Plant Ecological Treatment Technology for Livestock Wastewater: History
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抗生素在畜禽中使用后,一方面会形成选择压力,使畜禽肠道微生物产生抗药性,从而使畜禽粪便携带大量ARG;另一方面,大约30-90%的抗生素会随着畜禽粪便排放到环境中,进入环境的抗生素不仅会造成化学污染,最重要的是,可能会诱发环境中抗生素耐药细菌(ARB)和ARG的产生。畜禽废水中ARGs的来源可能有三个方面:(1)牲畜废水接收牲畜粪便中已经存在的ARG;(2)废水中的抗生素、重金属等污染物诱导微生物产生ARG;(3)微生物宿主细菌的增殖导致ARG的增殖。与传统化学污染物不同,ARGs由于其固有的生物学特性,表现出独特的环境行为,如可复制性、传播性和环境持久性,而ARGs则由质粒、转座子、整合子、插入序列公共区域和复杂集成子等移动遗传元件促进。这些ARGs通过水平基因转移(HGT)机制在环境介质中的不同微生物之间传播,并可能通过直接或间接途径进入食物链和人体,增加人体耐药性,危害人类公众健康。

  • plant
  • antibiotic
  • root
  • ARGs
  • 畜禽养殖

1. Effectiveness of Plant Ecological Treatment Technology on the Removal of Antibiotic Resistance Genes

Plant ecological treatment technology is gradually gaining popularity; therefore, more and more scholars are suggesting to use this technology for decentralized wastewater treatment systems [35]. Up until now, many valuable conclusions have been obtained by different researchers regarding the removal of ARGs (see Table 1). For example, Ávila et al. [32] established two ecological techniques for plant treatment, and the results showed good removal efficiencies for all five target ARGs: 46% to 97% for sul1, 33% to 97% for sul2, 9% to 99% for ermB, 18% to 97% for qnrS, and 11% to 98% for blaTEM. Chen et al. [36] used an Cyperus alternifolius L. constructed ecosystem to treat domestic wastewater, and the results showed that the removal rates of 18 target ARGs ranged from 50.0% to 85.8%. Du et al. [37] used rutabaga to treat pig farm wastewater, and the results showed that the average of sulI, sulII, sulIII, tetM, tetO, and tetW removal rates were 67.5%, 85.6%, 95.6%, 87.9%, 97.9%, and 98.5%, respectively. However, the action of plants on ARGs is selective, and ARGs of different mechanisms show different behavioral convergence during the same treatment, while the same ARGs may also show different extinction patterns in ecosystems of different plant types. For example, Chen [38] showed that, after Cyperus alternifolius L. treatment, the abundance of tetO and tetX in wastewater appeared to be enriched with a removal rate of −63.8% and −26.3%, respectively, while all other classes of ARGs showed better removal effects. The reason for this difference could be the different mechanisms of action or the transmission of resistance genes. For example, tetM is one of the most common tetracycline ARGs [39] which has been shown to possess the broadest bacterial host range [40], and it is usually associated with chromosomes, conjugates, and transposons of the Tn1545-916 family; therefore, tetM is ubiquitous in many systems and is widely disseminated in the environment, whereas tetO genes are mobile only on binding plasmids [41], which are theoretically less transmissible than tetM. Therefore, studying the mechanism of action specific to ARGs in combination with plant physiological properties will not only help enrich the knowledge of ARG removal mechanisms but also contribute to the sustainable development of the whole ecological treatment technology.

