Sediment Bacteria and Phosphorus Fraction Response, Notably to Titanium Dioxide Nanoparticle Exposure

Titanium dioxide nanoparticle (TiO2 NP) toxicity to the growth of organisms has been gradually clarified; however, its effects on microorganism-mediated phosphorus turnover are poorly understood. To evaluate the influences of TiO2 NPs on phosphorus fractionation and the bacterial community, aquatic microorganisms were exposed to different concentrations of TiO2 NPs with different exposure times (i.e., 0, 10, and 30 days). We observed the adhesion of TiO2 NPs to the cell surfaces of planktonic microbes by using SEM, EDS, and XRD techniques. The addition of TiO2 NPs resulted in a decrease in the total phosphorus of water and an increase in the total phosphorus of sediments. Additionally, elevated TiO2 NPs enhanced the sediment activities of reductases (i.e., dehydrogenase [0.19–2.25 μg/d/g] and catalase [1.06–2.92 μmol/d/g]), and significantly decreased the absolute abundances of phosphorus-cycling-related genes (i.e., gcd [1.78 × 104–9.55 × 105 copies/g], phoD [5.50 × 103–5.49 × 107 copies/g], pstS [4.17 × 102–1.58 × 106 copies/g]), and sediment bacterial diversity. TiO2 NPs could noticeably affect the bacterial community, showing dramatic divergences in relative abundances (e.g., Actinobacteria, Acidobacteria, and Firmicutes), coexistence patterns, and functional redundancies (e.g., translation and transcription). Our results emphasized that the TiO2 NP amount—rather than the exposure time—showed significant effects on phosphorus fractions, enzyme activity, phosphorus-cycling-related gene abundance, and bacterial diversity, whereas the exposure time exhibited a greater influence on the composition and function of the sediment bacterial community than the TiO2 NP amount. Our findings clarify the responses of phosphorus fractions and the bacterial community to TiO2 NP exposure in the water–sediment ecosystem and highlight potential environmental risks of the migration of untreated TiO2 NPs to aquatic ecosystems.


Introduction
Titanium dioxide nanoparticles are extensively employed in catalysts, composite materials, cosmetics, electronics, food, paints, plastics, sunscreens, and wastewater treatment processes due to their special physicochemical properties [1,2]. TiO 2 NPs, with explosive demand by society, inevitably migrate into aquatic and terrestrial ecosystems [2,3]. The occurrence of TiO 2 NPs in the environment has raised public concern about their potential influences on flora and fauna [4,5]. Prior studies have reported that TiO 2 NPs present distinct toxicities to human cells [6], reduce nematode survival [4], and affect the root split of the carrot [5]. However, the effects of the amount of TiO 2 NP and exposure time on the composition, diversity, coexistence pattern, and functional redundancy of microorganisms in aquatic ecosystems are poorly understood.
Aquatic microorganisms are responsible for the cycling of key nutrients (e.g., carbon decomposition, nitrogen fixation, and phosphorus mineralization) [7]. Recent studies have reported that TiO 2 NPs can accelerate methanogenesis in mangrove wetland sediments [8], and affect nitrogen transformation [3] and phosphorus adsorption [9]. However, it remains unknown how TiO 2 NPs affect phosphorus fractionation in the water-sediment ecosystem.
Excessive phosphorus in aquatic ecosystems is one of the greatest environmental concerns due to its significant contribution to cyanobacterial bloom [10]. The source of phosphorus in an aquatic ecosystem is mainly from external input (e.g., aquaculture wastewater and phosphorus-containing industrial wastewater) and internal input (e.g., phosphorus release from sediments mediated by phosphorus-cycling-related microorganisms) [5,11]. Previous literature studies have reported that engineered TiO 2 NPs can block phosphorus release from sediments to water by adsorption [9,12,13]. In addition, it has been reported that TiO 2 NPs show toxicity to planktonic microorganisms by damaging DNA and cells via producing reactive oxygen species [14][15][16], and this might lead to an increase in phosphorus content in the water. Therefore, knowing the environmental risks of TiO 2 NPs in aquatic ecosystems is important for estimating dynamic changes in phosphorus fractions and is beneficial to appealing to environmental protection policies for mitigating the migration of TiO 2 NPs to water.
Riding on the boom of high-throughput sequencing, knowledge regarding the composition and diversity of the bacterial community in response to metal oxide nanoparticles is quickly expanding. Most TiO 2 NP-related studies only report phosphorus adsorption characteristics by TiO 2 NPs rather than TiO 2 NP-mediated simultaneous changes in microbial diversity and phosphorus fractions [9,13]. This situation motivated us to estimate the potential environmental risks of TiO 2 NP migration to aquatic ecosystems. The objectives of this study were to investigate the effects of TiO 2 NPs on (i) phosphorus fractionation in the sediment-water system, (ii) reductase activity and phosphorus-cycling-related gene abundance, and (iii) composition, diversity, and functions of sediment bacteria. Considering that high-concentration TiO 2 NPs can induce cell-producing reactive oxygen [1], we hypothesized that bacteria might lose diversity under conditions with high TiO 2 NP concentrations. To achieve our targets and validate our hypothesis, we employed highthroughput sequencing for the bacterial 16S rRNA gene and determined the content of phosphorus fractions in both water and sediments.

