Insight Into Impacts of Sewage Discharge and River Self-Purication on Microbial Dynamics and Pathogenicity in River Ecosystem

Sewage directly discharge causes serious environmental problems. Here, the effects of treated and untreated sewage on the river ecosystem were investigated. The variations of microbial community structure, including infectious pathogenic bacteria and functional bacteria related to nitrogen, phosphorus, and COD metabolism were studied in detail. Bacterial diversity and richness were signicantly decreased, while, Proteobacteria containing various infectious pathogens, such as Vibrio and Helicobacter, signicantly increased after the discharge of raw sewage. Although the microbial structure was slightly restored and the abundance of most pathogenic bacteria was also slightly reduced through river self-purication, direct discharge of raw sewage caused severe and short-term irreversible damage to the river environment. Direct discharge also introduced various pollutants such as nitrogen, phosphorus, and COD, increasing the corresponding functional bacteria and their related genes. Furthermore, the high abundance of pathogenic bacteria of the drain outlet was mainly from raw sewage rather than bacteria reproduction caused by water deterioration according to the RDA analysis. With these results, direct discharge disturbed the ecological balance of the river. Therefore, more attention is needed to provide a hygienic situation for people and all sewage should be treated properly. In conclusion, all sewage should be treated properly before discharge into ecosystems to mitigate its negative impacts on receiving water bodies.


Introduction
With the massive worldwide population increase, water has been predicted to become one of the scarcest resources in the 21st century. Moreover, UN's World Water Development Report, 2017, said globally 80% of sewage (> 95% in developing countries) is directly released to the environment. Numerous questions have been raised about the ability of wastewater treatment programmes to remove pathogens from wastewater in which many waterborne diseases are associated supposedly treated water supplies. The untreated wastewater can threaten human and ecological system by enriching the pathogenic bacteria . Moreover, a body of literature has con rmed the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater (Ahmed et al., 2020; Haramoto et al., n.d.; Zhang et al., 2020). The SARS-CoV-2 possibly spreads through wastewater treatment network (Naddeo and Liu, 2020). When raw sewage contaminates the aquatic environment, these pathogens can be transmitted through water to those who use the water for swimming, boating and shing, causing a range of potential risks (Cornelisen et al., 2011). However, there exist many ways for untreated wastewater to enter rivers.
When sewage treatment can not treat too much wastewater or when it was rains heavily, raw sewage will be discharged directly into river channels, polluting the environment and making the water quality worse.
Natural disasters, such as earthquakes and oods, also make raw sewage ow into rivers, causing pathogens in rivers (Devane et al., 2014). Once sewage delivers to receiving waters, a series of physical and environmental factors changes will occur. Over time, river dilution, storage in sediments, and the intrinsic characteristics of the microorganisms and other related process may alter the destiny of the bacteria the pathogens of concern.
Page 3/18 The direct discharge of sanitary wastewater may cause environmental diversity changes. Sewage discharge without proper treatment signi cantly altered the concentrations of different organic and inorganic contaminants in the receiving water bodies (Duttagupta et al., 2020), especially NH 4 + , NO 3 − , total phosphorus (TP) and chemical oxygen demand (COD). The increase of these contaminants could trigger the aquatic organism by causing eutrophication of the catchment (Huelsen et al., 2016). David et al. (2014) found that the high NH 4 + concentration negatively in uenced the functional performance and taxonomic richness of the microbial community of 10 different WWTPs located across Switzerland (David et al., 2014). Moreover, phosphorus content was positively correlated with the microbial biomass (Lei, 2012) and occupied an important position among environmental factors that affect the microbial community of sediments (Jian, 2015;Yu et al., 2017).  (Velayati et al., 2015). Many diseases such as gastritis, peptic ulcer, and lymphoid proliferative gastric lymphoma were caused by Helicobacter pylori infection (Pereira and Medeiros, 2014). West et al. (1992) reported the capabilities of Helicobacter pylori to survive in various buffers at room temperature over a range of physical variables, which means that Helicobacter pylori may survive in a natural aquatic environment (West et al., 1992). Therefore, pathogenic bacteria in water environment will have direct or indirect effects on human beings or animals. The study research and analysis their microbial community composition and in uencing factors is urgent for future treatment measure.
The lack of a thorough understanding of the survival and persistence of different microbial types in different conditions and environments is one of the major gaps in the knowledge of pathogenic microorganisms in wastewater. Previous studies focused on the abundance of pathogenic bacteria and the relationship between the contaminants and pathogenic bacteria in rivers contaminated by raw sewage discharge (McCarthy, 1996;Miller et al., 2006). However, it is unclear whether the source of pathogenic bacteria in the river comes from sewage itself or is caused by the environmental change after sewage discharge. Also, a deep insight into microbial and genetic responses to river self-puri cation needs further investigation. Thus, the main target of the present study is to ll in this knowledge gap by investigating the environmental parameters and the microbial community structures of different sites along the Daxin River, representing treated and raw sewage discharge points. The speci c objectives of this study are to (1) provide more information about the water quality and microbial community structure before and after domestic wastewater pollution, (2) Figure 1 shows the distribution of sampling sites along the Daxin River regarding the discharge points of treated and raw sewage. The raw sewage was discharged between Site-1 and Site-2; therefore, Site-2 was directly polluted by raw sewage. The source of the sewage discharged from the drain outlet was domestic wastewater from the nearby neighborhood. Site

