The Effect of G-C3N4 on Bacterial Community in Sediment of Xiang River Under Tetracycline Pressure


 As photocatalysts applied more and more often to treat pollutants by photocatalytic reactions, they may enter the environment via water spreading. Although there are some investigations about their influence on different organisms, little is known about its impact on the ecological microenvironment. To understand how photocatalysts effect sediment ecological microenvironment in the process of pollution remediation, the impact of typical photocatalyst g-C3N4 (Graphitic carbon nitride) on rivered sediment community polluted by typical antibiotic tetracycline (TC) was investigated. The sediment samples were exposed to different concentrations of TC, g-C3N4 or TC/g-C3N4 (exposed to 60, 120, 180 mg/L TC, or 25, 75, 125 mg/kg g-C3N4, or 25, 75, 125 mg/kg g-C3N4 plus 60, 120, 180 mg/L TC, respectively), and sediment bacterial community were analyzed by Illumina sequencing. The results indicated that the dominant bacterial phyla in the samples were Acidobacteriota, Proteobacteria, Actinobacteriota, Chloroflexi. The diversity and richness of microorganisms in riverbed sediment were increased a little bit by g-C3N4 with different concentrations, which reached the highest value when exposed to 75 mg/kg g-C3N4. g-C3N4 lightly increased the percentage of relative abundance of Cyanobacteria. The bacterial communities’ structure of the samples treated with TC, g-C3N4 or TC/g-C3N4 were distinguishable. g-C3N4 alone had little effect on microbial structure, while TC/g-C3N4 had medium influence and TC had great impact on it. Under TC stress, g-C3N4 slowed down the growth of Cyanobacteria to some extent and restored the changes of bacterial community structure caused by TC, and reduced the residual TC in water body, thus eliminating the side effects of TC. The result shown that g-C3N4 could significantly reduce the residue of TC in riverbed sediment, without affecting the microbial ecology in the environment.


Introduction
Antibiotics are extensively used for human therapy, animal farming and for agricultural purposes (Martinez, 2009). Currently, antibiotics have been found at diverse residual concentrations in riverbed sediment, aquaculture, groundwater and so on (Archundia et al. 2017;Batt et al. 2006;Liu et al. 2018). In China, large quantities of various antibiotics are widely produced and used. Tetracyline (TC) is one of the typical antibiotics in livestock practice due to its high pharmacological activity and solubility in water (Chopra et al. 2001), and it is widely used for human therapy, animal husbandry, and aquaculture, which is ranked second worldwide and rst in China for production and usage (Chopra et al. 2001). Signi cant concerns have been raised over the presence TCs in aquatic environments (Ji et al. 2009). TC had signi cant negative effects on soil microbial community function and obviously effects Shannon's diversity, evenness of bacteria (Kong et al. 2006). It can in uence the microorganisms in the environment, inducing the emergence of dominant microbial populations and changing microbial community structure. It had been documented that under exposure of TC, the activity of nitrifying bacteria was prevented and an important Nitri er, Nitrospira, was inhibited (Du et al. 2018; Shu et al. 2015), and Bacteroidetes and Acidobacteria were increased, while Actinobacteria and Firmicutes decreased under antibiotics exposure (Uddin et al. 2019). TC also residues would signi cantly inhibit sediment N and C cycling rates and reduce the abundance of functional microbial groups (He et al. 2021).
Since TC was poorly biodegradable and toxic for microorganisms (Yahiat et al. 2011), various techniques have been developed to removal it from water, such as physical adsorption (Hu et  considered as potential method since it used light as energy and could mineralize TC. g-C 3 N 4 was a commonly used photocatalyst as it had some advantages, such as energy conservation, su ciently e cient, stable, inexpensive and capable of harvesting light (Wang et al. 2009). g-C 3 N 4 was widely applied in wastewater treatment, due to its low cytotoxicity and photoactivity with visible light (Luo et al. 2017), inspiring researchers to use it for the remediation of TC pollution (Yan et al. 2020). Previous studies have shown that g-C 3 N 4 displayed enhanced photocatalytic activity for TC degradation (Hong et al. 2016; Xue et al. 2015). Direct contact of g-C 3 N 4 and bacterial cells was indispensable for the cell inactivation (Deng et al. 2017), and h + was demonstrated as the dominant reactive species which could make the bacteria cells inactivated . g-C 3 N 4 not only exhibited striking bactericidal but also showed high e ciency in breaking down and preventing formation of new bio lms in vitro (Wang et al. 2017).
Sediment microorganisms played an important role in the benthic food web and the biogeochemical cycle of water ecosystems as they changed with environmental conditions and re ected the pollutant status of water sediment via variations in their abundance, diversity, and structure (Yang et al. 2013).
However, although there were many studies about the impact of TC on microorganism, there was still little information regarding g-C 3 N 4 application in sediment, especially for TC pollution control and the potential ecotoxicity.
In this study, in order to investigate the potential ecotoxicity of g-C 3 N 4 in the application of pollution remediation, TC was used as a typical environmental pollutant to explore the effect of g-C 3 N 4 on bacterial community structure in riverbed sediment while it photodegraded TC. The outcomes from this study may provide important information on the feasibility of applying g-C 3 N 4 photocatalysis for control of organism pollution.

