Recovery of subtropical coastal intertidal system

Recovery of subtropical coastal intertidal system prokaryotes from a destruction event and the role of extracellular polymeric substances in the presence of endocrine disrupting chemicalsRecovery of subtropical coastal intertidal system prokaryotes from a destruction event and the role of extracellular polymeric substances in the presence of endocrine disrupting chemicals
Yang, Lihua; Xiao, Sirui; Yang, Qian; Luan, Tiangang; Tam, Nora F.Y.
Published in: Environment International
Published: 01/11/2020
Document Version: Final Published version, also known as Publisher’s PDF, Publisher’s Final version or Version of Record
License: CC BY-NC-ND
Published version (DOI): 10.1016/j.envint.2020.106023
Publication details: Yang, L., Xiao, S., Yang, Q., Luan, T., & Tam, N. F. Y. (2020). Recovery of subtropical coastal intertidal system prokaryotes from a destruction event and the role of extracellular polymeric substances in the presence of endocrine disrupting chemicals. Environment International, 144, [106023]. https://doi.org/10.1016/j.envint.2020.106023
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Environment International
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Recovery of subtropical coastal intertidal system prokaryotes from a destruction event and the role of extracellular polymeric substances in the presence of endocrine disrupting chemicals
Lihua Yanga,b, Sirui Xiaob,c, Qian Yanga, Tiangang Luanc,, Nora F.Y. Tamb,
aGuangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510275, China bDepartment of Chemistry, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region c State Key Laboratory of Biocontrol, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China
A R T I C L E I N F O
Handling editor: Yong-Guan Zhu
A B S T R A C T
Intertidal sediments constitute the micro-environment for the co-existence of endocrine disrupting chemicals (EDCs) and biofilms consisting of the microbial community and extracellular polymeric substances (EPS). However, the interactions and the resulting eco-function of this community are complex and poorly char- acterized, especially after a destruction event. This study evaluates the re-construction of biofilms in terms of the abundance of prokaryotic cells and related EPS characterization in two destroyed sedimentary matrices from subtropical environments simulated by sterilization in the presence of EDCs and investigates the role of EPS. The results show that benthic prokaryotes recover from the deposition of active prokaryotes in natural seawater and form biofilms after sterilization. Sterilization triggers the release of polysaccharides and protein from lysed native microbial cells and bound EPS in sedimentary organic matter, thus increasing their concentrations. The increased portion of EPS also acts as a persistent stress on re-colonizing prokaryotes and leads to the overproduction of sedimentary EPS. Due to the protective role mediated by EPS, the effect of EDCs on biofilm composition in sterilized sediment is not significant. The sedimentary matrix is the most important determinant of the composition of the biofilm and the occurrence of EDCs. At the end of an 84-day experiment, the abundance of prokaryotic cells and the concentrations of polysaccharides and protein in mangrove sediment are 1.6–1.8 times higher than those in sandflat sediment, regardless of EDCs. Sandflat sediment exhibits higher concentrations of nonylphenol and bisphenol A but a lower concentration of 17α-ethinylestradiol than mangrove sediment. This study enhances our understanding of the role of sedimentary biofilms and the fate of EDCs in intertidal systems and highlights the benefit of a destructive event in enhancing ecosystem function, particularly tolerance to EDC adversity due to EPS production.
1. Introduction
Intertidal sediments, which lie at the interface between land and marine ecosystems, are dynamic micro-environments that undergo frequent flooding and aerial exposure due to incoming tides and ebbing of water, respectively (Middelburg et al., 1996). Sediment supplies an excellent substratum for microbial growth with the formation of biofilms. The characteristics of biofilms are strongly influenced by the environmental conditions of the sediment (Blanco et al., 2019). Man- grove and sandflat sediments, the two typical intertidal matrices, exhibit different textures (Yang et al., 2018b) and varied microbial alpha- diversity (Jiang et al., 2013), and thus, their biofilms are likely different
but have seldom been studied. The biofilms on the surface of sediment, often referred to as biofilm-sediment aggregates, are highly hydrated biofilm matrices containing a microbial community and extracellular polymeric substances (EPS) (Zhang et al., 2018; Yallop et al., 2000; Vert et al., 2012). The EPS molecules form a three-dimensional architecture from which cells might localize their extracellular activities and undergo cooperative/antagonistic interactions (Decho and Gutierrez, 2017).
EPS are primarily composed of protein and polysaccharides, followed by humic substances, lipids, nucleic acids and their combina- tions, such as glycolipids, glycoproteins, etc. (Gerbersdorf et al., 2009). The EPS could be operationally isolated from the sediment as colloidal
https://doi.org/10.1016/j.envint.2020.106023 Received 21 February 2020; Received in revised form 28 July 2020; Accepted 28 July 2020
Corresponding authors. E-mail addresses: [email protected] (T. Luan), [email protected] (N.F.Y. Tam).
Environment International 144 (2020) 106023
Available online 18 August 2020 0160-4120/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
(or slime, soluble, water-extractable, etc.) and bound (or capsular, sheaths, resin-extractable, etc.) fractions (Gerbersdorf et al., 2008). The former represents any EPS that is not tightly bound to the sediment but might be associated with cells, and the latter represents EPS that is more tightly bound to the sediment, probably through bridging with divalent cations (Laspidou and Rittmann, 2002; Stal, 2003). Based on the size; each fraction is further subdivided into low (LMW) and high molecular weight (HMW) sub-fractions (Yallop et al., 2000). The EPS creates weak forces, such as hydrogen and ionic bonds, van der Waals forces and hydrophobic interactions (Flemming et al., 2016), to aggregate sediment particles and increase sediment stability (Gerbersdorf and Wieprecht, 2015; Tolhurst et al., 2002). The EPS is also an important sources of dissolved/particle organic carbon (DOC/POC) and transparent exopolymer particles (TEP) in aquatic systems (Camacho- Chab et al., 2016); supplying a substantial pool of organic matter available to prokaryotes, especially heterotrophic bacteria and archaea. As a result, interactions of prokaryotes with EPS at the nanometer to millimeter scales are a fundamental problem in understanding how biofilms drive all biogeochemical processes on Earth (Flemming and Wuertz, 2019; Azam and Malfatti, 2007).
