Many deep soil environments are anoxic due to the scarcity of O2, wherein NO3-, SO42-, Fe3+, Mn4+, and HCO3- (CO2) can function as terminal electron acceptors (TEAs). Anoxic biodegradation of PAHs using NO3- and SO42- as TEAs has been the subject of extensive research. However, information related to the degradation of PAHs under methanogenic conditions using CO2 as TEA is poorly understood, although it is a critical pathway in elimination of PAHs from anoxic soils. However, the low degradation rate of PAHs under anoxic conditions is the primary bottleneck restricting the promotion and application of this technology. Therefore, in this study, a low -temperature (< 50 degrees C) thermal enhanced biodegradation microcosm experiment was conducted using three temperatures (15 degrees C, 30 degrees C, and 45 degrees C) under methanogenic conditions. The results revealed that the limited PAH removal was somewhat compensated for by elevated temperature (e.g., 45 degrees C) compared to lower temperature (e.g., 15 degrees C or 30 degrees C), and removal efficiency of 2-, 3-and 4-ring PAHs (except for benz(a)anthracene (BaA)) obeyed the order of 45 degrees C > 30 degrees C > 15 degrees C. Kinetic analysis and t-test confirmed this and indicated that the promotion of PAH removal is more significant when temperature is raised from 30 degrees C to 45 degrees C than from 15 degrees C to 30 degrees C. Soil microbial communities were significantly affected by both the incubation time and temperature, and the changes in bacterial and archaeal communities were enhanced with increasing temperature. Network analysis demonstrated that soil microbes tended to cooccur rather than coexclude. The bacterial and archaeal cooccurrence network became less complex after incubation. The numbers of nodes, edges, and modules declined after 250 days of incubation, indicating that microbial function tends to be simple with prolonged incubation time. These results provide new insights and a scientific basis for the bioremediation of PAH-contaminated sites.

In situ anoxic bioremediation is an easy-to-use technology to remediate polycyclic aromatic hydrocarbon (PAH)-contaminated soil. Degradation of PAHs mediated by soil bacteria and archaea using CO2 as the electron acceptor is an important process for eliminating PAHs under methanogenic conditions; however, knowledge of the per-formance and mechanisms involved is poorly unveiled. In this study, the effectiveness and efficiency of NaHCO3 (CO2) as an electron acceptor to stimulate the degradation of PAHs by bacteria and archaea in highly contam-inated soil were investigated. The results showed that CO2 addition (EC2000) promoted PAH degradation compared to soil without added CO2 (EC0), with 4.18%, 9.01%-8.05%, and 6.19%-12.45% increases for 2-, 3 -and 4-ring PAHs after 250 days of incubation, respectively. Soil bacterial abundances increased with increasing incubation time, especially for EC2000 (2.90 x 108 g(-1) soil higher than EC0, p < 0.05). Different succession patterns of the soil bacterial and archaeal communities during PAH degradation were observed. According to the PCoA and ANOSIM results, the soil bacterial communities were greatly (ANOSIM: R = 0.7232, P = 0.001) impacted by electron acceptors, whereas significant differences in the archaeal communities were not observed (ANOSIM: R = 0.553, P = 0.001). Soil bacterial and archaeal co-occurrence network analyses showed that positive correlations outnumbered the negative correlations throughout the incubation period for both treat-ments (e.g., EC0 and EC2000), suggesting the prevalence of coexistence/cooperation within and between these two domains rather than competition. The higher complexity, connectance, edge, and node numbers in EC2000 revealed stronger linkage and a more stable co-occurrence network compared to EC0. The results of this study could improve the knowledge on the removal of PAHs and the responses of soil bacteria and archaea to CO2 application, as well as a scientific basis for the in situ anoxic bioremediation of PAH-contaminated industrial sites.