Degradation of sulfonamide antibiotics in the rhizosphere of two dominant plants in Huixian karst wetland, Guangxi, China

Sulfonamide antibiotics (SAs) are synthetic antibacterial agents derived from sulfanilic acid. They are often added to veterinary feed or drugs in the form of additives to promote animal growth and disease prevention (Man et al. 2019). SAs are only partially metabolized in organisms, and 50–90% of the SAs in feces or urine will be discharged into the environment with their original structures intact, thus accumulating in various environmental media, such as surface water, groundwater, and soil (Zhang et al. 2014; Cui et al. 2020). Using plants to purify SA-polluted water has become a feasible technology (He et al. 2016). Phytoremediation is affected by rhizosphere microorganisms, rhizosphere soil contains a large number of SA-degrading bacteria that can use SAs as a designated carbon source for metabolic activities (Chen et al. 2016). Compared with oxidation processes, adsorption on activated carbon, and membrane filtration, phytoremediation has the advantages of high efficiency and low cost (Hu et al. 2019).

Karst aquifers are important sources of drinking water in many areas of the world. Because of the high hydraulic conductivity and short residence time of karst systems, antibiotics can be transported in the karst pipeline (Hillebrand et al. 2015), as a result, karst ecosystems are easily polluted by antibiotics. At present, antibiotics have been detected in the groundwater of karst areas in countries including Switzerland, USA, Germany, and France (Dodgen et al. 2017). The degradation of antibiotics in the environment is affected by environmental factors including temperature, pH, and ionic strength (Menció & Mas-Pla 2019). The hydrochemical characteristics of karst areas are quite different from those of non-karst areas. The long-term corrosion of carbonate rocks easily forms the special environment of ‘high calcium (Ca²⁺), alkali, high dissolved inorganic carbon (⁠⁠)’ found in karst areas (Li et al. 2015; Zhang et al. 2018). Therefore, the degradation of antibiotics in karst areas may be different from that in non-karst areas. With frequent human production activities (domestic sewage, medical wastewater, aquaculture and livestock farming, etc.), antibiotics, as a trace pollutant in the environment, are continuously discharged into natural water bodies such as wetlands, altering the dynamic balance of antibiotics in the original environment. A study by Conkle et al. (2008) showed that at initial concentrations of 4.090 and 0.068 ug L⁻¹ for sulfamethoxazole and sulfadiazine, respectively, the non-karst forest wetlands were less effective in removing the two sulfonamides and less effective in treating and diluting the antibiotics.

As the largest karst wetland in China, Huixian wetland plays important ecological roles in water conservation, climate regulation, water purification, biodiversity conservation, and flood storage in Li River. As part of its involvement in urban water recycling, the Huixian wetland is to some extent able to convert engineered reclaimed water into ecologically reclaimed water, a process that is particularly important in controlling the conversion of new pollutants such as antibiotics. It was reported that SAS pollution was detected in surface water, shallow groundwater, water near farms, and pond water of Huixian wetland (Qin et al. 2019). Therefore, the management of SAs in Huixian wetland has gradually attracted the attention of Chinese and international scholars. There are many types of vegetation in Huixian wetland. The plant coverage can reach 80–95%, and the dominant plants are Cladium chinense Nees, Typha orientalis Presl, Vallisneria natans, Phragmites australis, and Canna indica (Tu et al. 2019). P. australis is an emergent plant with strong resistance, fast growth, high yield, and strong adsorption, and is the preferred plant for purifying water quality (Lambertini et al. 2020; Wang et al. 2021a). V. natans is a perennial submerged plant that can effectively remove nitrogen, phosphorus, and heavy metals in polluted water (Yan et al. 2011). The investigation of dominant plant species can be used to assess biodiversity changes and ecosystem functions in wetland systems. It has been reported that the removal rate of SAs in plant systems was significantly higher than that in non-plant systems, and it was found that the degradation of SAs by plants was related to plant accumulation and stress resistance ability, and the degradation of antibiotics in plants was mainly related to rhizosphere microorganisms (Chen et al. 2016, 2021). It has also been reported that root exudates can play a very significant role in promoting microbial degradation of antibiotics (Zhi et al. 2019), and different kinds of substances in root secretions will increase rhizosphere microbial biomass and activity, and then affect how effectively wetland systems purify pollutants (Du et al. 2020).