Intertidal zones are a well-documented repository for terrestrial pollutants delivered from the catchment via fluvial transport, atmo- spheric deposition, and/or local wastewater discharge. Steroidal hormones and phenolic endocrine disrupting chemicals (EDCs), the most massive discharged emerging contaminants, are widely found in intertidal systems (Zoppini et al., 2018; Li et al., 2018) and even in biofilms and sediments (Writer et al., 2011). EDCs have potential harmful impacts on hormonal control, sexual differentiation, reproductive suc- cess and the community structure of indigenous wild animals and their offspring (Jobling et al., 2006; Rodgers-Gray et al., 2001). Evidence also exists that EDCs affect the sedimentary microbial community (Yang et al., 2014; Wang et al., 2014; Yuan et al., 2017); and sedimentary prokaryotes are the most effective microorganisms for biodegradation and thorough mineralization of EDCs (Ogata et al., 2013; Yang et al., 2018a).
The co-existence of EDCs, prokaryotes and EPS constitutes the most important micro-environment in intertidal sediments, but their interactions and resulting eco-function are complex and poorly character- ized. Our recent study reported the overproduction of sedimentary EPS and variation of the bacterial community in the presence of EDCs (Yang et al., 2018b; Yuan et al., 2017). Limited studies demonstrated the recovery of the microbial community after destructive events such as benthic microalgae following a beach nourishment event (Hill-Spanik et al., 2019) and recovery assessment of mangrove ecosystems impacted by refined-oil, fire and sea-level rise (Otitoloju et al., 2007; Smith et al., 2013). However, what will happen during the re-construction of biofilms in sediments after a destructive event that completely eradicates microorganisms and the role of EPS in the presence of EDCs in subtropical coastal ecosystems, which has seldom been researched. The impact of EDCs on the sedimentary EPS during such a re-construction process has also never been investigated. Therefore, this study evaluates the re-construction of microbial biofilms, in terms of prokaryotes and related EPS characterization, in mangrove and sandflat sediments after a destructive event simulated by sterilization, and the role of EPS in the presence of EDCs. These two sediments are commonly found in intertidal zones in subtropical coasts. The study also attempts to determine the effects of sterilization and EDC spiking on sedimentary prokaryotes and the related EPS composition and explores the relationships among cell abundance, EPS variations and EDC removal.
2. Material and methods
2.1. Chemicals and reagents
Standards including propylparaben (PP,> 99%), bisphenol A (BPA,> 99%), technical nonylphenol (NP,> 99%), 17α-
ethinylestradiol (EE2,> 99%) and 17β-estradiol (E2,> 99%) were purchased from Aldrich Chemistry Corporation (Milwaukee, WI). Internal standards including NP-d4 and EE2-d4 were obtained from Sigma-Aldrich (St. Louis, MO). The derivatization reagent N,O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA/TMCS) was obtained from Supelco (Supelco Park, PA). The fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), acetone and methanol of HPLC grade were all sourced from Sigma-Aldrich (St. Louis, MO). The modified bicinchoninic acid (BCA) protein assay kit and bovine serum albumin (BSA) were obtained from Sangon Biotech (Shanghai, China). Pyridine, glucose, phenol, sulfuric acid and other conventional reagents were of analytic grade and were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China).
2.2. Experimental set-up
Surface mangrove and sandflat sediments (0–1 cm) and foreshore seawater were collected from the Sai Keng mangrove wetland (22°25′N, 114°16′E) along the eastern coast of Hong Kong SAR in China. Mangrove sediment was sampled under the canopy formed by the leaves of mature mangrove trees at low tide, and sandflat sediment was collected from bare foreshore without any vegetation. The simulated water-sediment systems and the incubation condition of the batch tests were the same as those described in Yang et al. (2018b), except for sterilization of sediment at 121 °C for one hour. This sterilization pro- cedure was repeated three times every other day to ensure that the sterilization was thorough, which was immediately checked by counting the number of colony-forming units after plating on Luria- Bertani (LB) agar (Doghri et al., 2017). The particle size distribution of the sterilized sediment was analyzed by a Microtract S3500 laser particle size analyzer (MicrotractInc., CA, USA). Total organic matter (TOM) was measured by loss on ignition at 550 °C in a muffle furnace (Carbolite, UK). Total Kjeldahl nitrogen (TKN) and total phosphate (TP) were determined by a flow injection analyzer (FIA) (Lachat QuikChem Method 8000, USA). The sediment redox potential (Eh) and pH were recorded in situ using an Eh meter (TPS WP-81, Australia) and a pH meter (E.W. System Soil tester, Tokyo, Japan), respectively.
