Response of microbial community in the soil of halophyte after contamination with tetrabromobisphenol A

Coastal wetlands are subjected to increasing tetrabromobisphenol A (TBBPA) pollution, whereas knowledge of TBBPA degradation in marine environments is lacking. The changes of bacterial communities in TBBPA-polluted soil covered with halophytes were investigated. TBBPA could be degraded in the halophyte-covered saline-alkali soil in a microcosm experiment. Higher TBBPA removal occurred in the soil of Kandelia obovata compared with soils covered with Suaeda australis and Phragmites australis within 56 days of cultivation. The rhizosphere soils of S. australis, P. australis, and K. obovata mainly involved the classes of Bacteroidia, Gammaproteobacteria, Alphaproteobacteria, and Anaerolineae. Additionally, manganese oxidation, aerobic anoxygenic phototrophy, and fermentation functions were higher in the rhizosphere soil of K. obovata after TBBPA addition. This study supports that using suitable local halophytic plants is a promising approach for degrading TBBPA-contaminated coastal soil.


Introduction
Tetrabromobisphenol A (TBBPA; 4,4′-isopropylidenebis (2,6-dibromophenol)) is one of the most extensively used brominated flame retardants (BFRs), covering approximately 60% of the total worldwide usage [1]. It has been incorporated into numerous consumer products including plastics, textiles, and electronic circuit boards to protect materials against ignition [2]. TBBPA could act as hormones to interfere with the endocrine systems of organisms and humans due to its structural similarity to thyroxin [3]. However, it has been frequently detected in various environmental compartments and biota (ng/L-μg/L), and it is recognized as an important hazardous pollutant due to it may persist and bioaccumulate in the food chain [4][5][6][7].
Physicochemical studies have shown the fate of TBBPA in the aquatic environment [8,9] while the biotransformation process is usually considered to play a major role in determining the fate of this compound in the natural environment. Several biotic studies focused mainly on reductive debromination of TBBPA to yield less brominated bisphenol A (BPA) under ananerobic conditions, and further removal of BPA was not occurred [10,11]. However, TBBPA is probably long-term exposed to an aerobic environment before partitioning into anaerobic conditions. So far, only a few published reports on the aerobic biotransformation of TBBPA and mainly focused on freshwater sediment, river sediment, activated sludge, and soil [12][13][14]. Previous studies presented the first evidence for aerobic TBBPA biodegradation in terrestrial settings, whereas there is limited study on the degradation of this compound and community-level changes in the coastal marine environment [15].
Coastal marine environments have received land-based source, river flow, stromwater runoff, sewage effluent, and industrial wastewater [16]. Intertidal sediments, which are key transitional zones between terrestrial and aquatic environments, play an important role in biogeochemical cycles and ecosystem services [17]. It has been shown that TBBPA can be quite soluble under elevated pH [18]. On the other hand, a variety of industrial parks are located along the coastline in China, and varying degree concentrations of BFRs have been detected in sediments that were significantly associated with the urbanization and industrialization of the coastal zone [19]. Thus, if disposed of improperly, BFRs in the recycling sites adjacent to soil and river water could be finally transferred to the marine, which can exert potentially devastating effects on ecosystem structure and function. Yang et al. [20] found that Bacillus, Erythrobacter, Pseudomonas, and Rhodococcus were the main genus in a mangrove sediment supplemented with enzyme extract of spent mushroom compost under aerobic conditions within 80 d cultivation. Jiang et al. [21] demonstrated that the predominant genus in TBBPA (ca. 10 mg/kg dw) polluted mangrove species sediments during 3-month greenhouse pot experiment were Anaerolineae, Geobacter, Pseudomonas, Flavobacterium, and Azoarcus. Thus far, the degradation of this compound in coastal environments remains obscure.
We hypothesize that the intertidal sediments that undergo flooding with seawater and exposure to the atmosphere between high and low tides should have significant impacts on TBBPA elimination. The main study aims to compare the removal of TBBPA in the intertidal sediment under different types of vegetation coverages and then track changes in the bacterial community by using a 16S rRNA gene-based Illumina sequencing approach. This study will expand the understanding of the extent of whether indigenous marine microbes in the coastal marine environment are capable of biodegrading TBBPA and provide information on the fate of this compound.

Material and methods
Chemicals TBBPA (98 + % purity) for spiking was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). HPLCgrade methanol was obtained from Tedia Company Inc. (USA). All other chemicals were ACS grade or better.

Characterization of sediments
Species in a coastal wetland dominated by Suaeda australis (defined as "SUA" hereafter), Phragmites australis (PHR), Kandelia obovata (KAN), and bare mudflats (MUD) without plant coverage used in this study were collected in triplicate. These three plant species had a high survival rate and strong adaptability in the study area. Moreover, the study site has no known history of TBBPA contamination. A soil sample (0-20 cm) was also collected from each pot using a soil auger from the coast of Zhoushan Island (29°32′-30°04′N, 121°30′-123°25′), in May 2022. Large sand grains and plant debris in each plant species covered sediments were removed and manually homogenized. Sediments were placed on ice and transported to the laboratory immediately. The sediment was divided into two portions, one portion was stored at 4 °C until use: and the other portion was air-dried and sieved through a 2 mm sieve and used for physicochemical analysis. The characteristics of the sediments were determined, including pH, salinity, and soil organic matter (SOM) [22].