2. Drivers of Resistance Gene Elongation in Plant Ecological Treatment Systems

In terms of microenvironment, there are various factors that influence the behavioral attribution of ARGs during plant treatment of wastewater; microbial communities, mobile genetic elements, environmental factors, and other pollutants are closely related to changes in resistance genes. Indirect driving factors include socioeconomic and environmental factors that influence the use and dissemination of antibiotics and the development of antibiotic resistance. For example, most studies concluded that microorganisms are the host bacteria of ARGs and the growth and reproduction of microorganisms directly affect the changes in the abundance of ARGs [48]. Mobile genetic elements (MGEs) are important indicator elements for the horizontal transfer of ARGs among bacteria, and MGEs are closely related to ARG transmission. Other pollutants (antibiotics, heavy metals, etc.) and environmental factors (TN, TP, TOC, pH, etc.) can directly or indirectly affect the structure of microbial communities in wastewater treatment systems, thus affecting the changes in ARGs [49]. Currently, many studies have focused on the role of different factors in influencing changes in ARGs. For example, Zhu et al. [50] showed that microbial community structure explained 52.3% of the variation in ARGs, while MGEs explained only 7.8%. We recently showed [51] that MGEs within different systems explained most (>50%) of the ARGs, followed by microbial communities. In addition, antibiotic residues can also contribute to the horizontal spread of ARGs [52], but some studies have shown that antibiotics (OTC) have a weak effect on the distribution of ARGs in lettuce tissues, accounting for only 6.3% of the total variance, but significantly correlated with tetW, ermF, sul1, and intI1 (p < 0.05) [49]. Heavy metals (Cu, Zn, Cd, etc.) also induce the production and enrichment of ARGs and have a synergistic induction with antibiotics [53,54]. In turn, other environmental factors can act directly on microorganisms, thus indirectly influencing the dynamic pattern of ARGs [51]. Feng et al. [45] investigated the relationship between soluble organic matter (DOM) and ARG removal and showed that the removal rate of DOM was significantly correlated (p < 0.001) with the removal rate of ARGs during the purification of swine farm wastewater by Acorus calamus, but the removal rate of tetW was not significantly correlated with the removal rate of DOM. Thus, it is evident that determining the driving effect of each factor on ARGs is a hot topic of current research, and the conclusions for the driving effect of each factor on ARGs within different ecosystems vary.
From a macroscopic point of view, process conditions also influence the extinction pattern of ARGs, and, currently, many researchers have examined different process conditions for plant ecological treatment technologies. Direct driving factors conclude wastewater treatment processes such as activated sludge, biological nutrient removal, and membrane bioreactors. Operational parameters such as hydraulic retention time, temperature, and pH can also affect the removal of ARGs and chemical factors. For example, the presence of heavy metals. Moreover, plant type and filler type have a direct effect on ARG removal. Chen et al. [36] compared the removal effect of Thalia dealbata Fraser. and Iris tectorum Maxim. on 11 ARGs in wastewater through comparative experiments and pointed out that plant type significantly influenced ARG removal. Feng et al. [45] showed that dissolved oxygen has a significant effect on the removal efficiency of the whole plant ecological treatment process; therefore, aeration of the water body is favored by many researchers [30], but some studies have shown that increasing the oxygen capacity does not significantly contribute to the removal of ARGs [37]. The influent method can directly affect the degree of contact between the effluent and the plants and the turbulent flow pattern of the effluent within the system, thus influencing the overall pollutant removal [41,42]. In addition, hydraulic retention time and hydraulic loading are important factors in controlling the removal of pollutants from plant ecosystem effluent, and increasing the hydraulic retention time increases the contact time between pollutants and substrate biofilm, which theoretically contributes to the removal of ARGs [38]; however, excessive hydraulic retention time can lead to an increase in the overall process footprint.
From the above analysis, it can be seen that there are many factors affecting the removal rate of ARGs, and each factor interacts with each other. As shown in Figure 1, all process conditions can be considered as macro factors, and the setting of process conditions directly affects the parameters within the system (considered as micro factors), which theoretically cannot have significant effects on macro factors and therefore can be called “weak effects”. Macro factors ultimately affect ARGs by influencing micro factors. Macro and micro factors such as policy and regulatory frameworks, technological innovations, and funding and investment can also affect the development and implementation of wastewater treatment technologies and the capacity of communities and countries to address the challenge of antibiotic resistance. Therefore, it is not only necessary to clarify the influence of individual factors on ARGs but also to integrate the interactions between various factors in order to find the main factors affecting ARG removal.
Figure 1. Relationship diagram of different driving factors.