Soil treatment
The riverbed sediment sampling for this study was conducted on July 2, 2020, and it was collected from the Xiang River (112°94′71″E, 28°14′23″N) in Changsha, Hunan, China. The soil was collected from a depth of 10-20 cm by ve-point sampling method. After removing plant residues, the sediment sample kept in crushed dried ice boxes for transportation to the laboratory. The collected samples were homogenized and considered as fresh riverbed sediment (labelled as H), and divided into two parts for 16S rRNA sequencing and experimental design, respectively. The background physicochemical properties of the homogenized sediment samples, including pH, Total nitrogen (TN), Total phosphorus (TP), Total potassium (TK), Soil organic matter (SOM), Ammonium nitrogen (NH 4 + -N), Nitrate nitrogen (NO 3 − -N) and TC concentration, were detected as illustrated in Table S1. The concentration of TC was detected by high performance liquid chromatography (HPLC), and these results showed that the residues of TC in the riverbed of the middle Xiang River was low (Table S1) (Hou et al., 2020). After being loaded into a porcelain boat with a lid, and placed in a tube furnace, 5 g melamine was heated at 550 ℃ for 4 h under N 2 condition (rate: 5 ℃/min), and then cooled naturally to room temperature. The obtained g-C 3 N 4 was ground thoroughly into powder and stored at room temperature for further use.

Sediment exposure experiment
10000 mg/L TC solution was prepared by dissolving 1g of TC in 100 mL sterile water. 100 mg of H were paced into 500 mL asks with 100 mL water. 10000 mg/L TC solution and a certain amount of g-C 3 N 4 powder were added into the asks to achieve the nal exposure concentration of TC (60, 120, 180 mg/L), and the target exposure doses of g-C 3 N 4 (25, 75, 125 mg/kg), and then stirred for 3 min for even mixture.
The mixture containing 60, 120, 180 mg/L TC was marked as T L , T M , T H , and containing 25, 75, 125 mg/kg g-C 3 N 4 was labelled as P L , P M , P H , respectively. The sediment without TC or g-C 3 N 4 was utilized as the control. Each treatment was prepared for three replicated ( each set of treatment was respectively collected together and homogenized, 1-2 g of the homogenized sediment samples were stored at -80 ℃ for the analysis of bacterial community structure and diversity.

HPLC analysis for TC
The extraction method of TC in riverbed sediments was described in detail in the supplementary material.
After ltering through 0.22 µm microporous membrane and centrifuging at 13000 r·min − 1 for 10 min, 20 µL sediment precipitate was applied to detected TC concentration at 360 nm by high-performance liquid chromatography (HPLC).