However, few studies have revealed the degradation efficiency and mechanism of SAs in the rhizosphere of wetland plants in karst areas, and little attention has been paid to changes in the rhizosphere microbial community structure before and after antibiotic degradation. Therefore, this work selected Huixian wetland dominant plants (P. australis and V. natans) as the research object. In the simulated surface flow wetland reactor, the root exudates were extracted after 10 d of incubation under sulfadiazine (SD) stress. The root exudates were mixed with the rhizosphere soil to explore the degradation mechanism of SD by root exudates, and the composition and changes of bacterial communities in the rhizosphere soil before and after degradation were analyzed by high-throughput sequencing technology to reveal the mechanism of SD degradation in the rhizosphere so as to provide a theoretical basis for antibiotic treatment in Huixian wetland.

In the stress culture, according to the water consumption of the simulation system, referring to the results of Xia et al.'s (Xia et al. 2021) study and combining the detection limits of the actual instrument, SD was added only to the experimental group so that the aqueous solution contained 0.10 μg L⁻¹ SD, which was used as a stressor. After 10 d of cultivation, the plants in each group were taken out, and the root exudates of plants were extracted and analyzed by the root soaking method (Zhalnina et al. 2018).

The root exudates used were the original aqueous solutions collected from the two wetland plants after a 10-d cultivation under SD stress with a concentration of 0.10 μg L⁻¹. The collected original aqueous solution was filtered with 0.45 μm filter membranes to prepare a culture medium containing 0.10 μg L⁻¹ SD. The rhizosphere soil of each plant was collected by the root shaking method (Tu et al. 2019). One hundred grams of rhizosphere soil collected from each plant was weighed and placed in a conical flask, and 300 mL of the above antibiotic-containing culture medium was added. The soil and root exudates were fully mixed. After determining that the rhizosphere soil and root exudates of plants corresponded, they were recorded as P. australis experimental group (P), P. australis control group (CP), V. natans experimental group (V), and V. natans control group (CV). In the SD degradation experiment, the oscillation time was set at t = 0/2/8/16/30/48/96 h as seven time points, and the concentration of SD in the aqueous solution was measured at each time point to obtain the SD degradation rate in different time periods. In the degradation experiment, P. australis and V. natans were termed as PD and VD, respectively. The rhizosphere soil of 0 and 96 h samples collected to determine microbial community structure, and the aqueous solutions of 0 and 96 h samples were used to detect SD degradation products. The 0 h P. australis and V. natans groups were labeled as P₀ and V₀, respectively, and the 96 h P. australis and V. natans groups were P₉₆ and V₉₆, respectively.

The solution collected by the root soaking method was analyzed by gas chromatography–mass spectrometry (GC-MS) after treatment. The root washing solution was extracted with 300 mL CH₂Cl₂ three times and the CH₂Cl₂ extraction solution was concentrated to dry by rotary evaporation at 35 °C. Five milliliters of CH₂Cl₂ filtered by a 0.45-μm membrane was then added, the solution was dried with anhydrous Na₂SO₄, and finally, 0.5 mL of treated CH₂Cl₂ was taken for GC-MS analysis. The instrument used for determination was a GC-MS-type gas chromatography–mass spectrometer (Claus 600T Mass Spectrometer and Claus 680 Gas Chromatograph), Elite-5MS (30 m × 0.25 mm × 0.25 μm) chromatographic column.

SD was detected by solid phase extraction-high-performance liquid chromatography. The instruments used included the following: a Japan AQUAT race ASPE799 solid phase extraction instrument, Agilent 1260 high-performance liquid chromatography, a 24-bit nitrogen blowing instrument, an HLB extraction column, and an ultrasonic cleaner.