In this study, 12 sets of sediment tanks (28.2 cm × 20.6 cm × 7.3 cm) covered with approximately 2.9 L of sediment were prepared. Six tanks were filled with sterilized sandflat sediment, and the other six were filled with sterilized mangrove sediment. Approximately 7.9 L of collected seawater without any treatment were added into the seawater tray (38.3 cm× 26.3 cm× 13.2 cm). The sediment tank was immersed in the seawater tray for 12 h to simulate high tide and exposed to air for another 12 h to simulate low tide. After an acclimation period of one week, three seawater trays of each sediment were spiked with mixed EDCs by direct addition of EDCs into the water tray during high tide at a nominal concentration of 200 μg/L for each EDC compound. A mixture of EDCs was applied to mimic the combined effects of these contaminants in the real environment. The concentration simulated the severe point source contamination in intertidal systems with concentrations of PP, BPA, NP, EE2 and E2 reaching up to hundreds of μg/L (Yang et al., 2018b; Hopkins and Blaney, 2016). To maintain a steady EDC stress, EDCs were re-spiked every seven days with a total of 12 re-spiking events during the 84-day incubation. The remaining three sets of the same sediment did not undergo any EDC spiking. In all, four groups, namely, sterilized mangrove group (MGS), sterilized mangrove group with EDCs spiking (MGS + EDCs), sterilized sandflat group (SFS) and sterilized sandflat group with EDCs spiking (SFS + EDCs) were prepared, each in tripli- cate. On day 0, immediately after spiking of EDCs, as well as on days 7, 28, 56 and 84 (before re-spiking of EDCs), aliquots of 100 mL water were accurately sampled for analysis of EDCs. On days 0, 28, 56 and 84, surface sediments (0–1 cm) were also collected using small shovels at low tide, immediately before the next high tide.
L. Yang, et al. Environment International 144 (2020) 106023
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2.3. Analysis of EDCs
EDCs in water and sediment were extracted by organic solvent and analyzed by gas chromatography-mass spectrometry (GC–MS) (Yang et al., 2016a). In brief, water was filtered by GF/F glass fiber filters (Φ = 0.7 μm, Whatman International Ltd., Maidstone, England), added with internal standards (I.S.) of NP-d4 and EE2-d4, and submitted to solid-phase extraction with CNW Poly-Sery HLB cartridges (500 mg, 6 mL, CNW Technologies GmbH, Germany). The freeze-dried sediment was added with I.S. and subjected to accelerated solvent extraction (ASE 350, Dionex, USA). The extract was derivatized by BSTFA/ TMCS:pyridine (50:50) and analyzed by an Agilent 7890A GC coupled with a 5975C MS.
2.4. Determination of cell abundance.
The abundance of prokaryotic cells in the sediment was directly determined by the counting method under epifluorescence microscopy (Yang et al., 2018b; Porter and Feig, 1980). An accurate 1 mL of the sediment sample stained with 4′,6-diamidino-2-phenylindole (DAPI) was filtered through a black 0.22 μm polycarbonate filter (Millipore, USA) and washed by distilled water in a syringe filter. After cleaning, the filter was mounted in low-fluorescence immersion oil on a glass microscope slide after air drying. The cell number was counted using an epifluorescence microscope (Leica DM5000B, Wetzlar, German) with blue excitation light (450–490 nm) at a magnification of 10 × 100 under oil. The prokaryotes can be easily counted and distinguished from particles after DAPI staining. Because all of the cells in the diluted samples were retained in the filter, the recovery of cells was not eval- uated. To satisfy quality control standards, at least 30 randomly selected fields per slide containing 1000 cells for each sample were counted, and the mean was calculated as the abundance of prokaryotic cells.
2.5. Extraction and characterization of EPS
Sediment EPS was extracted immediately after collection according to the published Dowex resin method (Yang et al., 2018b; Takahashi et al., 2009). In brief, colloidal and bound EPS were separated by artificial seawater (ASW) and activated Dowex 50WX8 (hydrogen form, 200–400 mesh, Sigma-Aldrich, MO, USA), respectively. The LMW and HMW sub-fractions were segregated by ethanol at −20 °C. Therefore, sediment EPS were divided into four sub-fractions, i.e., colloidal-HMW, colloidal-LMW, bound-HMW and bound-LMW. These four sub-fractions were freeze-dried for characterization of EPS. The concentration of polysaccharides was determined by a phenol sulfuric acid assay using glucose as a standard (DuBois et al., 1956). The protein concentration was determined using a modified bicinchoninic acid (BCA) protein assay kit assay with bovine serum albumin (BSA) as a standard (Smith et al., 1987).
2.6. Statistical analyses
The statistical analyses and graphics were processed using Origin 8.5 software and the R program (https://www.r-project.org). The mean ± standard deviation (s.d.) of three replicates were calculated. The differences in sedimentary cell number, concentration of each EDC in water/sediment, and content of EPS composition (colloidal/bound LMW/HMW polysaccharide and protein) among sampling times and groups were tested by a parametric two-way analysis of variance (ANOVA). If the interaction between these two sources of variations was significant at p < 0.05, a one-way ANOVA followed by a Tukey post hoc test were applied to test the effect of sampling time in each group, and the difference among groups at each sampling time. To clarify the effect of sedimentary matrices, sterilization and EDC spiking, comparison between two counterpart treatment groups, i.e., sterilized
vs. non-sterilized, EDC spiking vs. non-spiking, or mangrove vs. sandflat sediment, was performed by paired sample t-tests. To evaluate the effect of sterilization, the data in the sterilized groups were compared with those in the non-sterilized groups reported by Yang et al. (2018b), because these two experiments were conducted simultaneously using the same sediments, except that the published work used fresh sediment without sterilization. The relationships of the multi-parameters, including EDCs contents and biofilm composition, were determined by Pearson correlation tests and principal component analysis (PCA).