Diversity of bacteria communities in riverbed sediment
In all studied 17 soil samples, 779,643 high-quality sequences were totally obtained from the V3-V4 region of 16S rRNA gene sequences clustered in 7,214 bacterial OTUs. The rarefaction curve of bacteria in the samples showed that when the number of reads reached about 3000, the Shannon indexes of all samples tended to be at (Fig. 1a), and the curves tended to be at, indicating that our sequencing depth was su cient and can truly re ect the bacterial community in the sediment sample.
Understanding biological diversity is very important as it associate the function and stability of ecosystems (Yu et al. 2020). The bacterial diversity of the riverbed sediment associated with untreated and treated sediments (TC, g-C 3 N 4 and TC/g-C 3 N 4 ) analyzed to identify whether different treatments shaped the environmental microbiome (Fig. 1b). The treatment way had a little in uence on the bacterial richness. The richness (Chao 1) of bacterial communities of H was 3901.70 lower than that of CK (4227.06), and lower than those of samples exposed to TC (4326.21-5068.42), g-C 3 N 4 (4563.89-4939.65) and TC/g-C 3 N 4 (4641.95-4962.17), respectively. However, the exposure of TC, g-C 3 N 4 and TC/g-C 3 N 4 almost not changed the diversity (Shannon index) of bacterial communities. Meanwhile, the Shannon in the samples containing TC/g-C 3 N 4 remained comparatively stable in the range 6.81-6.92.
Based on Bray-Curtis distance, PCoA was applied to examine the beta diversity of the samples to study the differences of bacterial community structure among them (Fig. 1c). The rst two axes (PCoA1 and PCoA2) explained 19.07% and 16.37% of the total variance in the sediment bacterial communities, respectively. A clear separation was observed among three sample groups exposed to TC, g-C 3 N 4 and TC/g-C 3 N 4 . The difference between H and the samples treated by TC/g-C 3 N 4 was littler than that between H and the sediments treated by TC alone, but larger than that between H and the samples handled with g-C 3 N 4 alone. It might conclude that major changes in bacterial diversities across the samples were attributed to the difference treatments, g-C 3 N 4 alone had little effect on microbial structure, while TC/g-C 3 N 4 had medium in uence and TC had great impact on it.
Five-Way Venn diagrams were constructed to understand the unique and shared OTUs among different treatments in each riverbed sediment (Fig. 1d). There was a high speci c OTUs number in each sample group and the shared OTUs (1,898) accounted for 26.31% of the total OTUs (7,214). The number of speci c OTUs in TC/g-C 3 N 4 treatment group (713) was highest, followed g-C 3 N 4 treatment group (228) and TC treatment group (181), suggesting that the speci c richness of bacterial communities in samples exposed to TC/g-C 3 N 4 was the higher than those exposed to TC and g-C 3 N 4 . These indicated that TC/g- Once external substances were added into sediment, there would be some corresponding changes in the environment, in order to cope with the varieties, the genetic characteristics, physiological and biochemical processes of some soil microorganisms will be changed. Thereby, some microbe was inhibited in sediment, while others in the environment may be stimulate to growth, which enhanced the generation and evolution of the microbes that had adapted to the conditions, corresponding promoted the number of them, and increasing the microbial diversity. At the same time, nutrient composition of sediment was changed by adding external material to the sediment, the structure of the bacterial community would be changed accordingly, thus varying the beta diversity. However, the changes in diversity induced by g-C 3 N 4 was basically stable and similar to those induced by other photocatalysts, including biochar (Wu et al.