Microcosm set-up
For a comprehensive understanding of in situ biological remediation, microcosms were adopted due to their authenticity, flexibility, and security which could reflect the fate of the target compound [23,24]. Two runs were carried out involving (i) sediment + plant only and (ii) sediment + plant amended with TBBPA. TBBPA was spiked into the microcosms at a final concentration of 20 mg/kg, and the microcosms were stirred vigorously with seawater using a stainless spatula to make homogenized sediments. Then, SUA, PHR, and KAN were separately planted in the microcosm, while sediments without plantation were used as control. For comparison, mudflats were also polluted with TBBPA, while mudflats without TBBPA contamination were used as control.

Soil DNA extraction and high-throughput sequencing
After 56 days of TBBPA contamination, the rhizospheric soils were collected separately, and the initial rhizospheric soils (i.e., without TBBPA contamination) were used as control. The total genomic DNA was extracted from the soils using the Soil DNA Kit (D5625, OMEGA) following the manufacturer's protocols. The concentration and purity of the DNA were quantified by Qubit®3.0 (Life Invitrogen). The integrity of the DNA was verified by running electrophoresis on 2% agarose gel. The V4-V5 regions of 16S rRNA genes of the samples were subjected to sequencing. The 16S rRNA genes were amplified using the primers 515F (5′-GTG CCA GCMGCC GCG G-3′) and 907R (5′-CCG TCA ATTCMTTT RAG TTT-3′) by PCR (95 °C for 5 min, followed by 27 ~ 30 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s and a final extension at 72 °C for 5 min). PCR reactions were performed in 30 μL mixture containing 15 μL of 2 × Phanta Master Mix, 1 μL of each primer (10 μM), and 20 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer's instructions and quantified using Qubit®3.0 (Life Invitrogen).
The amplicon library was paired-end sequenced (2 × 250) on an Illumina Novaseq 6000 platform (Nanjing GenePioneer Co. Ltd) according to the standard protocols. The PCR chimeras were detected and filtered out using the Vsearch uchime-denovo algorithm. The PE reads were overlapped to assemble the final tag sequences with the minimum overlap length as 10 bp with PANDAseq [25]. Quality filtering of MiSeq reads was carried out by PRINSEQ according to specific filtering conditions to obtain high-quality clean tags. The sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs) by Vsearch (Version 2.15.0). The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by uclust against the silva (SSU132)16S rRNA database using a confidence threshold of 90%. The rarefaction analysis based on QIIME (1.9.1) was conducted to reveal the diversity indices. Beta-diversity using both weighted and unweighted UniFrac was calculated by QIIME 1.9.1 [26]. The functional annotation of the bacterial taxa was performed using the Functional Annotation of Prokaryotic Taxa (FAPROTAX) database [27].

Soil characterization
The physicochemical properties of SUA, PHR, KAN, and MUD soils are summarized in Table 1. The soil properties differed between the plant-covered soils and the barren soil (i.e., MUD). The soils were alkaline, and the soil pH of MUD was higher than that of three plant-covered soils. The soil salinity was highest in the MUD soil (22.8 g/kg) and dramatically declined in the soils with different plant coverage. Compared with the barren soil, the above results suggested that these three halophytic plants (i.e., SUA, PHR, and KAN) could decrease soil pH and that desalinization occurred. Jing et al. reported that the cultivation of halophytes Suaeda glauca could assist in coastal soil desalination and altered soil properties [28]. The levels of SOM in all plant-covered soils were lower than that of MUD soil (28.0 g/kg) without plant coverage. The reduction of SOM was probably due to the continuous rainfall with only a small amount remained in the surface soil during the sampling period in May. It has been shown that the soil physicochemical properties extensively varied among halophyte vegetation types, and the soil bacteria-mediated functions involved are halophyte species specific [29,30].

TBBPA elimination in microcosm
The time profile of TBBPA removal in different plant-covered soils is shown in Fig. 1. Results showed that TBBPA degradation occurred in three plant soils as compared with the MUD soil without the plant. In plant-covered soils, KAN soil exhibited higher TBBPA removal, while TBBPA could be totally removed after 56 days of cultivation. Furthermore, the residual TBBPA concentrations were in the following order: SUA > PHR > KAN. Jiang et al. [21] demonstrated that TBBPA with an initial concentration of 10 mg/kg was almost degraded in Kandelia obovata-planted sediment during the 93-day growth period, which is in line with the present study. It was found that TBBPA biodegradation in  [20].