High-throughput 16S rDNA sequencing
The next-generation sequencing was applied to determine the microbial community structure based on the universal primer pair of 16S rDNA (Liu et al., 2018). The V4 amplicons were sequenced using primers 515F (GTGCCAGCMGCCGCGGTA) and 806R (GGACTACNVGGGTWTCTAAT) and the pair-ended method by Illumina Miseq sequencing platform (Lv et al., 2016). PICRUSt was used to predict the potential of a sample using 16S rRNA amplicon sequencing. KEGG Orthology (KO) was used to classify all genes that are homologous to a speci c gene whose function is known to the same category (Minoru et al., 2017).

Data analysis
The community richness was estimated by the Chao1 and community diversity was estimated by Simpson and Shannon indices using the MOTHUR software (

Water quality and environmental parameters of the sampling sites
The water quality parameters of the four sites were summarized in Table 1

Analysis of the community composition
Fifty-nine phyla were detected from the four sites, and ten predominant phyla were shown in Fig. 2  As described in Fig. 2  The Zoogloea and Pseudomonas genera whose main function is to degrade organic matters increased sharply at Site-2. The Pseudomonas genus, responsible for the removal of phosphorus, was also dominant in Site-2 coupled with Acinetobacter. Their enrichment may be due to a large amount of phosphorus in the raw sewage, as evidenced by the highest TP content in Table 1 at Site-2. Thiobacillus and Desulfovibrio are related to sulfur removal and Thiobacillus was enriched at Site-3 and Site-4.
The total obtained operational taxonomy units (OTUs ) were 28427,28354 32810, and 39105 for the four sites. Furthermore, four important functional microbes were extracted and classi ed as denitrifying bacteria (DNB), anammox bacteria, organic degrading bacteria (ODB), and phosphorus-accumulating bacteria (PAOs) as shown in Table 2. Three genera of DNB were detected in almost all samples. In particular, the relative abundance of Hydrogenophaga at Site-2 (3.333%) was more than 13-fold of that at Site-1 (0.253%); however, it suddenly decreased at Site-3 and Site-4. Azoarcus was absent at Site-1 and appeared at Site-2 (0.005%) then continued to grow in the latter two sites. Thauera also showed an increasing trend from Site-1 to Site-4. Table 2 shows that the discharge of raw sewage between Site-1 and Site-2 increased COD and nitrate contents, which provided sustainable and effective electron donors for denitri cation, promoting the growth of denitrifying bacteria. Anammox bacteria were also detected at the genus level, and its highest abundance was 0.59% at Site-3. Zoogloea has been reported with high versatile metabolism capabilities such as nitrogen xation, secretion of extracellular polymeric substance (EPS), and COD removal (Xia et al., 2018). The increase of ammonia nitrogen content at Site-2 may be related to the high Zoogloea abundance at this site. The contents of Zoogloea increased at Site-2 after the raw wastewater surged in and then decreased in the following two downstream sites. PAOs bacteria include Pseudomonas, Arthobacter, Nocardia, Beyerinkia, Ozotobacter, Aeromonas, Microlunatus, and Rhodocyclus . Only one PAOs genus was detected in all sites. The abundance of PAOs was much higher at Site-2 than that at Site-1, with a