The detection samples of SD degradation products were tested Guangxi Guilin RID Testing Co., Ltd. A triple quadrupole liquid chromatography–mass spectrometry (HPLC-MS) system was used with a chromatographic column: an Eclipse Plus C18 RRHD 2.1 × 50 mm, 1.8 μm. Sample treatment was conducted as follows: the sample solution was moved to a centrifuge tube, centrifuged at 4,000 r min⁻¹ for 5 min, and the supernatant was collected for filtration. One hundred milliliters of the sample solution were taken in a conical flask with a measuring tube. The sample solution was concentrated to 100 times by solid phase extraction and detected by HPLC-MS/MS.

The E. Z. N. ATM Mag-Bind Soil DNA Kit (Omega Bio-Tek Company, Guangzhou International Business Incubator) was used for DNA extraction, and then the Qubit 3.0 DNA detection kit was used for the accurate quantification of genomic DNA to determine the amount of DNA added to the PCR reaction. The PCR primers were fused with 16S V3–V4 primers on the sequencing platform. The PCR reaction conditions were 94 °C, 3 min → (94 °C, 30 s → 45 °C, 20 s → 65 °C, 30 s) 5 → (94 °C, 20 s → 55 °C, 20 s → 72 °C, 30 s) 20 →72 °C, 5 min → 10 °C. DNA extraction, PCR amplification and purification were performed by the Sangon Biotech Co., Ltd (Shanghai).

UPARSE software was used for operational taxonomic unit (OTU) sequence clustering of valid sample data. The Mothur method and SILVA's SSU rRNA database were used to perform species annotation analysis on the representative OTU sequences and obtain the community composition of the sample at each taxonomic level. The alpha diversity index was calculated using Qiime 1.9.1. A species abundance column diagram, degradation rate analysis diagram, degradation kinetics diagram, and dilution curve were analyzed and drawn by Origin 2018. The p-value was obtained using Welch's t-test. Abundance differences were plotted using STAMP software. Data analysis of the results was performed in SPSS 25.0.

Each measured peak (Supplementary Figure S1(a)–S1(d)) was searched in the atlas library to obtain the corresponding compounds (Supplementary Table S2). A total of 37 compounds were detected in the CP group, including alkanes, aldehydes, esters, alcohols, and amides. A total of 31 compounds were detected in group P, including alkanes and esters. A total of 35 compounds were detected in the CV group, including alkanes, aldehydes, esters, amides, and alcohols. A total of 34 compounds were detected in root exudates of group V, including alkanes and esters.

From the perspective of the most abundant component (Table 1), the most abundant component of the CP group was heptadecane, 2,6,10,15-tetramethyl, with a content of 8.46%, but that of the P group was 9-octadecenoic acid (Z)-, methyl ester, with a content of 31.88%. In the CV group, the content of 9-octadecenamide, (Z)- was 9.6%, and in the V group, the content of 9-octadecenoic acid (Z)-, methyl ester was 22.31%. According to the above results, the most abundant components of the two plant groups changed to esters under SD stress, and the content increased. Under material stress, the secretion of some compounds in plant root exudates was inhibited or changed (Rolfe et al. 2019). In this study, dichloromethane was used for extraction, and the obtained experimental results covered relatively complete root exudates.

Studies used different nitrogen and phosphorus concentrations to culture plants, and they concluded that the amount of compounds in the root exudates of the low nutrient treatment was higher than that of the high nutrient treatment (Wu et al. 2012). However, the detection of compounds in this study was higher than that in the above studies. Some scholars have found that the root exudates of rape seedlings mainly consist of compounds including hydrocarbons, alcohols, esters, and acids (Escolà Casas & Matamoros 2021). This finding is similar to the results in this study, but the specific characteristics of compounds are not consistent. After SD stress at a concentration of 0.10 μg L⁻¹, the species of compounds in the rhizosphere exudates of the two plants decreased, which may have been due to the fact that wetland plants would actively adapt to the environment by self-regulating the composition and quantity of secretions by roots under external stress (Duan et al. 2020).