3. Results
3.1. Sediment characterization
The medium particle size of sterilized sandflat sediment was 219.6 ± 7.5 μm, which was higher than that of sterilized mangrove sediment at 149.2 ± 16.1 μm. The texture of sterilized sandflat sediment was more sandy with 98.1% sand and 1.9% silt, whereas the sterilized mangrove sediment was more clayey and contained 60.1% sand, 39.7% silt and 0.2% clay. The concentrations of TOM, TKN and TP in sterilized mangrove sediment, at 3.3%, 2 mg/g dw and 0.4 mg/g dw, respectively, were higher than those in sterilized sandflat sediment with respective values of 0.6%, 1.0 mg/g dw and 0.1 mg/g dw. The pH in the two sterilized sediments were comparable, near 6.91 to 6.94. However, the redox potential varied dramatically between these two sterilized sediments, at −145.98 ± 63.25 mV in sterilized mangrove sediment and 129.40 ± 37.77 mV in sterilized sandflat sediment.
3.2. EDCs in water
The initial concentrations of EDCs in water of the MGS + EDCs and SFS + EDCs groups after spiking ranged from 136.4 to 246.8 μg/L except that BPA was found at a higher concentration of 559.4 to 710.9 μg/L (Fig. 1), probably due to its migration from polycarbonate bottles, as reported in previous research (Yang et al., 2018b). The aqueous concentrations of all EDCs decreased rapidly in the first 7 or 28 days and remained at relatively low concentrations until the end of the experiment. No significant temporal changes were observed from day 28 onwards; according to one-way ANOVA. At the end of the experiment on day 84, the concentration of BPA (73.1–79.3 μg/L) in water was the highest, followed by that of NP (1.8–7.3 μg/L), and the other three EDCs displayed low values, ranging from 0.3 to 1.5 μg/L. The two-way ANOVA results also showed the effects of times and groups, and the interaction of these two factors were significant for PP, BPA and E2 but insignificant for NP and EE2 (Table A1). The t-test results further revealed that the water concentrations of PP and BPA in SFS + EDCs on day 7 were significantly higher than in MGS + EDCs, but the former group had a lower E2 concentration on day 0 (Table A2). After slight differences in the first 7 days (preliminary stage) of the experiment, no significant differences were noted in the concentration of each EDC in water between the MGS+ EDCs and SFS + EDCs groups from day 28 onwards. All five EDCs, except for BPA, exhibited notably good removal percentages of> 95% during the experiment, although the spiked concentration was high (~200 μg/L on day 0 and subsequently once per week).
3.3. EDCs in sediment
The initial concentrations of EDCs before spiking represented the detected EDC concentrations in the samples collected from the field (Fig. 1). On day 0, the concentration of NP in sediments ranged from 1177.8 to 1562.4 ng/g, whereas that of EE2 was 15.4 to 20.9 ng/g, and the initial concentrations of these two EDCs did not show any significant difference between the MGS + EDCs and SFS + EDCs groups (Table A3). However, the initial concentrations of PP and E2 in SFS + EDCs sediment were significantly lower than those in
Fig. 1. Temporal variation of concentration of each EDC in water and sediment after EDCs spiking during 84-days incubation: (a) PP; (b) NP; (c) BPA; (d) E2 and (e) EE2. (different letters represent significant difference among sampling times in the same treatment group at p ≤ 0.05 according to ANOVA; small and upper cases in black represent samples in water of SFS + EDCs and MGS + EDCs groups, respectively; while the respective cases in sediment were in blue; ns indicates not significant at p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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MGS + EDCs sediment, whereas BPA showed the opposite trend with higher concentration in SFS + EDCs sediment. During incubation, the concentrations of PP in MGS + EDCs sediment decreased gradually, but the temporal change was not significant in SFS + EDCs sediment. Si- milarly, the temporal change of E2 in SFS + EDCs sediment was also not significant, although certain fluctuations were found, with a significant drop from days 56 to 84. However, the concentrations of NP, BPA and EE2 first increased, reached a peak on day 28 or 56, and subsequently decreased. The highest concentration of NP (12469.0 ng/ g) was found in SFS + EDCs sediment on day 56, and the highest concentrations of BPA (1633.3 ng/g) and EE2 (2008.4 ng/g) were detected in MGS + EDCs sediment on day 28.
The two-way ANOVA results showed that the effects of the times, groups and their interactions on the sedimentary EDC concentrations were all significant (Table A1). The temporal changes of EDC concentrations in the sediments differed between groups and EDC compounds. At the end of the 84-day experiment, the final concentrations of NP and BPA in SFS + EDCs sediment were significantly higher than those in MGS + EDCs sediment, but the opposite phenomenon was observed for EE2. Interestingly, the concentrations of PP (110.2–223.5 ng/g) and E2 (9.2–36.9 ng/g) were comparable between these two groups.