Predominant bacterial community
Microbial communities in coastal sediments play vital roles in biogeochemical cycles [31]. Bacterial communities in the rhizosphere soils of different plant coverage at the class and phylum levels are shown in Figs. 2 and 3, respectively. As shown in Fig. 2, more than 60% of total bacterial richness belonged to four classes: Bacteroidia, Gammaproteobacteria, Alphaproteobacteria, and Anaerolineae. A higher relative abundance of Bacteroidia was observed in MUD soil after being contaminated with TBBPA at the end of the experiment. The relative abundance of Bacteroidia was higher in SUA and PHR-covered soil as compared to the initial status. As for KAN-covered soil, a higher relative abundance of Bacteroidia and Actinobacteria was found after 56-day cultivation with TBBPA. At phylum levels, the major phyla were Bacteroidota, Proteobacteria, Gemmatimonadota, Chloroflexi, and Actinobacteriota after 56-day cultivation. Bacteroidota had the highest abundance in MUD soil with TBBPA. Similarly, this species had also dominated in plant-covered soils.
The previous report showed that Bacteroidota might participate in the decomposition of organic matter [32]. The phylum Proteobacteria played vital roles in the nitrogen fixation and nutrient cycling [33]. Gemmatimonadota might be related to plants and the rhizosphere [34]. Chloroflexi was directly associated with organic degradation [35]. Actinobacteriota played important roles in removing complex organic matter, nitrogen, and phosphorus [36]. Yang et al. [20] demonstrated that the classes of Gammaproteobacteria dramatically decreased and Alphaproteobacteria, Sphingobacteria, and Flavobacteria increased after the addition of TBBPA in mangrove sediments after 45 days of cultivation under aerobic conditions. The main phyla were Proteobacteria, Chloroflexi, Firmcutes, Fig. 2 Bacterial community composition at class-level classification. The panel labels represent the relative abundance of different bacterial species. Numbers 1-3 mean the same experimental set (without TBBPA) performed in triplicate, while the numbers 4-6 mean another experimental set (with TBBPA after 56 days) Actinobacteria, Bacteroidetes, Acidobateria, Nitrospira, and Cyanobacteria in the rhizosphere of A. marina and K. obovata after 93-day greenhouse pot experiment [21]. TBBPA removal might be due to microbial-driven biotransformation, bioaccumulation, and biodegradation by A. marina and K. obovata [21]. In our previous studies, Pseudoalteromonas sp. isolated from a TBBPA-polluted marine surface sediment played an important role in TBBPA biodegradation [15,37]. TBBPA degradation was an extracellular non-enzymatic process, and the extracellular biogenic siderophore, superoxide anion radical, hydrogen peroxide, and hydroxyl radical were involved in TBBPA removal [37]. The mechanism of TBBPA degradation by Pseudoalteromonas sp. presumably involved superoxide anion radical reduction and hydroxyl radical-based advanced oxidation [37]. Collectively, the results suggested that different plant species in coastal areas with different capacities for TBBPA elimination, probably involving the enrichment of potential TBBPA-degrading bacteria in the rhizosphere soils by these three halophytic plants.

Predictive functional profiling of KAN
The organic compounds degradation in environmental bioprocesses primarily results from microbial populations [38]. To further reveal the relationship between bacterial community and function in KAN-covered soil, ecological functions were predicted by using FAPROTAX (Fig. 4). Results showed that fumarate, nitrogen, and sulfur metabolisms were predicted in KAN soil prior to TBBPA contamination. A previous study has demonstrated that the carbon, nitrogen, and sulfur cycles were widespread in coastal wetlands [39], which is in line with this study. At the end of the 56-day cultivation with TBBPA, manganese oxidation, aerobic anoxygenic phototrophy, xylanolysis, fermentation, cyanobacteria, and oxygenic photoautotrophy functions were plentiful. It has been reported that Mn(III/IV) oxides were capable of oxidizing a wide range of compounds, including antibacterial agents, endocrine disruptors, and pesticides [40]. The aerobic anoxygenic phototrophy bacteria were widely distributed in marine environments, which played a unique role in the bioremediation of pollution [41]. The fermentation function might be related to bacterial debromination of TBBPA [20].

Conclusions
This study demonstrates that the coastal plant speciescovered soils were able to degrade TBBPA in a 56-day microcosm experiment. It was observed that the soil of K. obovate exhibited higher TBBPA degradation than that of S. australis and P. australis. More than 60% of total bacterial richness belonged to Bacteroidia, Gammaproteobacteria, Alphaproteobacteria, and Anaerolineae classes in the rhizosphere soils of these plants and, therefore, different impact on TBBPA removal in the sediment. The predicted function profiling of manganese oxidation, aerobic anoxygenic phototrophy, and fermentation played essential roles in TBBPA biodegradation in the rhizosphere soil of K. obovate. It was found that K. obovate would be a more preferred plant species when used for phytoremediation of TBBPA-contaminated coastal soil. This study is important for the assessment of the fate of TBBPA in coastal ecosystem and possibly for designing bioremediation strategies.
Author contribution CG and FZ conceived and designed the study. QXS and RWG conducted the literature search and performed the experiments. WKL was involved in the analysis and interpretation of data. CG drafted the manuscript. QS: The study was supervised and tutored. All authors read and approved the final manuscript.
Funding This work is supported by a grant from the Postdoctoral Advance Programs of Zhejiang Province (ZJ2021022 and ZJ2021048).

Conflict of interest
The authors declare no competing interests.