Abstract:

Volatile organic sulfur compounds (VOSCs), which are recognized as atmospheric pollutants, are critical targets for environmental and health risk assessments because of their chemical reactivity and potential toxicity. Bioaerosols such as airborne particulate matter containing microorganisms or their derivatives have recently emerged as a growing concern for atmospheric pollution. This is because VOSCs and bioaerosols are frequently co-released from liquid or solid phases into the atmosphere during sewage treatment, waste transfer, and landfill operations. The emission from pollutants of both VOSCs and bioaerosols provides complex exposure scenarios for the risk assessment of ecosystems and human beings. Co-release during these processes has been widely reported, but the effect of VOSCs on microorganisms, as well as the response of the microorganisms during the release process, remain poorly understood. Dimethyl disulfide (DMDS) and ethanethiol (ET) are employed as typical VOSCs, frequently detected in sewage treatment plants, waste transfer stations, and landfills, determine their influence on the viability, structural integrity, and metabolic activity of microorganisms in bioaerosols during their release processes. Therefore, this study aimed to investigate the dose-dependent effects of DMDS and ET on the viability, morphology, structural integrity, and growth characteristics of bacteria in bioaerosols. By elucidating the potential molecular regulatory mechanisms of bacteria activated under VOSC exposure, we provide critical insights into assessing health risks and developing control strategies in environments with VOSC bioaerosol pollution. Herein, DMDS and ET were tested at environmentally relevant concentrations of 0.2, 2.0 and 4.0 mmol∙m⁻³, corresponding to simulate open-air, semi-closed and fully enclosed exposure scenarios, respectively. These concentrations were selected based on the field monitoring results and reflected gradient thresholds. Furthermore, gentamicin-resistant Escherichia coli has been employed as a representative bacterium in bioaerosols because of its prevalence in waste-associated environments and as a conditional pathogen capable of causing opportunistic infections in immunocompromised populations. Bacterial viability in the bioaerosols was quantified via colony-forming unit (CFU) counts combined with quantitative polymerase chain reaction (qPCR) and flow cytometry with live/dead staining. Membrane integrity and cellular morphological structure were assessed by scanning electron microscopy (SEM). Bacterial growth curves were generated through optical density (OD₆₀₀) measurements over 36 h. Reverse transcription-qPCR was used to validate the relative expression levels of genes associated with oxidative stress, cell membrane damage repair, DNA damage repair, metabolic phosphorylation, electron transfer, and efflux pumps in bacteria in bioaerosols exposed to individual DMDS and ET. Results demonstrated that exposure to DMDS at 0.2-4.0 mmol∙m⁻³ did not significantly alter culturable/total bacterial ratio (average 32.66%) or mortality rate (average 47.79%) compared with the untreated control (31.6% and 48.9%, respectively). These results suggest the absence of acute cytotoxic effects of DMDS on bacteria in bioaerosols. Furthermore, structural analysis via SEM confirmed the above results regarding the non-cytotoxic effects of DMDS at environmentally relevant concentrations. That is, most of the DMDS-exposed bacterial cells retained intact membranes and preserved a surface structure indistinguishable from that of the control. These results suggest that DMDS at the tested concentrations did not directly impair microbial survival or structural stability. In contrast, the inhibitory effect of ET exposure on bacterial viability was concentration-dependent. Exposure to ET at concentrations of 0.2, 2.0 and 4.0 mmol∙m⁻³ for 10 min induced a significant reduction in culturable/total bacterial ratio (0.7%, 0.2% and 0.01%, respectively) and mortality rate (60.5%, 64.7% and 67.0%, respectively) of bacteria in bioaerosol. These results demonstrate that ET exposure induces dose-responsive damage to bacteria in bioaerosols, ultimately leading to irreversible cell death or transition to a viable but non-culturable (VBNC) state, further suggesting impaired metabolic activity and irreversible cytotoxicity. SEM analysis identified significant cell membrane structural damage as the principal cause of irreversible cell death in bacteria in bioaerosols after exposure to ET. Concomitantly, the observed transition from the characteristic rod-shaped to truncated cellular morphology strongly indicated that a substantial proportion of the bacterial population entered the VBNC metabolic state. The specific growth rate (μ) of bacteria in bioaerosol exposed to 0.2, 2.0 and 4.0 mmol∙m⁻³ DMDS and ET decreased significantly to 0.4-0.2 h⁻¹, respectively, compared with the control group (0.4 h⁻¹). Correspondingly, the generation time (G) exhibited a significant prolongation to 1.4-3.1 h under these exposure conditions, in contrast to the control group (1 h). This demonstrated that both DMDS and ET exposure induced measurable reductions in the proliferative capacity of bacteria in bioaerosols. Exposure to both DMDS and ET led to a decrease in the growth capacity of bacteria in bioaerosols, which gradually decreased with increasing concentrations. In contrast, ET exposure significantly reduced bacterial growth in bioaerosols. However, the growth curves of the surviving bacterial populations in both the control group and those exposed to DMDS and ET demonstrated sustained proliferative capacity, which achieved OD₆₀₀ values of more than 1.1 at the stationary phase of microbial growth. This indicated that the final biomass accumulation of bacteria in bioaerosols remained unimpaired after VOSC exposure, with a preserved capacity for continuous proliferation under environmental stress conditions. However, the surviving bacterial populations retained their proliferative potential, indicating that bioaerosols pose a serious health risk due to VOSC exposure. Notably, RT-PCR analysis revealed no significant changes in the expression of genes associated with the oxidative stress response (rpoS and soxS) under DMDS and ET exposure. The typical cellular stress response pathways of bacteria in bioaerosols did not induce activation, and no significant correlation was observed between the VOSCs-mediated damage and oxidative stress mechanisms. The expression of genes related to electron transfer (ccmD and yodB) was upregulated in bacteria exposed to DMDS. Cell membrane damage repair genes and DNA damage repair genes were also partially activated. Bacteria in bioaerosols exposed to DMDS demonstrated resistance to cell damage by regulating energy balance, coupled with the activation of DNA repair and membrane repair genes to achieve antagonism to DMDS damage and maintain cell survival. Genes related to metabolic phosphorylation (clpV, atpA, and pckA) and electron transfer (ccmD) of bacteria in bioaerosols were significantly upregulated under ET exposure, with expression levels demonstrating a concentration-dependent elevation with increasing ET concentrations. Meanwhile, membrane damage repair genes and DNA damage repair genes were activated under ET exposure. Energy metabolism triggers a stress response mechanism through metabolic phosphorylation and electron transfer, thereby facilitating a rapid adaptive response to ET stress. These adaptive responses enable bacteria to maintain essential functions despite VOSC-induced damage. This study clarifies microbial stress responses to VOSC exposure and provides foundational data for health risk assessment and control strategies for both pollutants in environments with combined VOSC-bioaerosol pollution.