relative abundance of 2.69% and 0.08%, respectively. The relative abundance of all the above-mentioned functional bacteria at Site-2 is higher than those at Site-1, while only four bacteria related to COD and nitrate removal showed a continuous increase between Site-3 and Site-4. In other words, the discharge of both raw and treated wastewater caused changes in river water quality, which further altered the microbial community structure. The changes in water quality and microbial community at Site-2 might be mainly due to the high nutrients in the discharged wastewater. Site-4 subjected to the discharge of treated wastewater with high temperature, which further affected the microbial species and abundance. The Chao1, Simpson, and Shannon indexes were calculated to determine the richness and diversity of the microbial community . The Chao1 index of sediment samples at Site-1 (874) was higher than that of Site-2 (869), as shown in Table 4; the same results were registered for Simpson and Shannon indexes with insigni cant uctuations. This may be because the direct discharge of sewage altered the environmental conditions of the river, caused changes in the microbial community, and eventually led to a decrease in species richness and diversity. The results indicated that the richness and diversity of the bacterial community were higher in Site-1 compared to Site-2. The values of Chao1 kept increasing from Site-2 to Site-4 indicating the increase of the richness of the microbial community. Discharging the treated sewage provided a relatively suitable environment for microorganisms in the river. Therefore, the species richness increased again during the process from Site-3 to Site-4.

Pathogenic gene abundance based on KEGG database and qPCR results
Pathogenic bacteria in water affect human and animal health. Pathogens could live in untreated wastewater for longer periods and spread further through water bodies (Casanova et al., 2009). Figure 3.
shows the gene abundance of infectious diseases at four sites according to the forecast of the KEGG database ( Fig. 3 (A)) and the relationship among four sites based on qPCR results (Fig. 3 (B)). There were signi cant differences in infectious diseases between Site-1 and Site-2, while no obvious variation was observed between Site-3 and Site-4. After polluted by raw sewage, the gene abundance of the infectious diseases increased sharply. The increased gene was related to Vibrio cholera pathogenic cycle process, whose gene amount increased from 4055 to 23556 reads. The gene abundance of Helicobacter pylori infection increased from 3797 to 13163 reads. The gene abundance of Tuberculosis also increased from 13545 to 19887 reads. The four kinds of pathogenic genes had the most remarkable variation among all the genes related to infectious diseases between Site-1 and Site-2. On the other hand, there is no remarkable big gap between the gene abundance of samples at Site-3 and Site-4. The values of these four infectious disease abundance were veri ed by qPCR, as shown in Fig. 3(B). Compared to Site-1, the abundance of these four infectious diseases in Site-2, Site-3, and Site-4 increased. The abundance of Staphylococcus aureus at Site-2 far exceeded that of Site-1. The abundance of these four infectious diseases decreased at Site-4, compared to Site-3.