Sample Collection, TiO 2 NPs Suspension, and Expose Experiment
Sediments were collected from eutrophic Lake Nanhu (Wuhan, China) by using a sludge sampler in July 2019. The visibly large objects (e.g., stones, leaves, and glasses) were removed, and sediments were stirred (to mix them evenly). The sediments contained 3.67% of total carbon, 0.29% of total nitrogen, and 1.21 mg/g of total phosphorus. Lake water was simultaneously collected by using a water sampler (LB-800). The water contained 1.26 mg/L of total phosphorus and 25.33 mg/L of total nitrogen.
Commercially available TiO 2 NPs (25 nm, purity ≥ 99.8%, anatase) were purchased from Aladdin (Shanghai, China). To prepare 1000 mg/L of TiO 2 NP stock suspension, 1 g of TiO 2 NP was added to 1.00 L of Milli-Q water and then the mixture was sonicated at 250 W under 20 • C for 1 h [17].
About 100 g of sediments were added to 250 mL wide-mouthed glass bottles and were spread evenly. Subsequently, 200 mL of lake water was slightly added into the wide-mouthed glass bottles, and we let the mixtures stand for 1 h. According to TiO 2 NP concentrations used in previous studies [18][19][20], we added different volumes of TiO 2 NP stock suspensions to these bottles. The final concentrations of TiO 2 NPs were 5 mg/L (Amount 2 or A2), 10 mg/L (Amount 3 or A3), 20 mg/L (Amount 4 or A4), and 50 mg/L (Amount 5 or A5). The bottles without TiO 2 NP additions were treated as blank controls (Amount 1 or A1). The bottles in the experimental and control groups were placed in a dark place and incubated at room temperature. These five treatments contained three replicates, and bottles were incubated at room temperature for a total of 30 days. During incubation, we used spoons to collect about 5 g of sediments on days 1, 10, and 30. The collected sediments were stored at −80 • C for subsequent analysis.

Detection of TiO 2 NPs
To investigate whether TiO 2 NPs could be adsorbed by aquatic microorganisms, we added 10 mL of 1000 mg/L TiO 2 NP stock into 10,000 mL of lake water. The lake water without the TiO 2 NP addition was used as the blank control. The mixture was incubated at room temperature for 24 h; we then collected planktonic microorganisms by filtering the mixture through a 0.22-µm polycarbonate membrane (Millipore Corporation, Billerica, MA, USA). The collected planktonic microorganisms were washed 5 times using sterile water, freeze-dried, and processed further for the detection of TiO 2 NPs.
Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used to characterize cell morphology and the existence of the Ti element by applying a SU8010 Scanning Electron Microscope (HITACHI, Japan). The X-ray diffraction (XRD) patterns of the collected planktonic microorganisms were recorded on a Bruker D8 Advance diffractometer (Bruker, Germany) with graphite monochromatized Cu Ka radiation.

