Formatted Title

Mechanisms of Thermal Treatment Coupled with Biostimulant Enhanced Biodegradation of PAHs and BTEX Mixture in Contaminated Soil by Native Microbial Consortia

Background/Objectives

Mixed contamination refers to the occurrence of multi-type pollutants in a given environment. Mixed contamination exists in most industrialized countries mainly because of leakage of underground storage tanks and landfills, accidental surface spills, industrial and military activities, and uncontrolled waste disposals. The majority of the mixed organic pollutants found at contaminated sites is of great concern for human health and safety due to the increased production of synthetic organic chemicals over the past few decades. Contaminated sites with multi-type contaminants pose technical challenges associated with each class of contaminants (e.g., PAHs and BTEX) along with the remediation problems that arise due to the presence of two or more classes of contaminants having different physicochemical characteristics. Soil contamination with polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene, and xylene (BTEX) has become a serious problem in most industrialized countries. According to the Agency for Toxic Substances and Disease Registry, PAHs and BTEX are among the top priority hazardous contaminants that cause carcinogenicity and teratogenicity. Therefore, it is essential to remediate these contaminants safely to avoid their toxic influence on the ecosystem.

Approach/Activities

This research is one full package evaluating the biodegradation of PAHs and BTEX mixed contaminants, mechanisms of biostimulant (BS), thermally enhanced biodegradation (TEB), and their coupled TEB-BS of PAHs and BTEX, which is one of the challenging tasks for environmental scientists and engineers. In general, many factors affect the biodegradation of different contaminants, among which, lack of substrate and lack of contaminants bioavailability are the primary bottlenecks restricting the efficiency of biodegradation. Therefore, several studies were conducted to explore the mechanisms of different biostimulants (BS), thermally enhanced biodegradation (TEB), and their coupled (TEB-BS) strategies' influence on the biodegradation of PAHs and BTEX mixed contaminated soils. The soil was collected from a steel-making factory in Hangzhou, Zhejiang province. It has been reported that the site is contaminated with mixed PAHs and BTEX, however, the contaminant concentrations were not detected compared to our level of contamination, therefore, we did the experiment on artificially contaminated soil with 50 mg benzene and BaP kg-1 soil, respectively. In the first experiment, three biostimulants including methanol, ethanol, and vegetable oil were used. The concentrations of biostimulants were selected according to the previous studies. Changes in the benzene and BaP concentration, native microbial composition and diversity, along with the abundance of total bacteria (16S rRNA gene), and enzymatic activities were analyzed under different treatments. In the second experimental setup, four different thermal treatments were used, including 15, 30, 45, and 70 °C during the thermally-enhanced biodegradation (TEB). Benzene and BaP concentration variation with time, PAH-RHDα gene in gram-positive and gram-negative bacteria, changes in native microbial composition and diversity, along with the abundance of total bacteria (16S rRNA and ITS gene), enzymatic activities, ROS generation, and soil properties were analyzed under different treatments.  In the final experiment, a biostimulant-coupled TEB experiment was carried out to assess the availability of substrates and enhance contaminants' bioavailability influence on contaminants' biodegradation.

Results/Lessons Learned

The results revealed that biostimulation significantly improved the biodegradation of both contaminants under co-contaminated conditions. A trend of a significant reduction in the bioavailable BaP contents was observed in all biostimulant-enhanced groups, implying that the bioavailable BaP was the preferred biodegradable BaP fraction. Furthermore, the enzymatic activity analysis revealed a significant increase in lipase and dehydrogenase activities, as well as a reduction in the catalase and polyphenol oxidase, suggesting that the increased hydrolysis of fats and proton transfer, as well as the reduced oxidative stress, contributed to the enhanced benzene and BaP biodegradation in the vegetable oil treatment. In addition, the microbial composition analysis results demonstrated that the enriched functional genera contributed to the increased biodegradation efficiency, and the functional genera in the microbial consortium responded differently to different biostimulants and competitive growth was observed in the biostimulant-enhanced treatments. In addition, the enrichment of Pseudomonas and Rhodococcus species was noticed during the biostimulation of benzene and BaP co-contamination soil and was positively correlated with the dehydrogenase enzyme activities, indicating that these species encode dehydrogenase genes which contributed to the higher biodegradation.

A set of comprehensive experiments were conducted to explore the effects of temperature on biodegradation, bioavailability, and generation of reactive oxygen species (ROS) by thermally enhanced biodegradation (TEB) under benzene and BaP co-contaminated conditions. The biodegradation rates of benzene increased from 57.4% to 88.7% and 84.9%, and the biodegradation efficiency of BaP was enhanced from 15.8% to 34.6% and 28.6%, when the temperature was raised from the ambient temperature of 15°C to 45°C and 30°C, respectively. In addition, the bioavailability analysis results demonstrated that the water- and butanol-extractable BaP increased with elevated temperatures. High enzymatic, microbial activities and polycyclic aromatic hydrocarbons-ring hydroxylating (PAH-RHDα) gene in gram-positive bacteria favored the elevated temperatures (30 and 45°C) compared to gram-negative bacteria. Moreover, ROS species (O2•− and •OH) generation was detected which were scavenged by the increased superoxide dismutase and catalase activities at elevated temperatures. Properties (pH, TOC, moisture, total iron, Fe3+, and Fe2+) were affected by the temperature treatments, revealing that metal-organic-associated reactions occurred during the TEB of benzene–BaP co-contamination. The results concluded that biodegradation of benzene–BaP co-contamination was greatly improved at 45°C and that high biodegradation was the composite effect of microorganisms and ROS generation and degradation.

The constraints of lacking available substrate and contaminant bioavailability are the primary bottleneck restricting the efficient biodegradation of PAHs and BTEX co-contamination in soil. Therefore, in this study, the composite effects of vegetable oil usage as a biostimulant to increase the available substrate coupled with the application of thermal treatments coupled with biostimulant enhanced biodegradation (TEB-BS) to increase the bioavailability of the contaminant for enhanced biodegradation of PAHs and BTEX mixed contaminated soils were investigated. The results revealed a significant improvement in the biodegradation potential of BTEX, where the biodegradation followed the order toluene>o-xylene> p-xylene>m-xylene≥ethylbenzene>benzene. Moreover, toluene was completely remediated at 30 and 45°C thermal conditions, and 45°C followed by 30°C temperature showed good performance in remediation of BTEX. On the other hand, PAH biodegradation rates followed the number of rings pattern, where 3-ring PAH (phenanthrene) was highly biodegraded, followed by 4-ring PAH (pyrene), whereas the lowest biodegradation was recorded for 5-ring PAH (BaP). Potential microbes, including Meiothermus, Effusibacillus, and Brevibacillus, were highly abundant at 45°C thermal condition, indicating that these microorganisms might play a crucial role in the fast biodegradation of PAHs and BTEX under mixed contamination. In addition, the polycyclic aromatic hydrocarbon-ring hydroxylating (PAH-RHDα) gene in GP and GN bacteria revealed that nidA, a PAH-RHDα gene in GP bacteria abundance increased at the elevated temperature conditions (15 to 30 and 45°C) further confirming the involvement of GP bacteria in the enhanced biodegradation of PAHs and BTEX. Metabolic pathways revealed that all of the biodegradation occurred via the meta-cleavage pathway confirmed by the increased abundance of the catechol 2,3-dioxygenase gene. Moreover, the metabolic pathways of PAHs and BTEX were confirmed via the abundance of genes and biochemical reactions that can be useful in the application of the TEB-BS technique under mixed contaminated conditions.