The relationship of the infectious pathogens and functional bacteria to water quality parameters
The relationship between microbes and environmental variables was evaluated by redundancy analysis (RDA) (Fig. 4). Based on Fig. 4, there are signi cant differences between Site-1 and the other three sites.
The seven water quality parameters have diverse behaviors in affecting the distribution of the infectious pathogens in the four sites. Site-2 has higher COD, TP, and NH 4 + concentrations than other locations.
Meanwhile, TP and NH 4 + got a strong positive correlation with each other among all the environmental factors and they contributed to a large proportion in affecting the abundance of Mycobacterium tuberculosis. COD had less correlation to other parameters but strongly in uenced the abundance of Mycobacterium tuberculosis. Site-4, having the highest temperature and NO 3 − concentration, had a low similarity with the other sites. Few functional bacteria were located in Site-1, while the discharge of raw sewage led to the most abundant and multiple distributions of bacteria at Site-2 ( Fig. 4 (B)). Many bacterial species at Site-2 were affected by COD, NH 4 + , and TP. Moreover, NO 3 − and temperature contributed to the microbial community diversity at Site-3 and Site-4. The functional microbes at Site-3 were in uenced by pH and NO 2 − .
4. Discussion 4.1 The directly discharged sewage altered the microbial community structure According to our analysis, the abundance of pathogens, which belong to Proteobacteria, witnessed a noticeable growth after raw wastewater owed into the river, as shown in Fig. 2. The taxonomic analysis found that Proteobacteria, Bacteroidetes, and Firmicutes were the most abundant phyla in all samples. The abundance of Proteobacteria decreased at Site-3 by the dilution or degradation of pollutants. The Proteobacteria also contains some bacteria whose function is to x NH 4 + , and they had a positive correlation with NH 4 + concentration (Naddeo and Liu, 2020). As shown in Table 3, all indexes were higher at Site-3 and Site-4, because the owing water carried a large amount of sediment and bacteria to the river downstream. The most signi cant change between Site-1 and Site-2 among the functional bacteria was Pseudomonas, which can remove low concentrations of TP from wastewater (Li et al., 2012). RDA results suggested that functional bacteria, such as Zooloea, Pseudomonas, and Hydrogenophaga, were closely related to COD, NH 4 + , and TP. The DNB, which can restore NO 2 − , was enriched sharply at Site-2.
The possible reason may be that NO 2 − , and NH 4 + provide nutrition for DNB (Akizuki et al., 2015). 4.2 Raw sewage increased the pathogens content and the risk potential of human health This paper revealed that raw sewage remarkably contributed to the enrichment of pathogens in sediment samples of the river. From the results of KEGG, the gene contents of four infectious diseases indeed increased after raw wastewater was discharged into the river, while most gene contents decreased after the discharge of treated wastewater. Further analysis proved that the KEGG prediction was in accord with the qPCR results, similar to a study conducted by Langille et al. (2013) (Langille et al., 2013). In general, the discharge of raw sewage into rivers changed the water quality of rivers and caused the growth of pathogenic bacteria in these rivers. The number of pathogenic genes of Site-2 showed a signi cant increase, which was higher than the other three sites. With the puri cation of the river, the water quality gradually changed and the abundance of pathogenic genes decreased in Site-3. When the treated wastewater was discharged into the river, however, the pathogenic genes of site-4 did not change dramatically. In the case of sediments being disturbed, it is likely that microorganisms, including pathogens, would remigrate into the water column. Medema et al. reported that protozoa can settle from the water column into the sediment and, due to their size, remain undisturbed for long periods (Medema et al., 1998). Thus, re-suspension events increase the potential risk potential to human health for those who are involved in activities in the aquatic environment. A suitable temperature is also an essential factor for the growth of pathogenic bacteria. When the pathogen is sown in pasteurized sewage, it can be infectious for up to 7 days, in deionized water at 25°C for 22 days, and in these media for up to 4 weeks or more at lower temperatures (< 4°C) (Casanova et al., 2009 4.3 Pathogenic bacteria content increased mainly due to raw sewage discharge rather than water quality change The discharge of pollutants from industrial processes causes diverse adverse environmental impacts on aquatic ecosystems, such as eutrophication of water bodies, the introduction of pathogens, reassembling of the microbial community structure in rivers, and so on (Liao et al., 2019;Marassi et al., 2020). With the change of water quality, mostly COD, in the river from Site-2 to Site-3, it was more suitable for Mycobacterium tuberculosis to be enriched than other bacteria. However, except for the Mycobacterium tuberculosis that related to COD, the correlation between other pathogenic bacteria and environmental factors was not noticeable. Through RDA analysis, functional bacteria are closely related to water quality changes, but most pathogenic bacteria most are inconsistent. This indicates that the sudden increase of pathogenic bacteria at Site-2 was probably since these pathogenic bacteria were contained in the sewage itself. In other words, the pathogenic bacteria in the Daxin river probably came from untreated wastewater directly rather than environmental changes.