3.4. Recovery of prokaryotes in sediment
The traditional plating method revealed that culturable microflora were not found in sterilized sediment at the beginning of the acclimation, confirming that the sterilization was thorough. Bright blue was also not detected by DAPI staining under epifluorescence microscopy, which indicated that double-stranded DNA in dead cells were destroyed by sterilization. After an acclimation period of 7 days (considered as day 0 in the 84-day incubation test), the prokaryotes in sediment were recovered with abundances varying from 0.66 to 0.86 × 108 cells/g, and no significant difference was observed among four treatment groups (Fig. 2). The cell abundance continued to increase from day 7
onwards, indicating that the prokaryotes gradually re-colonized and recovered from sterilization. During the re-construction process, several distinct growth phases of sedimentary prokaryotes were recorded in the MGS and SFS groups. The abundance exhibited rapid propagation with an amplification coefficient (the divisor of concentration) of 4.6 to 6.0 times from days 0 to 28 but subsequently slowed to 1.5 to 3.1 times from days 28 to 56. The cell abundance was comparable among the four groups on day 28 but exhibited significant differences on day 56. The two-way ANOVA results showed that the effects of times, groups and interactions on the cell abundance in sediments were significant (Table A4). The t-test revealed that EDC spiking did not have any significant effect on cell number in the sandflat and mangrove sediments during the experiment, except that the number in the MGS + EDCs group was significantly lower than in the corresponding group without EDCs on day 56 (Table A5). From days 56 to 84, the amplification coefficient of cell number was only 1.0 to 1.6. For mangrove sediment, prokaryotes in the EDC-spiked group reached the highest abundance on day 84, but the abundance on day 56 was comparable to that on day 84 in the corresponding group without EDCs (Fig. 2). At the end of the experiment (on day 84), the cell number in mangrove sediment (15.3 × 108
cells/g) was 1.8 times higher than in sandflat sediment (8.4 × 108
cells/g).
3.5. Extracellular polysaccharide concentrations
The concentration of total extracellular polysaccharide on day 0 in SFS group was 68.6 ± 5.9 µg/g, only 15.7% of that in the MGS group, which had a mean and standard deviation of 437.5 ± 12.4 µg/g (Fig. 3). Among the four sub-fractions, the colloidal and bound sub- fractions of LMW constituted 84.1% of total polysaccharides in the SFS group, whereas in the MGS, the bound-LMW sub-fraction was more dominant and accounted for 35.3%, followed by the colloidal- and bound sub-fractions of HMW at 25.5% and 27.6%, respectively. The total polysaccharide concentrations in these two groups decreased gradually with incubation time, and the final concentrations on day 84
Fig. 2. Temporal variation of cell abundance in sediments during 84-days incubation (different letters in small case indicate significant difference among the four treatment groups at the same sampling time, while different letters in upper case represent significant difference among sampling times in the same treatment group at p ≤ 0.05 according to ANOVA).
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in the SFS and MGS groups were only 34.1 ± 4.4 µg/g and 58.8 ± 3.6 µg/g, respectively, significantly lower than the initial values. The four sub-fractions in the MGS group also decreased significantly with time. The temporal trends in the SFS group depended on the sub-fraction, with decreases in the colloidal- and bound-LMW sub- fractions but increases in those of HMW. The concentrations of polysaccharides in all four sub-fractions in MGS group were higher than those in the SFS group during the entire experiment. The two-way ANOVA results showed that the effects of times, groups and their interactions on extracellular polysaccharide concentrations were all significant (Table A4). Despite the significant group effect, spiking of EDCs did not change the concentrations of total polysaccharides in the sediments during the experiment because no significant differences were observed between the MGS and MGS + EDCs groups or between the SFS and SFS + EDCs groups. However, the concentrations of total polysaccharides in the colloidal-HMW sub-fraction of the EDCs spiking groups (MGS + EDCs and SFS + EDCs) were significantly lower than in the corresponding non-spiking groups (MGS and SFS) on day 84 (Table A6).
3.6. Extracellular protein concentrations
The total protein concentration on day 0 was 100.2 ± 7.3 µg/g in the SFS group, which was only 25 percent of that in the MGS group at 394.8 ± 35.9 µg/g, and the concentrations were significantly higher in the MGS group than in the SFS group during the experiment from days 28 to 84 (Fig. 4). The ratios of the four sub-fractions were substantially different between these two sedimentary matrices. The bound-HMW sub-fraction in the SFS group on day 0 was the most dominant (51.8%), followed by the colloidal-LMW (21.0%) group, and was the least for the colloidal-HMW (14.0%) and bound-LMW (13.3%) sub-fractions. How- ever, the constituent ratios of the four sub-fractions in the MGS group on day 0 were relatively uniform, ranging from 21.8 to 29.1%. The effects of times, groups and their interactions on extracellular protein concentrations were all significant according to the two-way ANOVA results (Table A4). The trends of the changes in total protein concentrations from days 0 to 28 were the opposite between the sandflat and mangrove sediments, with concentrations decreased to 55.6–58.5 µg/g in the former sediment (SFS group), but increased to
SF S
Bound-LMW Colloidal-LMW Bound-HMW Colloidal-HMW
Day 84Day 56Day 28
Fig. 3. Temporal variation of extracellular polysaccharide concentrations in four sub-fractions of sediment biofilms during 84-days incubation (different letters in small case indicate significant difference among the four treatment groups at the same sampling time, while different letters in upper case represent significant difference among sampling times in the same treatment group at p ≤ 0.05 according to ANOVA).
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481.3–553.5 µg/g in the latter, MGS group (Fig. 4). The concentration in the SFS group maintained a range of 63.8–119.8 µg/g until day 84, whereas the MGS group exhibited a further decrease from days 56 to 84 with final concentrations near 200 µg/g. The temporal variations of the four sub-fractions in the SFS group were minor, and the concentrations of the three sub-fractions, except for bound-LMW in the MGS group, decreased significantly during incubation. Bound-LMW in the MGS group exhibited a peak concentration of 462.7 µg/g at day 28 and subsequently decreased to 125.0 µg/g at day 84, but this sub-fraction still constituted 64.8% of the total protein. Similar to the total polysaccharide concentrations, EDC spiking did not have any significant effects on total protein concentrations, and no significant differences were noted between the MGS and MGS + EDCs or between the SFS and SFS + EDCs groups (Fig. 4, Table A7).