Key words: bioaerosol, volatile organic sulfur compounds, release, vitality of microorganisms, response mechanism

摘要：

挥发性有机硫化物（VOSCs）和微生物会在污水处理、垃圾中转及填埋等过程中同时被释放到大气环境中。微生物会形成生物气溶胶，VOSCs可能影响生物气溶胶上微生物的活力，目前有关这方面的研究未有报道。该研究旨在揭示在不同物质的量浓度二甲基二硫醚（DMDS）和乙硫醇（ET）对生物气溶胶中细菌的活力、形态结构及其生长特性的影响，并阐明其潜在的分子调控机制。结果表明，暴露于DMDS时，生物气溶胶中细菌的可培养/总数占比和死亡率均与对照组无显著差异，分别稳定维持在32.7%与47.8%的平均水平。而ET暴露对细菌活力的抑制作用呈现浓度剂量效应，同时引发显著的细菌膜结构损伤，导致可培养/总数占比降低至0.7%-0.01%，死亡率为60.5%-67.0%。虽然VOSCs暴露下的生物气溶胶中细菌活力受到损伤并且生长速率受到抑制，但仍然可以保留继续生长的能力。生物气溶胶中细菌通过调控代谢、能量合成、细胞修复等相关基因改变，驱动生物气溶胶对VOSCs的防御损伤响应机制。该研究阐明了VOSCs胁迫下生物气溶胶的响应机制，可为VOSCs和生物气溶胶复合污染环境下的健康风险评估及污染防控提供理论依据。

关键词: 生物气溶胶, 挥发性有机硫化物, 释放, 微生物活力, 响应机制