}+\text{OH}\leftrightarrow\ \text{HO}_{2\ }+H_{2}\text{O\ }\)equation (4) Apart from the ’Fenton’- driven hydroxyl radicals, there is another short-living oxidative intermediate, namely, superoxide [\(O_{2}^{-}]\) that are prominent in oxidation reactions involving hydrogen peroxide 23.
The importance of anaerobic free radical formation has been underestimated in the field of wastewater treatment. Anaerobic microorganisms can encounter free radical formation even under strictly anaerobic/microaerobic conditions, and they have developed radical scavenging mechanisms (i.e, SOR; superoxide oxidoreductases) to neutralize such radicals 44. In the current study, the oxidative stress induced by hybrid (MW-H2O2) pretreatment was analyzed by the measurement of superoxide and the corresponding sludge bioactivity by the measurement of TTC-DHA activity (Figure 5). TTC-DHA activity helps to identify the biological activity of anaerobic digestion, through the respiratory activity of the active microbial population. Surprisingly, on day 1, the intracellular superoxide s[\(O_{2}^{-}]\) levels of the untreated digester were observed to be 81% higher than the hybrid (MW-H2O2) pre-treated digester (Figure 5a). A plausible explanation of this phenomenon could be the formation of more reactive hydroxyl radicals [OH] through Haber-Weiss type reaction (equation 5), in the treated digester that could account for the decrease in superoxide levels45.
\(O_{2}^{-}+H_{2}O_{2}\ \rightarrow\ \text{OH}+HO^{-}+O_{2}\)equation (5)
The mechanism of Haber-Weiss reaction makes use of Fenton chemistry and the catalysis requires the presence of ferric ion in sludge. Mixed sludge being a source of various indigenous metal ions becomes a suitable target for Haber-Weiss reaction and accelerates the production of the highly reactive OH radicals 46. The high s[\(O_{2}^{-}]\) levels in untreated digester in day 1 could be due to the oxidative stress experienced by aerobic living cells of activated sludge when they are exposed to anoxic conditions. In the subsequent days, s[\(O_{2}^{-}]\) decreases in untreated digester as the anaerobic microbial population takes over the entire digestion. A delayed increase in s[\(O_{2}^{-}]\) levels was observed in day 3, in the treated digester similar to our previous reported results 22, which confirms that the oxidative stress exerted by hybrid (MW-H2O2) pre-treatment has a prolonged effect in anaerobic digestion (Figure 5a). On comparison of the TTC-DHA activity, the treated digester exhibited 127% higher bioactivity during the initial thermophilic phase, which is concurrent with the increased methane production and process stability achieved in the treated digester (Figure 5b, 1 and 2a). On transfer to the mesophilic anaerobic digester, an improvement in methanogenic activity is observed in both the systems (Figure 5b). And s[\(O_{2}^{-}]\) levels are diminished during this peak methanogenesis (day 6 to 12) period in both the systems (Figure 5a). Moreover, an increasing s[\(O_{2}^{-}]\) trend is observed as the digestion comes to completion which is marked by decreasing TTC-DHA activity (Figure 5). Briefly, the generation of s[\(O_{2}^{-}]\)radicals are downregulated during active methanogenesis and observed to increase at dormant sludge activity.