3.7. Correlation analyses
Principal component analysis (PCA) was performed to clarify the correlation of EDC concentrations in water and sediment. The first two principal components (PCs) of PCA, showing the correlation of EDC
concentrations in water and sediment in the EDCs spiking groups, contributed 75.22% and accounted for the main portion of the total variance, with 59.32% by PC1 and 15.90% by PC2 (Fig. 5a). The score plots in PC1 showed that the EDC data for samples collected on day 0 were clearly separated from those on days 28, 56 and 84, indicating the effect of incubation time. However, PC2 showed the effect of sedimentary matrices, and the samples in the SFS and MGS groups were well separated. The EDCs in water were clustered in PC1, indicating their positive correlation, which was also shown by Pearson correlation tests (Fig. A.1a). All EDCs in water were positively correlated to PP in sediment but negatively correlated to EE2 in sediment. The concentrations of EE2 and BPA in sediment were also positively correlated.
In terms of cell number and EPS composition, the two axes of PCA ordination accounted for 82.48% of total variations, with the first and second axes explaining 66.33% and 16.15%, respectively (Fig. 5b). The score plots showed that the samples collected on day 0 in the MGS group were clearly separated from the other samples by PC1, whereas the samples in the SFS and MGS groups were well differentiated by PC2. The sedimentary cell number was positively correlated to the concentration of bound-LMW protein but negatively correlated to that of
Fig. 4. Temporal variation of extracellular protein concentrations in four sub-fractions of sediment biofilms during 84-days incubation (different letters in small case indicate significant difference among the four treatment groups at the same sampling time, while different letters in upper case represent significant difference among sampling times in the same treatment group at p ≤ 0.05 according to ANOVA).
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colloidal-LMW protein as well as those of polysaccharides in colloidal- HMW, colloidal-LMW and bound-LMW sub-fractions (Fig. A.1b). With the exception of bound-LMW protein, positive correlations were found among the concentrations of both polysaccharides and protein in nearly all the four sub-fractions.
4. Discussion
4.1. Recovery of prokaryotes and re-construction of biofilms in sediment with enhanced resistance towards EDC stress
The re-construction of biofilms in sterilized sediments was due to
Fig. 5. Principal component analysis for the combined data set in the factorial plane PC1-PC2. (a) concentration of each EDC in water (W) and sediment (S); (b) sedimentary cell number (S.B.N) and EPS compositions (C.H.C, B.H.C, C.L.C and B.L.C indicate polysaccharide of colloidal-HMW, bound- HMW, colloidal-LMW and bound-LMW sub-fractions, respectively; C.H.P, B.H.P, C.L.P and B.L.P indicate protein of colloidal-HMW, bound-HMW, colloidal-LMW and bound-LMW sub-fraction, respectively). Different colors represent sampling days (green: day 0, yellow: day 28, red: day 56 and blue: day 84) and different shapes of symbols represent treatment groups (: MGS, : MGS + EDCs, : SFS and : SFS + EDCs). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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active intrinsic microorganisms in natural seawater that could randomly attach to the surface of sediment particles via the physical forces of microbial deposition (Lai et al., 2018). Stal et al. (2019) considered that the initial attachment was reversible and became irreversible through adhesion by the appendages of microorganisms and/or by the exudation of sticky EPS (Stal et al., 2019). The growth stages observed in model bacteria such as Escherichia coli (Bacun-Druzina et al., 2011) and the mixed bacterial population in sediment (Owabor et al., 2011) were also found in this study, with prokaryotic cells dividing at a maximum rate exponentially during the first month and subsequently entering a period of stationary growth. The initial abundance of the cells (on day 0) in the MGS and SFS groups was only approximately 13.4–20.5% of that in the corresponding non-sterilized groups, but at the end of this experiment, the prokaryotes in both groups recovered, with numbers comparable to those in the non-sterilized counterparts (Yang et al., 2018b). The ranges of microbial abundance recorded in this study were comparable to those in the sediments of the Njoro River, Kenya (Muia et al., 2003) and the fresh water sediments of reservoirs and groyne fields (Gerbersdorf et al., 2009). Our previous results also demonstrated that the prokaryotes recovered from sterilization had similar taxonomic composition as the non-sterilized groups (Yuan et al., 2017) at the phylum level, with Proteobacteria as the predominant phylum, followed by Cyanobacteria, Chloroflexi, Planctomycetes and Acidobacteria, but sterilization led to lower microbial diversity.
In this study, the stress of EDCs did not have any significant effects on the cell numbers and the concentrations of total polysaccharides and protein in the two sterilized groups (MGS + EDCs vs.MGS, SFS + EDCs vs. SFS) during the recovery process of prokaryotes in sediments. These findings were different from our previous research in that EDCs caused a delay in bacterial growth in non-sterilized mangrove (MG) and sandflat (SF) sediments (Yang et al., 2018b); suggesting that the resistance of prokaryotes towards EDC stress was enhanced during re- construction of biofilms in sediments after sterilization. The increased EPS by sterilization in this study could alleviate EDC stress in sterilized sediments because polysaccharides and protein contributed to detox- ification of toxic chemicals in environments by acting as a protective barrier via the interaction of sorption/binding (Yan et al., 2019; Flemming and Wingender, 2010). Biofilm developed in artificial soil exhibited increased microbial community diversity and enhanced metabolic activity, as demonstrated by Wu et al. (2019). Such a benefit of biofilm re-construction in ecosystem function might also apply to similar destruction events and other unpredictable conditions of any natural ecosystem.