Determination of Phosphorus Fractions and Sediment Enzyme Assay
Water samples in five groups with different TiO 2 addition amounts were filtered through a 0.22-µm polycarbonate membrane; part of the water was used to measure soluble reactive phosphorus (SRP). The remaining part was digested by potassium persulfate to determine the total soluble phosphorus (TSP) [21]. Water total phosphorus (WTP) was directly digested by potassium persulfate without filtering through a 0.22-µm polycarbonate membrane. Sediment total phosphorus (TP), Olsen P, inorganic phosphorus (IP), organic phosphorus (OP), total apatite inorganic P (AP; P bound to calcium), and non-apatite inorganic P (NAIP; P bound to iron and aluminum oxyhydroxides) were extracted based on standard protocols described in a prior study [22]. Microbial biomass phosphorus (Pmb) was extracted by applying the chloroform fumigation-extraction approach, and a detailed description was reported in a previous study [23]. Phosphorus content was determined by using molybdenum-blue colorimetry. The activities of catalase and dehydrogenase were estimated based on previous studies [24,25].

DNA Extraction, Gene Quantification, MiSeq Sequencing, and Data Processing
The total genomic DNA was extracted from 0.5 g of freeze-dried sediments using the ISO-11063 standardized DNA extraction approach [26]. The extracted DNA was purified, employing a DNA-EZ Reagents M Humic acid-Be-Gone B kit (Sangon Biotech, Shanghai, China) following the manufacturer's instructions. DNA concentrations were measured applying a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All extracted DNA samples were stored at −80 • C.
The absolute abundances of phosphorus-cycling-related genes in sediment bacteria were determined by employing quantitation polymerase chain reaction (PCR) with the SYBR green mix. Quantitation PCR conditions and primer sequences for amplifying the inorganic phosphorus-solubilizing-related gcd gene, organic phosphorus-mineralizingrelated phoD gene, and inorganic phosphorus-transporting-related pstS gene are summarized in Supplementary Method 1.
Universal primers 338F (5 -ACT CCT ACG GGA GGC AGC A-3 ) and 806R (5 -GGA CTA CHV GGG TWT CTA AT-3 ) were used to amplify bacterial 16S rRNA gene targeting V3-V4 region [27]. The PCR was performed in a thermal cycler (ABI 9700, Thermo, USA) and conducted under the following conditions: a pre-denaturation at 95 • C for 5 min; 30 cycles at 95 • C for 30 s, 58 • C for 40 s, and 72 • C for 40 s; and then a final extension at 72 • C for 10 min. The PCR products were sent for Illumina MiSeq sequencing at the Personal Biotechnology Co., Ltd. (Shanghai, China).
The raw reads were processed using the pipeline of QIIME with the help of Vsearch [28]. To minimize the influences of any random-sequencing errors, we removed (i) sequences that contained ambiguous bases call; (ii) sequences that did not exactly match barcodes and primers; (iii) sequences with average quality scores less than 20; and (iv) sequences with maximum homopolymers less than 10 base pairs. The purified sequences were clus-tered into operational taxonomic units (OTUs) at 97% similarity levels against the SILVA v132 reference.

Data Analysis
Significant differences, if not specially-stated, were analyzed by analysis of variance, and Tukey's test was applied to compare the mean values for each variable when data followed a normal distribution (p < 0.05). A Venn diagram and nonmetric multidimensional scaling were built to reflect the community composition similarity of bacteria among five groups with different TiO 2 NP addition amounts by using the "VennDiagram" and "vegan" packages, respectively. Analysis of similarity was applied to estimate whether there were significant differences in the community compositions among the five groups. Permutational multivariate analysis of variance (PERMANOVA) was used to quantitatively evaluate the effects of the TiO 2 NP amount and exposure time on the phosphorus components, composition, diversity, and function of the bacterial community. Co-occurrence networks were constructed based on Pearson's correlations between bacterial phyla and the false discovery rate-corrected p-values were less than 0.05 [29]. The networks were visualized by using Gephi v. 0.9.2 (https://gephi.org/ (9 June 2022)). Functional prediction of bacterial taxa was performed by applying the "Tax4Fun2" package in R, and the functional redundancy index (FRI) of each sample was computed according to the 16S rRNA gene sequence similarity [30].