Composition of bacterial communities determined by Illumina MiSeq
According to the annotation and abundance of OTU, a relative abundance table for phyla was obtained (Table S2) The effect of TC on bacterial community structure in sediment was illustrated in Fig. 2a and Table S3. Compared to CK, the abundance of some microorganism were differ from the samples treated by TC, especially the dominant bacteria, such as Actinobacteriota, Acidobacteriota and Firmicutes, that is TC had a greater impact (p < 0.05) on them. However, between different concentrations of TC, the changes of relative bacteria abundance at phyla level was insigni cant (p > 0.05) between different concentrations of TC. The average abundance of Actinobacteriota, Acidobacteriota and Firmicutes was signi cantly decreased in TC treatment (p < 0.001), and its abundance was 17.33%, 14.14% and 6.04%, respectively, however, in CK, the relative abundance was 25.05%, 16.07% and 8.68%, respectively, so these three phyla were regarded to be susceptible to TC application. The results were consistent with other reports, Alexandrino et al. (2017) also found that Actinobacteriota and Firmicutes were sensitive to TC.
The in uence of g-C 3 N 4 on bacterial community structure in sediment was demonstrated in Fig. 2b and Table S3. Compared with the CK, the presence of g-C 3 N 4 signi cantly inhibited (p < 0.001) the growth of Firmicutes and Actinobacteriota in riverbed sediment. But the growth of Acidobacteriota (20.89%) was signi cantly improved (p < 0.001) by g-C 3 N 4 , and the average abundance was increased about 4.82% in comparison of CK. In the P L treatment group, the biomass of Cyanobacteria (0.88%) was signi cantly lower (p < 0.001) than that of CK (3.50%), this was indicated that g-C 3 N 4 might inhibited the growth of Cyanobacteria. Based on the above results, it could be concluded that Firmicutes, Actinobacteriota, Acidobacteriota and Cyanobacteria were susceptible to g-C 3 N 4 exposure. Cyanobacteria was photoautotrophic organisms in aquatic ecosystems and ubiquitous in riverbed sediment, as well as a typical microorganism in the identi cation and detection of environmental pollution (Teta et al., 2019). Acidobacteria had the potential to degrade polymeric carbonaceous complexes and actively participate in the cycling of organic matter, and it played an important role in maintaining the structural stability of microorganism in the environment (Kalam et al. 2020). Cyanobacteria proliferate in large quantities, which could affect the microbial structure in the environment (Chen et al. 2020). Our results showed that g-C 3 N 4 could promoted Acidobacteriota, inhibited Cyanobacteria, and was bene cial to environmental pollution remediation. However, The growth of Cyanobacteria was effected by g-C 3 N 4 concentration, its abundance of P H treatment (6.34%) was higher than that of P L treatment (0.88%), it might due to the automatically agglomerate of g-C 3 N 4 at high concentration, inactivating its function.
The impact of TC/g-C 3 N 4 on bacterial community structure in sediment was illustrated in Fig. 2c and Table S3. Compared with Actinobacteriota (25.05%) and Firmicutes (8.68%) in the CK treatment group, the average abundance of Actinobacteriota (16.48%) and Firmicutes (4.97%) was signi cantly decreased (p < 0.001) in TC/g-C 3 N 4 treatment. But, compared with the CK treatment groups (16.07%), the average abundance of Acidobacteriota increased signi cantly (p < 0.05) in TC/g-C 3 N 4 treatment groups (20.71%).
When the same does of g-C 3 N 4 was added, the biomass of Cyanobacteria treated with low concentration of TC (T L P L = 4.96%, T L P M = 4.14%, T L P H = 3.81%) was higher than that treated with high concentration of TC (T H P L = 3.71%, T H P M = 2.76%, T H P H = 2.16%). Meanwhile, the biomass of Chloro exi (11.79% − 13.73%) and Gemmatimonadota (3.23% − 5.32%), which were the dominant microorganism in the sample, could be maintained in a stable range with the addition of both TC and g-C 3 N 4 , and the biomass of them were similar to that of H group, respectively. Acidobacteria was very important for the environment to keep the structural stability of microorganism as it potentially involved in the degradation of polymeric carbonaceous complexes and the cycling of organic matte (Kalam et al. 2020). Therefore, it was regarded as a kind of potential microorganism for environmental bioremediation and biotechnological applications (Kielak et al. 2017). Our study indicated that g-C 3 N 4 could signi cantly slow down the growth of Cyanobacteria and promote the growth of Acidobacteriota in the environment under TC pressure, thus it might bene cial for sediment to remediate TC pollution and haven't adverse effect on environment.

