Oxidative stress: An unutilised mechanism for whole-cell biocatalysis of emerging wastewater micropollutants

Abstract

The inadequate removal of organic micropollutants (OMPs) such as pharmaceuticals, industrial chemicals, pesticides and personal care products by biological wastewater treatment processes into aquatic environments is an issue of global environmental concern. To minimise the release of these substances into the environment, I placed a special emphasis on investigating the microbial ability to degrade OMPs by exploiting an in-built mechanism that has so far not been utilised by microbial community. Cells growing aerobically experience oxidative stress created by the production of reactive oxygen species (ROS) – superoxide anion radical (O2•-), hydrogen peroxide (H2O2) and hydroxyl radical (OH•-), during aerobic respiration. To cope with the existing oxidative stress, microbes produce a host of enzymes (oxidoreductases such as peroxidases and cytochromes P450) to destroy ROS. These enzymes are known to efficiently destroy a wide range of OMPs. The goal of my study was to elucidate the regulation of oxidative stress mechanisms by inducing oxidative enzyme gene expression and the associated synthesis of enzymes for enhanced transformation of OMPs. My study on the adaptive response of microbes to oxidative stress was performed to answer different research questions using multiple high throughput ‘omics’ (genomics, proteomics and metabolomics), qPCR, spectrophotometry and chromatography techniques. First, what is the impact of dynamic variation in oxygen concentration on enzymatic OMP transformation by representative farm wastewater microbial consortia. Second, how does the characteristics of perturbations in dissolved oxygen (DO), such as DO stress intensity and variation in oxygenation and deoxygenation phases over the time, effects the physiology and functionality of bacterial communities. Third, what magnitude of oxidative stress is necessary for inducing the adaptive responses in mixed and pure microbial species by direct exposure to differential levels of ROS (paraquat and hydrogen peroxide). Fourth, how does the perturbation index reprogram the intracellular carbon metabolic pathways to enhance the oxidative stress genetic responses in Pseudomonas putida KT2440. The last component of my research explored a new interdisciplinary field, microbial endocrinology, to correlate what influence a neuroactive compound, L-norepinephrine (L-NE), has on the generation of oxidative stress and enhanced bacterial growth in farm and municipal sludge. A potential of cyclic oxygen perturbations on farm and municipal wastewater microbial studies demonstrated the distribution of core proteobacterial families (Pseudomonadaceae, Rhodocyclaceae and Comamonadaceae) over the DO perturbation ranges with a maximum abundance in cultures treated with 0.25 cycles/hour-DO at continuous high-DO aerobic range. The higher relative abundance of OxyR and SoxRS oxidative stress genes justifies the upregulation of oxidoreductases (peroxidases and cytochrome P450) in mixed cultures treated with 0.25 cycles/hour-DO. The significant removal (70-90%) of hard-to-degrade OMPs mixture by mixed cultures at above DO range explains the enhanced enzymatic expressions during the interphase of aerobic and microaerobic zones. The threshold of ROS mediated oxidative stress in mixed wastewater microbial cultures and pure species (bacterial and fungal) was determined by quantifying the expression and abundance of soxRS and oxyR oxidative stress genes and regulation of oxidoreductases activities with increasing ROS levels (2.5 – 150 μM). The expression of SoxRS- and OxyRmediated antioxidant proteins (peroxidases, superoxide dismutase, catalases and cytochrome P450) were elevated in cultures exposed to ROS ranging from 10-150 μM. This reveals that high ROS is required for triggering the disturbance in intracellular ROS steady state that activates and elevates the expression of antioxidant functions consequently improving the biodegradation of OMPs (80-90%). The expression of different oxidoreductases in Pseudomonas putida KT2440 during oxidative stress is intrinsically linked to intracellular ROS and electron flow balance. Metabolite profiling of different carbon oxidation pathways activated during transitions from aerobic to microanoxic phases provides the electron equivalents required for univalent reduction of oxygen to ROS. Higher ROS levels and higher relative abundance of oxidative stress genes (oxyR) and upregulation of anti-oxidative stress biocatalysts such as peroxidases, laccases and cytochrome P450 was significant in continuous DO perturbations compared to discontinuous perturbations. Hence, it indicates that continuous perturbations best explain the ability of bacteria to cope with oxidative stress by enhanced protein translations. Correlation of increasing bacterial growth in mixed farm and municipal sludge cultures to the generation of oxidative stress in the presence of a neuroactive compound (5×10-5 M L-NE) coincided with an increase in the concentrations of autoinducers of growth and higher expression of oxidoreductases. I confirmed a higher relative abundance of autoinducer synthesising LuxS genes in exponentially growing cultures at 24 h. The expression of oxyR genes justifies the maximum oxidoreductase activities at 5×10-5 M L-NE. The abundance of major oxidoreductase producing and OMP degrading proteobacterial families (Pseudomonadaceae, Rhodocyclaceae, Enterobacteriaceae, Sphingomonadaceae and Comamonadaceae) correlates to their influence in OMP degradation under L-NE treatment. My research concludes that exposing wastewater microbial species to various source of oxidative stress can affect the frequent occurrence and abundance of microbial species across different DO perturbations, induce de novo synthesis of oxidative biocatalysts and subsequently enhance OMP removal. My thesis identified and provides a new perspective for improving the underutilized biological mechanisms for wastewater treatment plants. Moreover, enhancing the performance of existing wastewater treatment systems under conditions that provide directed environmental stresses that result in over-production of beneficial OMPdegrading biocatalysts.