The experimental results showed that the removal rate of antibiotics with these species was higher than that of other constructed wetlands or non-karst wetland plants (Yan et al. 2019). Owing to the long-term erosion of carbonate rocks, the Huixian wetland ecosystem contains a large amount of Ca²⁺ (Li et al. 2017), and the form of calcium that occurs in the soil used in this study is mainly exchangeable, with an ECa/TCa value of 52.82–69.48% (Supplementary Table S3). The high content of exchangeable calcium indicates that calcium in the soil is active in migration and bioavailability, and Ca²⁺ can form a complex with antibiotics and then be adsorbed by the soil matrix (Liang et al. 2018). However, SAs are a typical amphoteric compound. When the soil is alkaline, antibiotics are difficult to adsorb, and when the pH is close to neutral, the removal effect of antibiotics is the best (Kurade et al. 2019). Huixian wetland soil is rich in calcium and alkaline, and its water quality is mostly weak alkaline. In this study, the rhizosphere soil pH was 7.16–7.22 (Supplementary Table S3), which indicated that SD was difficult to adsorb by the soil matrix in the karst wetland, and probably existed in the form of complexes. In addition, the removal mechanism of SD in Huixian wetland may have been mainly dependent on the interaction between plants and rhizosphere microorganisms.

The R₁² values of PD and VD were higher than R₂² and MSE₁ was lower than MSE₂, indicating that the fitting degree and correlation of the first-order equation of degradation kinetics were higher than those of the second-order equation, and the first-order kinetic model was more reasonable to express the degradation of SD in this study. This result was consistent with the results of research on the photodegradation, Fenton-oxidation degradation, and chlorination degradation of antibiotics (Dirany et al. 2010). Different plants had different pollutant removal capacities, and the selection of plant species was a key part of pollutant removal (Rezania et al. 2015). Therefore, the cultivation of P. australis was of great significance for the prevention and control of SD in Huixian wetland.

Microorganisms can degrade the phenyl portion of SD molecules, but the pyrimidine ring of SD is stable and produces an equal molar amount of 2-aminopyrimidine. When 2-aminopyrimidine is formed, it will be transformed into 4-hydroxy-2-aminopyrimidine by bacterial metabolism (Deng et al. 2016). The intermediates of SD also include sulfonamide aniline acid, p-aminobenzenesulfonic acid, 2-hydroxypyrimidine, aniline, and pyrimidine-2-sulfonamide acid. The degradation pathways of the intermediates may be one of three parallel pathways: first, the S–N bond, N–C bond, or C–S bond is broken, and then hydroxylation, formylation, and acetylation occur (Mohring et al. 2009). In this experiment, the two plant groups obtained the same three metabolites (Supplementary Figure S2(a) and S2(b)): m/z 171.2 (p-aminobenzenesulfonic acid), m/z 114.1 (4-hydroxy-2-aminopyridine), and m/z 224.2 (phenyl sulfoxide).

Root exudates can be transported by cells and excreted around the rhizosphere, creating a unique environment for root microorganisms (Hu et al. 2018), thus affecting the degradation of SAs. This work found that after SD stress culture, the highest component of root exudates was changed to organic acid esters. In the presence of acid or alkali, organic acid esters can be hydrolyzed to organic acids or alcohols. Many studies have shown that plant roots secrete organic acids, amino acids, sugars, and other substances that contribute to microbial growth (Li et al. 2019). Biodegradation has been proved to be the main pathway for SA degradation in plants (Chen & Xie 2018). The contribution of plant roots and pollutants to microbial growth is different. Plant root exudates can increase the activity of microorganisms, and some microorganisms can promote the activity of plants, helping them resist pollutant stress (Cristaldi et al. 2017). Therefore, the calcium-rich and alkali-rich characteristics of karst wetland soil will lead to the hydrolysis of some organic acid esters in root exudates to produce organic acids, which indirectly affects the rhizosphere microbial metabolism, and thus regulates the degradation of antibiotics.