This study focuses on the abundance of prokaryotic cells and the associated EPS composition during the recovery process because they are essential characteristics of biofilms in sediments. Nevertheless, other parameters such as the abundance and diversity of eukaryotic microbes and the spatial structure and roughness of the sediment are also important for a complete understanding of the re-construction process of biofilms. The abundance of indigenous diatoms in the two intertidal sediments were up to 5.7–15.5 × 106 cells/g with similar dominated species of Cyclotella sp., Navicula sp., and Nitzschia sp. (unpublished data). Schmidt et al. (2016) also reported the biofilm developed in fine sediments was dominated by Nitzschia sp. (Schmidt et al., 2016). Biofilms developed in artificial soil were found to exhibit aggregates at sizes of micro-meter (Wu et al., 2019). The high density of binding sites of EPS could connect to sediment particles; bridge the void space and increase sediment stability (Gerbersdorf and Wieprecht, 2015). Biofilm formation has a profound effect on the physical structure of the clay, such as interlayer contraction and expansion (Alimova et al., 2009).
4.2. Sterilization emits EPS and induces overproduction in sediments
Sterilization of sediment by autoclaving releases cellular compounds from dead and lysed native microbial cells, as evidenced by the
significantly higher concentrations of initial polysaccharides and protein in the sterilized MGS and SFS groups than the corresponding non- sterilized groups (Yang et al., 2018b). Polysaccharides and protein were the main components of microbial cells such as microalgae and bacteria in natural ecosystems (Pivokonsky et al., 2014). The destruction of microbial cells was also evidenced by the discussed absence of bright blue observed from the DNA-DAPI complex in fresh sterilized sediments under epifluorescence microscopy. In addition to cellular compounds, sterilization also led to the release of EPS bound in sediments, depending on the sediment types. Compared with the fresh sediment without sterilization (non-sterilized groups), the increases in bound- HMW polysaccharides and colloidal-HMW protein in MGS reached 16.0-fold and 15.0-fold, respectively, whereas the respective increases of these two sub-fractions in SFS were only 1.8-fold and 11.5-fold. These variations between MGS and SFS might relate to the different binding ability and forms of EPS in the two sedimentary matrices. The adsorption of EPS on soil clay minerals is via binding with amide, carboxyl and phosphoryl functional groups, which depends on the concentrations of electrolytes (Cai et al., 2018). As EPS vary in composition and structure, their interactions with soil particles will also change accordingly (Costa et al., 2018). The increase in initial colloidal- HMW protein due to sterilization in both sediments was significantly higher than that in the other three sub-fractions, probably due to the decreased binding capacity with structural alteration and protein de- naturation during autoclaving.
In this study, the initial high concentrations of polysaccharides in both sterilized mangrove and sandflat sediments gradually decreased, and protein maintained a relatively stable concentration during incubation. This result implied that the components of the increased EPS released by sterilization underwent different processes during the re- construction of biofilms, with polysaccharides consumed by microorganisms while protein was relatively persistent. The negative correlation between the colloidal/bound polysaccharides in both the LMW and HMW fractions and sedimentary cell number further supported the observation that the increased polysaccharides might be used by prokaryotes during re-construction of biofilms in sterilized sediment. Wang et al. (2015) also showed that polysaccharides could be assimilated by soil bacteria, particularly members of the phylum Planctomycetes (Wang et al., 2015) and that EPS could act as potential carbon reserves (Decho and Gutierrez, 2017). Selected EPS could present as highly labile carbon forms, but other forms appeared to be quite refractory to degradation (Decho and Gutierrez, 2017). Similar to polysaccharides, many proteins and peptides were easily hydrolyzed by heterotrophic microorganisms, but a subset could be quite refractory to degradation (Decho and Gutierrez, 2017) because protein occurred in a variety of molecular forms such as peptides, amino-sugars, glycoproteins, pro- teoglycans and amyloid proteins (Ram et al., 2005). The steady concentration of protein during incubation in this study was probably due to the high portion of refractory protein in the increased EPS.
Overproduction of EPS is a common response of living microorganisms in biofilms when exposed to exogenous chemicals such as heavy metals, organic acids, chlorophenols, nanoparticles and EDCs (Yang et al., 2018b; Sheng et al., 2005; Dogan et al., 2014; Verneuil et al., 2015). At the end of the 84-day experiment, the concentrations of total polysaccharides in the sterilized mangrove sediment (MGS group), as well as the total protein concentrations in both the MGS and SFS groups, were significantly higher than in the corresponding non-sterilized group (Yang et al., 2018b). These results suggested that the portion of EPS increased by sediment sterilization, especially refractory protein, could exert an effect as an exogenous chemical during the re- construction of biofilms and might act as an additional stress, leading to overproduction of the EPS. To the best of our knowledge, this is the first report of such an observation. However, the increased EPS by sterilization not only alleviated EDC stress in sediments but was also fa- vorable for sediment stability, increasing the erosion threshold and decreasing the erosion rate (Tolhurst et al., 2002; Meng et al., 2019).
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Sediment stability is the key ecosystem function for the highly dynamic system in intertidal marine systems with periodic fluctuations in environmental parameters (Decho, 2000). These findings suggest that proper interruption of a natural ecosystem might be beneficial because it enhances ecosystem function through the emission and overproduction of EPS.