Taxonomy-based comparisons of microbiota groups
To identify the biomarkers in sediment samples, the microbial communities in different treatments was compared based on taxonomy, and the results were showed in (Fig. 3), in which LEfSe was applied to determine each group that was revealed in cladograms and histogram of LDA scores of 2.5 or more. The larger the score of LDA was, the more remarkable the difference caused by species abundance was. As depicted in the gure, there were obvious changes in dominant bacteria in sediment treated with TC, g-C 3 N 4 and TC/g-C 3 N 4 at the level of phylum, class, order, family and genus level. In the sediment treated with TC, more than 20 microbes including Actinobacteriota, Desulfuromonadia, Myxococcia were signi cantly enriched (p < 0.05). Similarly, 8 groups of bacteria including Brocadiae, Entotheonellia, Dependentiae were obviously enriched (p < 0.05) in riverbed sediment treated with g-C 3 N 4 . However, in the sediment treated with TC/g-C 3 N 4 , only 4 microbes were signi cantly enriched (p < 0.05), such as Babeliae, Subgroup_25 and c_unclassi ed_p__Actinobacteriota. These indicated that the selected biomakers could clearly distinguish (p < 0.05) these three treatments and the difference caused by TC was the most, orderly followed by g-C 3 N 4 and TC/g-C 3 N 4 . TC mainly affected the bacteria with high abundance, while g-C 3 N 4 , TC/g-C 3 N 4 mainly in uenced the low abundance ones. The results suggested that g-C 3 N 4 might mitigate the side effect caused by TC on sediment bacteria by reducing the difference of microbes in the environment.

TC residue and Changes in species at genus level caused by TC and g-C 3 N 4
In order to study the effect of TC residue on bacteria community structure, we analyzed the TC concentration in sediment with different treatment, as well as the relative dominant bacterial genus (Fig. 4). As illustrated by Fig. 4a,   , which was much higher than that in our experiment. This phenomenon might attribute to two facts: Firstly, the light intensity under our experimental condition was weak, so only a small number of photon were available for the degradation. Secondly, the composition of the sediment system was complex, which effected the light transmittance and further effected the photocatalytic activity of g-C 3 N 4 . Figure 4b and Under TC exposure, g-C 3 N 4 was bene cial to enrichment of norank_f__Vicinamibacteraceae and norank_f__Gemmatimonadaceae, belonging to the bacteria phyla of Acidobacteriota and Gemmatimonadota, respectively Previous research had showed that RB41 was actively participated in the carbon cycle in sediment (Ito et al. 2019), RB41 was the dominant and sensitive microorganism in contaminated soil and played a positive role in environmental ecosystem (Ai et al. 2018;Shen et al. 2018). Based on the fact that its abundance increased in the treatment of TC and g-C 3 N 4 , it should be sensitive to them, and more to g-C 3 N 4 . Bacillus could fully degrade organic matter and eutrophic substances in sewage ), at the same time, Bacillus was also a kind of bene cial bacteria that was bene cial to soil microecological stability. It could inhibit or kill pathogenic bacteria in the environment (Nicholson et al. 2002). It had been reported that antibiotics induced oxidative stress and inhibited the growth of Bacillus cells (Sannasimuthu et al. 2020). Based on the fact that compared to CK,the degree of decline in the content of Bacillus in T H was greater than that of samples contained g-C 3 N 4 , it was suggested that TC had obvious toxic effect on Bacillus, while g-C 3 N 4 could reduce the toxic effect of TC on Bacillus. Hence, one inference was that the g-C 3 N 4 might reduce the effect of TC on Bacillus in sediment.
In our experiment, we founded that the addition of g-C 3 N 4 would reduce TC residue in sediment, and this trend was strengthened with the increase of the concentration of g-C 3 N 4 (Fig. 4a). These results indicated that 1) g-C 3 N 4 could degrade some organic substances, including TC, and changed the nutrient composition of sediment, thus affecting the structure of microorganisms; 2) g-C 3 N 4 could act on some microorganisms, change the growth of sensitive bacteria, thus affecting their ecological niche in the sediment. Based on the effect of g-C 3 N 4 on dominant bacteria RB41 and Bacillus, it was indicated that g-C 3 N 4 could improve the ability of sediment remediation and reduce the toxic effect of TC on bene cial bacteria, which was bene cial to the ecological health of sediment.