Responses of Phosphorus Fractions to TiO 2 NP Addition
Phosphorus fractions in both water and sediments varied in different samples with different TiO 2 NP addition amounts and exposure times (Table S1) Figure S1). For instance, SRP was significantly positively correlated with TSP, TP, Olsen P, OP, and AP, and noticeably negatively correlated with WTP and Pmb. According to the results of PERMANOVA, the TiO 2 NP addition amounts showed significantly greater effects on the phosphorus fractions (R 2 = 55.60%, F = 56.71; p < 0.001) than the exposure times (R 2 = 3.00%, F = 1.53; p > 0.05) ( Figure 1). Consequently, the ten phosphorus components presented significant differences among five groups with different TiO 2 NP addition amounts ( Figure 2). The content of SRP, TSP, TP, Olsen P, OP, IP, and AP increased with greater TiO 2 NP addition amounts, while WTP and Pmb decreased with elevated TiO 2 NPs. Previous literature has reported that TiO 2 NPs can enhance phosphorus adsorption by sediments [12]. Moreover, NPs can lead to algae aggregation and precipitation [15,31]. Therefore, the decreased WTP might be via two pathways: (i) direct adsorption by sediments and/or TiO 2 and (ii) indirect immobilization by TiO 2 NPs.   Subsequently, we used SEM, EDS, and XRD techniques to investigate the distribution of TiO 2 NPs. The surfaces of aquatic microorganisms presented smoothly under conditions without the TiO 2 NP addition and rough under conditions with the TiO 2 NP addition based on the SEM results ( Figure 3A). The Ti element was presented in cell surfaces by the EDS analysis ( Figure 3B). The collected microorganism had spinous peaks at 2θ of 25.3 • , 36.9 • , 39.5 • , and 47.9 • , which corresponded to anatase (JCPDS 83-2243) ( Figure 3C). It was reported that TiO 2 NPs possess high protein adsorption capabilities [14] and TiO 2 NPs can bind to algae cells and show rough surfaces [15].
Subsequently, we used SEM, EDS, and XRD techniques to investigate the distribution of TiO2 NPs. The surfaces of aquatic microorganisms presented smoothly under conditions without the TiO2 NP addition and rough under conditions with the TiO2 NP addition based on the SEM results ( Figure 3A). The Ti element was presented in cell surfaces by the EDS analysis ( Figure 3B). The collected microorganism had spinous peaks at 2θ of 25.3°, 36.9°, 39.5°, and 47.9°, which corresponded to anatase (JCPDS 83-2243) ( Figure 3C). It was reported that TiO2 NPs possess high protein adsorption capabilities [14] and TiO2 NPs can bind to algae cells and show rough surfaces [15].  Additionally, it is worth mentioning that the increase in the content of SRP and TSP might be due to the disintegration of aquatic microorganisms. Prior studies have reported that TiO 2 NPs impose toxicity to cyanobacterium Synechocystis sp. by affecting the expression levels of genes (e.g., psbA, psbD, and petF) [16]. The addition of TiO 2 NPs shows oxidative damage on Microcystis aeruginosa by increasing the content of reactive oxygen species and malondialdehyde [15]. In addition, previous literature has also reported that the decomposition of cyanobacteria can contribute to the formation and distribution of iron-bound phosphorus [32]. Therefore, the increases in TP, Olsen P, OP, and AP in sediments might be attributed to phosphorus transfer from water to sediment via algae decomposition and phosphorus precipitation, which can also be proved by significant correlations between sediment phosphorus components and water phosphorus fractions ( Figure S1). The increased soluble reactive phosphorus and total soluble phosphorus could be potential phosphorus resources for cyanobacteria growth. Prior studies have reported that TiO 2 NPs can weaken and even lose their toxicity to organisms by binging other substrates and aging [17,33]. From an environmental risk viewpoint, TiO 2 NPs might have both positive and negative contributions to cyanobacterial blooms.
