1. Introduction:
Fruit and vegetable wastes (FVW) constitute one of the major organic wastes production from different sources and in Western Australia, around 30% of production end up as waste 1. The disposal of FVW in large quantities in landfills is difficult due to its high biodegradable organic content and subsequent environmental impacts2,3. On the other hand, anaerobic digestion has been proven to be an efficient technology for the treatment of organic solid wastes and energy recovery. Therefore, one of the alternatives to landfills of these large quantities of FVW is utilization and valorization of the residue in the production of biogas and high-quality organic-rich digestate through anaerobic co-digestion process. Anaerobic digestion involves biochemical degradation of organic wastes by microorganisms in an anoxic environment with the production of methane and carbon dioxide as major gases. In recent years, various researches are undertaken to address the challenges and process optimization of anaerobic digestion for its efficient application in waste management4. Anaerobic digestion involves four major biochemical reactions, viz, hydrolysis, acidogenesis, acetogenesis and methanogenesis; separation of respective microbial communities and facilitating them with optimum conditions help to reduce the limitations of anaerobic digestion 5.
Fermentation of FVW in anaerobic digestion faces severe acidification issues that inhibit the growth of methanogenic population due to its high sugar content and volatile solids (VS) fraction6. Moreover, the high C/N ratio of FVW leads to nitrogen depletion in anaerobic digestion and requires an additional substrate that can sustain fermentation efficiency 3. For these reasons, co-digestion of FVW with complementary substrates such as kitchen waste, agricultural waste and sewage sludge have been tried out 3. The co-digestion of sewage sludge with FVW have shown improvement in biogas yield and agronomic digestate quality 7,8. Furthermore, various authors have investigated two-stage anaerobic digestion under mesophilic conditions to improve the biogas production and sustain high OLR (organic loading rate) 9–11. Phase separation of microbial population offers better control over the respective phases, increases the specific activity of methanogens and also improves the suspended solids removal efficiency 12. Besides, different reactor configurations for phase separation of the acidogenic and methanogenic population have also been studied 13,14. In most of the studies, an acidifier (pre-methanizer) reactor helps in stabilization of methanogenic population in methanizer reactor while handling solid wastes with high organic content. And, the overall retention time can also be reduced in such two-phase systems compared to conventional anaerobic digestions.
Besides the novelties of reactor configurations and co-digestions, various pre-treatments of solid wastes have also been studied to overcome the rate-limiting hydrolysis step and enhance anaerobic digestion performance 15. Pre-treatments breakdown macromolecular structures and facilitate sludge hydrolyzation. In the case of polysaccharide-rich solid wastes (FVW), the lignin barrier present in skins and seeds makes bacterial hydrolysis difficult16. Among various FVW pre-treatments, thermal hydrolysis has been established as a prominent pre-treatment technique with enhanced sludge solubilisation, digestion performance and improved dewaterability 17,18. Recently, microwave enhanced advanced oxidation process has gained huge research attention as a pre-treatment technique for its effectiveness in hydrolyzation, biogas production and effluent digestate quality 19,20. The synergetic effect of microwave and hydrogen peroxide (MW-H2O2) have been shown to improve sludge solubilisation and also significantly affect biogas production from activated sludge samples 20–22. Apart from the initial sludge solubilisation through thermal effect, microwave enhanced advanced oxidation process (MW-H2O2) also imparts oxidative stress on the microbial population mediated by reactive oxygen species (ROS) 22,23. During the oxidation process, highly reactive oxidative intermediates such as hydroxyl [OH] and superoxide radicals [\(O_{2}^{-}]\) are generated, that oxidize the organic pollutants and also intermediate intracellular oxidative stress in the microbial population 23. Evaluation of such oxidative intermediates helps us to understand the effect of hybrid (MW-H2O2) pre-treatment on microbial sludge activity and expands the knowledge on oxidative stress in sludge microenvironment.
Previous studies on hybrid (MW-H2O2) pre-treatment were mainly focused on anaerobic digestion of activated sludge samples under mesophilic condition. The effects of hybrid (MW-H2O2) pre-treatment differs dependent on the nature of the feedstock. And also, hybrid treatment of activated sludge under acidic conditions has shown improved methane production in mesophilic anaerobic digestion 20. The acidic nature of FVW has the potential to enhance the effects of hybrid pre-treatment in anaerobic co-digestion with activated sludge. Hence, in the present study, the impact of hybrid (MW-H2O2) pre-treatment on anaerobic digestion efficiency of FVW co-digested with mixed activated sludge is studied. Besides, a two-stage anaerobic digester was operated to separate hydrolysis-acidogenesis in phase I (thermophilic condition) and methanogenesis in phase II (mesophilic condition). Two different mixing ratios of FVW and mixed activated sludge were evaluated based on biomethanation and process stability, followed by the studies of hybrid (MW-H2O2) pre-treatment. The impact of hybrid (MW-H2O2) pre-treatment on anaerobic digestibility of co-digestion is evaluated based on biogas production, biogas quality, sludge hydrolyzation and process stability. In addition, the effects of pre-treatment on the oxidative status of anaerobic digestion mediated by superoxide radicals were studied along with sludge bioactivity to establish knowledge on reactive oxygen species in anaerobic digestion.