Redundancy analysis (RDA) of bacterial community at the phylum level and class level
The relationships between TC, g-C 3 N 4 and bacterial community was analyzed based on RDA score plot and depicted in Fig. 5. The impacts of each factor (TC or g-C 3 N 4 ) on bacterial community were represented by the length of arrows, and the cosine angle between arrows illustrated their relationship (smaller angle indicated more signi cant correlation). As shown in Fig. 5a, at the phylum level, axis 1 and 2 of the RDA plots explained up to 19.37% and 3.45%, respectively. Speci cally, TC positively affected (p Using Spearman correlation analysis, the relationship between the top 30 bacterial genus and TC or g-C 3 N 4 were examined to further investigated the in uence of TC or g-C 3 N 4 on bacteria community (Fig. 2s). According to the heatmap, TC and g-C 3 N 4 signi cantly correlated with the major phyla and genus. Acidobacteriota was the main phyla positively affected by g-C 3 N 4 (p < 0.01), Chloro exi was the main phyla affected by TC (p < 0.05). RB41 (p < 0.05) and Ellin6067 (p < 0.001) were the main genus signi cantly associated with g-C 3 N 4 . These results were consistent with the results obtained with the RDA.

Conclusion
To our knowledge, this was the rst-time research on the potential ecological toxicity of typical ptotocatalyst g-C 3 N 4 by analyzing the change of sediment microbial community under TC exposures. The bacterial community and TC residue in the sediment under different conditions were examined. According to the results, compared with CK, TC, g-C 3 N 4 and TC/g-C 3 N 4 treatments could increase microbial richness in the riverbed sediment, but kept microbial diversity basically stable. Beta diversity analysis showed that TC had greater in uence on bacterial community structure, while g-C 3 N 4 and TC/g-C 3 N 4 had less in uence on it. These indicated that g-C 3 N 4 had less effect on species diversity and might be safer for environment. TC could inhibit the growth of Actinobacteriota, Acidobacteriota and Firmicutes in the riverbed sediment. g-C 3 N 4 could signi cantly slow down the growth of Cyanobacteria and promote the growth of Acidobacteriota in the environment under TC exposure, thus it might bene cial for sediment to remediate TC pollution and reduce its adverse effect on environment. The LEfSe analysis showed that there were 20 classes, 8 classes and 4 classes of biomarkers in the sediments treated by TC, g-C 3 N 4 and TC/g-C 3 N 4 , respectively, g-C 3 N 4 might mitigate the side effect caused by TC on sediment bacteria by reducing the difference of microbes in the environment. Meanwhile, in sediment g-C 3 N 4 could decrease TC residues and promoted the mass reproduction of RB41 as well as inhibited the toxic effect of TC on Bacillus. So, g-C 3 N 4 would not adversely affect the ecological function of the revered sediment, and was an environmentally friendly photocatalyst. In conclusion, g-C 3 N 4 might be expected to be used for TC   Bacterial communities structure analysis by principal coordinates at the phyla level, only top (relative bacterial abundance > 1.5%) bacterial taxa are shown. (a) Microbial community structure of sediment exposed to g-C3N4 at 30 days, (b) Microbial community structure of sediment exposed to TC at 30 days, (c) Microbial community structure of sediment treated with different amounts of TC/g-C3N4 at 30 days.

Supplementary Files
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