Large differences in the bacterial community composition were found among five groups with different TiO2 NP addition amounts ( Figure 6). A total of 29,633 OTUs were found in five groups, and they shared 4788 OTUs ( Figure 6A). These OTUs were classified into 42 phyla. The Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi dominated in 45 sediment samples, with relative abundances ranging from 20.70% to 40.29%, from 20.62% to 34.36%, from 7.34% to 20.08%, and from 5.20 to 15.88%, respectively ( Figure 6B). Additionally, Firmicutes, Bacteroidetes, Gemmatimonadetes, Rokubacteria, Patescibacteria, and Planctomycetes were secondary dominant phyla. Significant differences were found in relative abundances of Actinobacteria, Acidobacteria, Firmicutes, Gemmatimonadetes, Rokubacteria, Patescibacteria, and Planctomycetes among five groups ( Figure S2). Moreover, distinct differences in the bacterial community composition were found among five groups based on the nonmetric multidimensional scaling plot, and the analysis of similarity confirmed the differences were significant (R = 0.694, p < 0.001) ( Figure 6C). According to the PER-MANOVA results , exposure time (R 2 = 20.84%, F = 6.35; p < 0.001) exhibited a greater effect on the bacterial community composition compared to the TiO2 NP addition amount (R 2 = 10.54%, F = 6.12; p < 0.001) (Figure 1). Sediment total phosphorus showed a larger Figure 5. Differences in absolute abundances of phosphorus-cycling-related genes (i.e., gcd, phoD, and pstS) among five TiO 2 NP addition amounts (i.e., A1, A2, A3, A4, and A5). Lowercase letters above the columns represent significance (p < 0.05). Table 1. Pearson's correlations between phosphorus fractions and dehydrogenase activity, catalase activity, and taxonomic diversity (Shannon-Wiener index), and the effects of phosphorus fractions on bacterial community composition based on PERMANOVA. Asterisks denote significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Abbreviations of phosphorus fractions are defined in the "Materials and Methods" section. Large differences in the bacterial community composition were found among five groups with different TiO 2 NP addition amounts ( Figure 6). A total of 29,633 OTUs were found in five groups, and they shared 4788 OTUs ( Figure 6A). These OTUs were classified into 42 phyla. The Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi dominated in 45 sediment samples, with relative abundances ranging from 20.70% to 40.29%, from 20.62% to 34.36%, from 7.34% to 20.08%, and from 5.20 to 15.88%, respectively ( Figure 6B). Additionally, Firmicutes, Bacteroidetes, Gemmatimonadetes, Rokubacteria, Patescibacteria, and Planctomycetes were secondary dominant phyla. Significant differences were found in relative abundances of Actinobacteria, Acidobacteria, Firmicutes, Gemmatimonadetes, Rokubacteria, Patescibacteria, and Planctomycetes among five groups ( Figure S2). Moreover, distinct differences in the bacterial community composition were found among five groups based on the nonmetric multidimensional scaling plot, and the analysis of similarity confirmed the differences were significant (R = 0.694, p < 0.001) ( Figure 6C). According to the PER-MANOVA results, exposure time (R 2 = 20.84%, F = 6.35; p < 0.001) exhibited a greater effect on the bacterial community composition compared to the TiO 2 NP addition amount (R 2 = 10.54%, F = 6.12; p < 0.001) (Figure 1). Sediment total phosphorus showed a larger influence on the bacterial community composition than other phosphorus components based on PERMANOVA (Table 1). Previous literature studies have reported that the TiO 2 NP addition affected the microbial community composition [1,17,39,40]. Apart from TiO 2 NPs, other NPs (e.g., ZnO NPs, CuO NPs, and Ag NPs) also showed distinct effects on the microbial community composition [41,42]. This phenomenon might be due to NP toxicity to the activity and living of microorganisms, which in turn affect the microbial community composition.