3. Transmission Pathways and Distribution Characteristics of Antibiotic Resistance Genes in Plant Tissues

Throughout the ecological treatment system based on plant uptake, plants play a crucial role in the extinction of ARGs, and the fugitive values of ARGs within different plant tissues determine the risk and probability of ARG transmission to the next level of the food chain. The distribution characteristics of ARGs within plant tissues are a hot topic of research within the ecological transformation system. Notably, many studies have shown that ARGs can be distributed in plant tissues such as roots, stems, and leaves. For example, Yang et al. [56] showed that the plant tissues of celery, cabbage, and cucumber contained culturable bacteria resistant to cefadroxil after cefadroxil selection pressure was applied to the plant growth environment in various tissue sites of plants. The size order was soil samples > leaf peripheral samples > root endophyte samples > leaf endophyte samples. However, not all ARGs can migrate through the plant root system to all tissues of the plant. For example, Duan et al. [49] showed that sul1, sul2, ermF, and ermX can migrate from the root endophyte to the leaf part of lettuce, but tetracycline ARGs were very low in the leaf part, where tetW was not detected in the stem and leaf tissues. However, Ye et al. [57] showed that sulfonamide resistant bacteria or resistance genes (sul1 and sul2) were not detected in new lettuce leaf tissues, while they were detected in old leaf tissues (10–7 to 10–9 copies/16S copies). This shows that ARGs are unevenly distributed in different parts of the plant; moreover, different species of ARGs have different distribution characteristics.

4. Mechanism of Removal of Antibiotic Resistance Genes

ARGs, as an emerging pollutant, exhibit a different behavior and fate in different plant ecological treatment systems in terms of species and abundance. A schematic diagram about the removal mechanism of ARGs within the whole plant ecological treatment system is shown in Figure 2. Overall, the removal pathways of ARGs in plant ecological treatment systems include the following three aspects: (1) Biological role: Microorganisms play an important and complex role in ARG removal because they are not only related to the propagation and proliferation of ARGs but may also play a role in degrading ARGs [58,59]. Chen et al. [36] showed that the process of reflectant domestic wastewater microorganisms play a major role in ARG removal (73.7–95.2%). (2) Substrate sorption: Substrate sorption also plays an important role in ARG removal, and the abundance of ARGs in substrate materials showed accumulation in different plant treatment processes, which indicates that substrate materials can sorb ARGs from wastewater to achieve ARG removal [36,43]. Chen et al. [30] clearly pointed out that substrate sorption and microbial degradation are the two main mechanisms of action for ARG removal. (3) Plant uptake: Plant uptake is also an aspect of ARG removal that cannot be neglected. Studies have shown that plant tissues are not completely immune to ARGs and plant root endophytes can acquire some ARGs from root surface stomata and mechanical damage and spread them with plant endophytes so that ARGs reach the stems and leaves [60]. Although some studies have shown that microbial degradation plays a relatively large role in ARG removal while substrate sorption and plant uptake play a relatively small role [36], the role of the latter two is inextricably linked to microbial degradation, and the substrate and plant root system can provide attachment sites for pollutants (ARGs) and microorganisms, thus allowing the microorganisms to more fully contact the pollutants and achieve a better degradation effect [36]. In particular, inter-root microorganisms specific to plant roots may also be important for the removal of ARGs [61,62]. Plant root surface tissue secretions, which regulate root surface pH and redox conditions, provide suitable growth conditions for interfacial microorganisms and also increase microbial activity, thus enhancing the overall biodegradation process.
图2.ARG的去除机制示意图。
内生细菌如何获得ARG以及ARG如何在植物组织之间传播的问题一直是一个困难的研究问题。许多研究表明,植物组织的内生菌落与根系外围环境中的微生物群落之间存在较大的重叠[5063,64],这表明植物(尤其是根组织中)的微生物群落组成在很大程度上受外部环境微生物群落的影响;然而,对于远离根组织的叶片等部分,其微生物群落与根系内生菌的微生物群落差异更大。例如,Zhang等人[65]表明,根微生物群落中只有12个细菌OTU(II组)可能进一步扩散到叶片内生菌中,这表明与叶片内生菌的微生物群落存在差异。此外,这种微生物传播能力直接影响ARG传播的概率。例如,Duan等[49]表明,厚壁门厚壁菌纲(ARG的潜在宿主细菌)无法从根系迁移到茎叶组织,这可能是tetW不能迁移到茎叶部分的主要原因。其他研究还表明,外部环境可以作为根系内微生物群落的种子库,植物内生菌大多从外部环境水平获取,而不是通过种子或花粉从亲本植物垂直传播[6667]。因此,重要的是要明确ARG是否从浮萍的废水迁移到根系。如果存在这样的迁移,ARGs是否会在浮萍的不同组织之间迁移?如果是这样,它们如何迁移和传播?澄清这些问题是控制ARGs向食物链下一级扩散的瓶颈,有助于为控制ARGs的二次传播和评估ARGs进入食物链的风险提供参考依据。

This entry is adapted from the peer-reviewed paper 10.3390/ijerph20054357

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