4.3. Sedimentary matrix is the determinant for biofilm composition and EDC occurrence
In terms of cell abundance and EPS concentrations, the biofilm composition in mangrove sediment was higher than in sandflat sediments after sterilization and during the re-construction process. The growth rate of prokaryotes in mangrove sediment was also faster. However, EDC stress did not exhibit any significant effect on the biofilm composition in both MGS and SFS sediments at the end of the 84-day incubation. These findings indicated that the sedimentary matrix was a greater determinant factor in prokaryotes and EPS concentrations than the stress of EDCs. Mangrove and sandflat sediments had highly different textures and TOM contents, as well as varied bacterial alpha- diversity (Jiang et al., 2013; Yuan et al., 2015). Different types of microphytobenthos colonized on the surface of fine and coarse sediment depending on the sedimentary composition (Stal, 2003). From the analysis of 20 biofilms collected in situ from five contrasting extreme environments, Blanco et al. (2019) also highlighted the strong influence of environmental conditions on the characteristics of biofilm (Blanco et al., 2019). Sedimentary properties affected the difference in EPS sub- fractions between mangrove and sandflat sediments in this study. HMW polysaccharide was the main component binding to the sedimentary organic matter in mangrove sediment, probably due to its higher TOM content than sandflat sediment. Sedimentary matrices further influenced the fractionation of the increased EPS by sterilization. At the end of the 84-day experiment, the increased polysaccharide concentration in mangrove sediment (MGS group) primarily contributed to the HMW of either the colloidal or bound fraction, whereas the increased protein concentration in the SFS and MGS groups was from the colloidal-HMW and bound-LMW groups, respectively.
On day 28 or day 56 in this study, some EDCs exhibited higher concentrations in sterilized sediment than the corresponding non-sterilized sediment (Yang et al., 2018b). For instance; higher concentrations of PP, BPA, E2 and EE2 were found in sterilized mangrove sediment (MGS) than in the non-sterilized one (MG), which might be partially due to the lower microbial abundance caused by sterilization because EDCs were biodegraded by the living microorganisms in sediment (Ogata et al., 2013; Yang et al., 2018a). However, the concentrations of each EDC in both sterilized mangrove and sandflat sediments at the end of the 84-day experiment were the same as those in the corresponding non-sterilized sediments (Yang et al., 2018b), probably because the biofilm re-constructed in the sterilized groups with the recovery of prokaryotes for biodegradation. The final concentrations of all EDCs in sediment, except PP and E2, differed significantly between the two sedimentary matrices. Our recent work showed the efficient removal of PP and E2 in water, and the controlling removal process was abiotic degradation mediated by hydroxyl radicals in the presence of an environmental concentration of dissolved organic matters (unpublished data). Among the three relatively persistent EDCs with low water so- lubility, the concentration of NP was higher than those of BPA and EE2 in sterilized mangrove and sandflat sediments, which followed the order of hydrophobicity (expressed as octanol-water partition coefficient, Log Kow) with NP (5.99) > EE2 (4.12) > BPA (3.64) (Yang et al., 2016a; Yuan et al., 2015). Sedimentary NP and BPA concentrations were higher in SFS than in MGS but the opposite for EE2; with a lower concentration in SFS. This result further confirmed that the sedimentary matrix contributed to the variations in EDCs in sediments. Compared with sandflat sediment, mangrove sediment exhibited higher concentrations of TOM, prokaryotes and EPS sub-fractions, but a
smaller particle size. The non-specific hydrophobic interactions between EDCs and colloid, organic matters or inorganic minerals might lead to their partition from water to the suspended particles and sediments (Wu et al., 2019; Sun and Zhou, 2015). The differences in the structure, accessibility and microporosity of these two sediments might affect the adsorption of EDCs onto the sedimentary matrix (Yeh et al., 2014). The EDCs in these two sediments could also be removed with varying degrees by the interactions of biotic and abiotic processes (Yang et al., 2016b), such as photo-degradation, biodegradation by living microorganisms, oxidative degradation by ferro-manganese ma- terials, and/or adsorption onto sedimentary inorganic and organic matter. The details of the mechanisms by which sediment matrices affect the composition of biofilms and concentrations of EPS, as well as the major removal process, migration and bioavailability of EDCs in sediment, are still limited.
5. Conclusions
This study reveals that sterilization of sediments triggers the release of EPS from lysed cells and/or those bound to sedimentary organic matter. During the re-construction of sediment biofilms, prokaryotes (and especially heterotrophic bacteria) completely recover, as shown by their abundance and the overproduction of sedimentary EPS. The protective role of the increased EPS enhances the resistance of the re- constructed biofilms to EDC stress. The increased portion of EPS polysaccharides is consumed by the prokaryotic cells recovered after sterilization. However, EPS protein might be a persistent stress to prokaryotic cells in the sediment, leading to overproduction of sedimentary EPS. These results indicate that proper interruption of microorganisms in the sediment by processes such as autoclaving might be beneficial because it increases the resistance of microorganisms towards EDC stress.
Author statement
The work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Key Research and Development Program of China (grant number 2018YFD0900604); the National Science Foundation of China (grant numbers 21876211, 21625703); a grant from Hong Kong Research Grants Council (grant number UGC/IDS(R)16/19); and Hong Kong Scholar Program (grant number XJ2011048).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.envint.2020.106023.
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Introduction
Statistical analyses
Extracellular polysaccharide concentrations
Extracellular protein concentrations
Recovery of prokaryotes and re-construction of biofilms in sediment with enhanced resistance towards EDC stress
Sterilization emits EPS and induces overproduction in sediments
Sedimentary matrix is the determinant for biofilm composition and EDC occurrence
Conclusions

Documents

Recovery of subtropical coastal intertidal system