Microorganisms 2022, 10, x FOR PEER REVIEW 9 of 14 influence on the bacterial community composition than other phosphorus components based on PERMANOVA (Table 1). Previous literature studies have reported that the TiO2 NP addition affected the microbial community composition [1,17,39,40]. Apart from TiO2 NPs, other NPs (e.g., ZnO NPs, CuO NPs, and Ag NPs) also showed distinct effects on the microbial community composition [41,42]. This phenomenon might be due to NP toxicity to the activity and living of microorganisms, which in turn affect the microbial community composition. The taxonomic α-diversity represented by the Shannon-Wiener index (6.5-7.3) showed significant divergences among five groups with different TiO2 NP addition amounts (p < 0.05; Figure 6D). The TiO2 NP addition amount (R 2 = 69.67%, F = 98.88; p < 0.001) exhibited significant effects on the bacterial diversity compared to exposure time (R 2 = 0.78%, F = 0.57; p > 0.05) (Figure 1). The increased TiO2 NP addition amount decreased the sediment bacterial diversity, which is inconsistent with prior studies describing that TiO2 NPs increased bacterial α-diversity [43,44]. Moreover, it has been reported that the toxicity of TiO2 NPs can decrease the diversity and richness of phosphate-accumulating organisms [17]. The divergences in the effects of TiO2 NPs on bacterial diversity might be greatly due to the discrepancy in the bacterial community composition in different habitats. Microorganisms show different adaptation capabilities to the environment [45,46]. Furthermore, some microorganisms can produce and release reductase and, thus, might weaken the oxidative stress of TiO2 NPs on other microorganisms [15].
We found divergences in coexistence patterns of bacteria in sediments with different TiO2 NP addition amounts, showing different topological properties (e.g., node, edge, and Relative abundance of the top ten phyla. Numerical numbers (i.e., 1, 10, and 30) denote the incubating time (B). The nonmetric multidimensional scaling plot reflects the bacterial community composition among the five groups. Asterisks denote significance (***, p < 0.001) (C). Differences in bacterial taxonomic α-diversity (Shannon-Wiener index) among five groups with different TiO 2 NP addition amounts. Different letters denote differences (p < 0.05) (D).
The taxonomic α-diversity represented by the Shannon-Wiener index (6.5-7.3) showed significant divergences among five groups with different TiO 2 NP addition amounts (p < 0.05; Figure 6D). The TiO 2 NP addition amount (R 2 = 69.67%, F = 98.88; p < 0.001) exhibited significant effects on the bacterial diversity compared to exposure time (R 2 = 0.78%, F = 0.57; p > 0.05) (Figure 1). The increased TiO 2 NP addition amount decreased the sediment bacterial diversity, which is inconsistent with prior studies describing that TiO 2 NPs increased bacterial α-diversity [43,44]. Moreover, it has been reported that the toxicity of TiO 2 NPs can decrease the diversity and richness of phosphate-accumulating organisms [17]. The divergences in the effects of TiO 2 NPs on bacterial diversity might be greatly due to the discrepancy in the bacterial community composition in different habitats. Microorganisms show different adaptation capabilities to the environment [45,46]. Furthermore, some microorganisms can produce and release reductase and, thus, might weaken the oxidative stress of TiO 2 NPs on other microorganisms [15].