Root exudates have been shown to affect the composition of rhizosphere microbial communities. For example, salicylic acid can induce systemic resistance in plants and inhibit the growth of pathogens (Badri et al. 2013), benzoic acid in peanut root exudates increases the relative abundance of Burkholderiaspp in rhizosphere soil (Liu et al. 2017), and ferulic acid in watermelon root exudates can promote the formation and germination of Fusarium oxysporum spores (Hao et al. 2010). In this experiment, it has been shown that a large amount of organic acid alcohols is produced in the rhizosphere secretions during SD degradation. Organic acid alcohols are hydrolyzed in acid or alkaline environments to produce corresponding acids and alcohols. Some strains exhibit significant absorption of amino acids, organic acids, sugars, and quaternary amines during root growth (Zhalnina et al. 2018).

The selective influence of plants on the composition and structure of the inter-rooted microbial community is always dominant, mainly due to the secretions of the plant roots that regulate the structure of the inter-rooted microbial community, which differs in root exudates before and after SD degradation. These root secretions can induce and stimulate the growth of specific bacterial groups and influence the abundance and diversity of inter-rooted microorganisms, which in turn has an impact on degradation efficiency and products. In a study of SD degradation dynamics, it was found that the areas with the highest degradation rates were those with the highest microbial abundance, and that the community structure changed before and after the experiment, probably because the crop roots secreted large amounts of organic matter into the soil, and these root secretions in turn directed the microbial community toward reducing external stresses, promoting SD degradation, causing the inter-rooted microbial community structure to respond.

As shown in Table 2, the number of OTUs in the samples was 2,541–3,353, and the coverage was over 98% (coverage: 98–99%). It can be seen from the table that for the Shannon P₀ > P₉₆ > V₀ > V₉₆ and for the Simpson V₉₆ > V₀ > P₉₆ > P₀; thus, the bacterial diversity of the rhizosphere in the P. australis group was always higher than that in the V. natans group before degradation (0 h) and after degradation (96 h). In the above analysis of the degradation rate, that of the P. australis group was also greater than that of the V. natans group, which was consistent with the expression of diversity analysis. The main bacterial groups involved in SA degradation may be resistant to antibiotics, thus contributing to SA biodegradation (Yang et al. 2016).

It can also be found from the table that the rhizosphere bacterial diversity of P. australis and V. natans decreased after 96 h of SD degradation. Chao1 was P₉₆ > P₀ > V₉₆ > V₀ and Shannoneven was P₀ > P₉₆ > V₀ > V₉₆, indicating that the degradation of SD increased the total number of bacteria in the rhizosphere of plants, but decreased the uniformity of bacterial distribution. Wang et al. (2021b) found that adding root exudates reduced microbial community diversity, but increased community abundance. This may have been due to the presence of a large amount of organic matter in plant root exudates. This leads to the evolution of the microbial community toward the reduction of external stress, which promotes the degradation of SD and induces and stimulates the growth of specific bacterial communities, thereby affecting the abundance and diversity of rhizosphere microorganisms (Yuan et al. 2017).

In order to reveal the sulfonamide degradation mechanism of the wetland plant rhizosphere, we compared the composition of rhizosphere exudates of P. australis and V. natans under 0.10 μg L⁻¹ SD stress in this study. The degradation of SD by rhizosphere exudates extracted under stress was also observed. We found that SD stress could increase the content of organic acid esters in plant roots. In the degradation experiment, we found that the degradation effect of the P. australis group was stronger than that of the V. natans group. During the degradation process, the relative abundance of rhizosphere bacteria will change, the diversity will decrease, and new genera will be introduced. In addition, we conclude that the degradation of SD in plants in karst areas may be controlled indirectly by increasing the content of organic acid esters in roots. Under weak alkaline conditions, organic acid esters break down to produce organic acids that affect the rhizosphere microbial metabolism.

This research was funded by the Guangxi Natural Science Foundation (grant number 2022GXNSFFA035033), the National Natural Science Foundation of China (grant number 52260023 and 51878197); the Basic Ability Enhancement Program for Young and Middle-aged Teachers of Guangxi (grant number 2021KY0265); and Innovation Project of Guangxi Graduate Education (YCBZ2022117).

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