Aerobic Toluene Degraders in the Rhizosphere of a Constructed Wetland Model Show Diurnal Polyhydroxyalkanoate Metabolism

Abstract

Constructed wetlands (CWs) are successfully applied for the treatment of waters contaminated with aromatic compounds. In these systems, plants provide oxygen and root exudates to the rhizosphere and thereby stimulate microbial degradation processes. Root exudation of oxygen and organic compounds depends on photosynthetic activity and thus may show day-night fluctuations. While diurnal changes in CW effluent composition have been observed, information on respective fluctuations of bacterial activity are scarce. We investigated microbial processes in a CW model system treating toluene-contaminated water which showed diurnal oscillations of oxygen concentrations using metaproteomics. Quantitative real-time PCR was applied to assess diurnal expression patterns of genes involved in aerobic and anaerobic toluene degradation. We observed stable aerobic toluene turnover by Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis was upregulated in these bacteria during the day, suggesting that they additionally feed on organic root exudates while reutilizing the stored carbon compounds during the night via the glyoxylate cycle. Although mRNA copies encoding the anaerobic enzyme benzylsuccinate synthase (bssA) were relatively abundant and increased slightly at night, the corresponding protein could not be detected in the CW model system. Our study provides insights into diurnal patterns of microbial processes occurring in the rhizosphere of an aquatic ecosystem. IMPORTANCEConstructed wetlands are a well-established and cost-efficient option for the bioremediation of contaminated waters. While it is commonly accepted knowledge that the function of CWs is determined by the interplay of plants and microorganisms, the detailed molecular processes are considered a black box. Here, we used a well-characterized CW model system treating toluenecontaminated water to investigate the microbial processes influenced by diurnal plant root exudation. Our results indicated stable aerobic toluene degradation by members of the Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis in these bacteria was higher during the day, suggesting that they additionally fed on organic root exudates and reutilized the stored carbon compounds during the night. Our study illuminates microbial processes occurring in the rhizosphere of an aquatic ecosystem. C onstructed wetlands (CWs) are well-established treatment systems for the bioremediation of contaminated waters (1), including industrial effluents originating from petroleum refineries and their associated contaminated sites (2, 3). Aromatic hydrocarbons present in those wastewaters can effectively be removed in CWs (4-7), largely driven by the interplay of plant physiology and microbial degradation processes in the rhizosphere (8, 9). As in any planted habitat, the rhizosphere of CWs is a complex environment with increased abundance and activity of microorganisms compared to those in the surrounding zones (10). It is shaped by the roots and plant-derived exu...

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model

“…Within this PFR, a circulating flow regime prevents the formation of large chemical gradients and an efficient turnover of organic carbon occurs [13]. In previous studies on a PFR running for several years with toluene as sole external carbon source, it was shown that toluene was mainly degraded aerobically by members of the Burkholderiales [14, 15]. However, several cultivation-independent analyses suggested that microbes able to degrade toluene anaerobically were also plentiful.…”

Microbial Community Assessment in Wetlands for Water Pollution Control: Past, Present, and Future Outlook

“…Perhaps the most important observation is the overall increase in TW studies directly investigating microbial communities over the last four years. Given this exceptional trend and overall research activity, the author suggests that the field of TWs is working to move beyond the black-box understanding which has been referenced as recently as 2016 (see Lünsmann et al [32]). Following this first known activity assessment, the first study utilizing enumeration was found in 1994, the first study utilizing a structural analysis was found in 2001, and the first microbial community function study in 2004.…”

“…Perhaps the most important observation is the overall increase in TW studies directly investigating microbial communities over the last four years. Given this exceptional trend and overall research activity, the author suggests that the field of TWs is working to move beyond the black-box understanding which has been referenced as recently as 2016 (see Lünsmann et al [32]). …”

“…The microarray was based on DNA, and therefore potential function was assessed, however the authors followed up with stable isotope analysis to provide a second line of evidence pointing to the ring monooxygenation pathway for toluene degradation in their systems. The second study requiring mention is that of Lünsmann et al [32], which involved a functional analysis of microbial communities in lab-scale TWs also treating toluene contaminated water. Lünsmann and coauthors describe a rigorous assessment of toluene degrading function through mRNA extraction, cDNA construction and subsequent qPCR.…”

“…The study of Lünsmann et al [32] was completed in lab-scale, well controlled TWs. Perhaps this assisted in creating a matrix where mRNA extraction could be successful.…”