Decreased pathogenic bacteria abundance may due to self-puri cation
After the raw sewage enters the river, the self-puri cation in uenced the river's environmental conditions and then affected the further migration and deposition of pathogenic bacteria. The content of COD, NH 4 + , NO 3 − , and TP gradually decreased from Site-2 to Site-3, as well as the abundance of genes related to Vibrio cholerae, Staphylococcus aureus, and Helicobacter pylori, as shown in Fig. 3. The response of the ecosystem to chemically-driven environmental changes is modulated by physical and biological phenomena. Most pathogens, which came from raw sewage, could also be absorbed by animals, plants, and sediments in the river. Self-puri cation could the maintenance or restoration of river ecology and in uencing factors may include seasonal uctuations, the microbiological community present (Liao et al., 2019), and temperature (Wagner and Zalewski, 2016). Moreover, Silva et al. revealed that the selfpuri cation potential increased in the dry season, in uenced by the wastewater load .
So the temperature in the Daxin river also contributed to self-puri cation. These biological processes are mainly carried out by endogenous and exogenous microorganisms that can remove organic and inorganic pollutants in contaminated water bodies, essential for maintaining a stable ecosystem dynamics state, in terms of function and structure (Clements and Rohr, 2009;Wagner and Zalewski, 2016). Another concern is that microbes could be shielded by physical embedding in organic matter, suspended particles, and occlusion of a bio lm, making them less susceptible to the inactivation action of disinfectants . So it is urgent to further focus on the migration and transformation mechanism of pathogenic bacteria in sediments.

Signi cance of this research
This study contributes to an improved understanding of the effects of sewage discharge on the changes of microbial communities and the abundance of pathogenic bacteria in river sediments. Despite water and sewage treatment technologies continue to achieve rapid progress in recent years, waterborne pollution remains a major threat to public health worldwide. This paper brings to the fore the need for comprehensive research into the movement and behavior of these microorganisms in river sediment after raw sewage is contaminated. And besides, with the evidence of increased pathogen abundance, we are here to demonstrate and highlight the risks of direct sewage discharge. Wastewater as a secondary source of transmission is not given full attention, especially in third-world countries. If wastewaters act as the source of transmission, it will be hard to break the chain of pathogen transmission in the third world countries and the consequence will be faced by developed nations as well. There is a common practice to discharge wastewater directly into rivers, canals, and lakes without any treatment in third-world countries. The wastewater carried pathogens can go to a big population of the world and create population crises in the world. So, measures should be taken as soon as possible in third-world countries to break the chain of pathogen transmission. Strict surveillance of wastewater treatment is suggested to stop the spread of pathogens in the human community.

Conclusion
This study shows that raw sewage discharge into rivers indeed changed the microbial community structure and the polluted water is a potential source of the spread of infectious diseases. Raw sewage discharged directly into the river decreased the richness and diversity of the microbial community and introduced pathogenic bacteria. Although the self-puri cation of water slightly reduced the pollution, sewage directly discharge still caused irreversible effects. In conclusion, direct discharge of wastewater should be forbidden with regards to the negative impact on entering the ecosystem, especially on the lives that depend on the ecosystem for sustenance.

Declarations
Daxin River sampling points locations Microbial composition in four sites at the phylum (A) and genus (B) levels.

Figure 3
Prediction of abundance of genes related to infectious diseases (A) and the multiple relationship among them by qPCR (B) at four sites