Potential Effects of Abiotic and Biotic Factors on Phosphorus Fractionation
The addition of TiO 2 NPs causes phosphorus (i.e., TP, Olsen P, OP, IP, and AP) accumulation in sediments and affects the diversity, composition, and function of the bacterial community. The activity, diversity, and function of the bacterial community might affect phosphorus fractionation in the water-sediment ecosystem. Phosphorus components, except NAIP, were significantly correlated with dehydrogenase and catalase (Table 1). Community diversity was significantly negatively correlated with SRP, TSP, TP, Olsen P, IP, OP, and AP, and dramatically positively correlated with WTP and Pmb ( Table 1). The abun-dances of the top 10 phyla (e.g., Acidobacteria, Firmicutes, and Rokubacteria) showed different correlations with phosphorus components (Table S3). Additionally, community functions showed significant correlations with phosphorus components in the water-sediment ecosystem, including lipid metabolism, xenobiotics biodegradation and metabolism, and translation and transcription based on KEGG pathway level 2 (Table S4).

Potential Effects of Abiotic and Biotic Factors on Phosphorus Fractionation
The addition of TiO2 NPs causes phosphorus (i.e., TP, Olsen P, OP, IP, and AP) accumulation in sediments and affects the diversity, composition, and function of the bacterial community. The activity, diversity, and function of the bacterial community might affect phosphorus fractionation in the water-sediment ecosystem. Phosphorus components, except NAIP, were significantly correlated with dehydrogenase and catalase (Table 1). Community diversity was significantly negatively correlated with SRP, TSP, TP, Olsen P, IP, OP, and AP, and dramatically positively correlated with WTP and Pmb ( Table 1). The abundances of the top 10 phyla (e.g., Acidobacteria, Firmicutes, and Rokubacteria) showed different correlations with phosphorus components (Table S3). Additionally, community functions showed significant correlations with phosphorus components in the water-sediment ecosystem, including lipid metabolism, xenobiotics biodegradation and metabolism, and translation and transcription based on KEGG pathway level 2 (Table S4).
Prior literature studies have reported that microbial activity (e.g., phosphatase and phytase) contributed to organic phosphorus mineralization in a farmland ecosystem [50] and compost system [23]. Bacterial diversity mediates phosphorus immobilization in the South China Sea [51] and can facilitate phosphorus transfer from sediments to water [29]. Some functional bacteria, including phosphate-accumulating bacteria, phosphate-solubil- Prior literature studies have reported that microbial activity (e.g., phosphatase and phytase) contributed to organic phosphorus mineralization in a farmland ecosystem [50] and compost system [23]. Bacterial diversity mediates phosphorus immobilization in the South China Sea [51] and can facilitate phosphorus transfer from sediments to water [29]. Some functional bacteria, including phosphate-accumulating bacteria, phosphate-solubilizing bacteria, and phosphate-mineralizing bacteria are responsible for phosphorus transformation in both aquatic and terrestrial ecosystems [11,22,23]. We guess that phosphorus components could affect the diversity, composition, and function of the sediment bacterial community, whereas the bacterial community might affect phosphorus fractionation in the water-sediment ecosystem. Therefore, the effects of TiO 2 NPs on phosphorus accumulation in sediments might be via two pathways: (i) showing toxicity to planktonic microorganisms and resulting in phosphorus precipitation, and (ii) blocking phosphorus transfer from sediments to water by affecting activity, diversity, composition, and function of the bacterial community in sediments.

Conclusions
In this study, we characterized the distribution of TiO 2 NPs and estimated the effects of TiO 2 NPs on phosphorus fractionation and diversity, composition, and function of the bacterial community in sediments. We found that TiO 2 NPs could bind to cell surfaces of planktonic microorganisms based on SEM, EDS, and XRD techniques. The addition of TiO 2 NPs could lead to the accumulation of phosphorus (e.g., total phosphorus and available phosphorus) in sediments, and significantly affect the composition, coexistence pattern, and function of the bacterial community. The absolute abundances of phosphoruscycling-related genes and bacterial diversity decreased with the increased TiO 2 NP addition amount, and significantly correlated with phosphorus components. Our findings are of significance for understanding the environmental risks of TiO 2 NPs. Given the undeniable toxicity of TiO 2 NPs to organisms, we will conduct more experiments to explore the effects of TiO 2 NPs on functional microorganisms at both pure culture and community levels in the future.

Data Availability Statement:
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at https://www. ncbi.nlm.nih.gov/, PRJNA735373.

Conflicts of Interest:
The authors declare that they have no conflict of interest.