Urbanisation Reshapes Freshwater Microbiomes: A Systematic Review of Ecological Patterns and Functional Shifts

urbanisation, freshwater microbiome, systematic review, antimicrobial resistance, 15 urban waterbodies, lakes, rivers, functional traits 16 17 18 Table of Contents: 19 Graphical Abstract 2 20

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25 Number of Studies 26 Geographic Distribution of Studies 27 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 2 Study Sites 28 Microbial Functional Traits Studies 29 Anthropogenic Activities 30 Microbial Diversity 31 Microbial Diversity across Anthropogenic Activities 32 Effect of urbanisation across studies on microbial diversity 33 Correlation of Microbial Diversity across Anthropogenic Activities 34 Principal Component Analysis 35 Relative Stressor Composition 36

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of the papers used for data extraction: 40 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 3 41 Graphical abstract 42 43 Waterbodies in urban areas function as convergence platforms for anthropogenic and 44 environmental microbiomes. Runoffs, wastewater and effluents contain antimicrobial resistance 45 genes and other pathogens that survive in water due to inadequate treatment. Disposal, use, and 46 overflow of wastewater cause restructuration of microbial communities, proliferation of 47 opportunistic microorganisms, and spread of antimicrobial resistance in aquatic ecosystems. 48 49

50 Rapid urbanisation has profoundly shaped microbial diversity across different ecosystems. 51 Freshwater microbiomes are particularly affected by urbanisation activities, such as 52 eutrophication, pollution, runoff, and sewage. This is of significant concern as marginalised 53 communities often depend on waterbodies for their livelihood. Freshwater bodies play a crucial 54 role in maintaining both human and ecological health at population level. Currently, we lack a 55 3 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 4 systematic understanding of the global impacts of urbanisation on freshwater microbiomes in 56 relation to human health, ecosystem functioning, and sustainability. 57 We identified 90 eligible papers from the last 25 years after screening based on the inclusion 58 exclusion criteria. We extracted data that examined changes in the functional traits such as 59 antimicrobial resistance (AMR), nutrient cycling of the microbiome in urban waterbodies and 60 several other factors. Data were extracted by a thematic analysis followed by a narrative 61 synthesis on specific functional traits. This systematic review presents a comprehensive analysis 62 on the changes and challenges brought about by urbanisation on freshwater bodies. 63 Our results indicate that urbanisation leads to reduced bacterial diversity of urban waterbodies, 64 with a striking increase in reporting of Proteobacteria, Cyanobacteria and Coliform bacteria. 65 These insights will help inform public health strategies and sustainable urban planning. 66

67 Cities are the fastest growing ecosystems globally with 81 per cent of the world’s population 68 already residing in urban areas(United Nations, 2025). Urban ecosystems are also highly 69 heterogeneous with a complex mosaic of natural and built environments consisting of buildings, 70 parks, gardens, remnant forests, roads, pavements and waterbodies(Jones et al., 2022). It is no 71 surprise that we live on a planet that is consistently shaped by human activity and cities, the hub 72 of human activity-are rarely designed keeping flora or fauna in mind. Consequently animals 73 often enter cities as their natural habitats are reformed or displaced(Schell et al., 2020). 74 Additionally, microbes, which are fundamental to public health as human commensal microbiota 75 and invisible biodiversity(Matthews et al., 2024), rarely feature in our thinking of urban fauna. 76 This is particularly interesting despite a long history of evidence showing their influence on 77 urban life. John Snow’s seminal work on cholera has shown how the urban dwellers' interaction 78 with microbes can be very different from those of their rural counterparts(Tulchinsky et 79 al.,2018). 80 In the last 100 years with the rising use of antibiotics, pollution and the urban rural divide the 81 relationship between humans and microbes has become more complex if not diversified(Kabwe 82 et al., 2020). We are now surrounded by more novel antibiotic resistant genes(ARG) than ever 83 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 5 before. Another growing risk is antimicrobial resistance(AMR) as a recent study predicted that in 84 2050 the global bacterial AMR burden will spell particularly high mortality rates for South Asia, 85 Africa and the Caribbean(Naghavi et al., 2024). 86 Microbial ecology depicts how ecosystems respond to human-driven climate change. One Health 87 proposes that the well-being of humans is linked to the health(World Health Organization of the 88 ecosystem and the thread that holds it all together are microbial contributions(WHO), 2025). 89 Many studies discuss microbe host associations such as plants, humans and animals, as an eco-90 holobiont or an extended second genome(Banerjee & Heijden, 2023). Even recently microbial 91 research would heavily focus on pathogens affecting microbial hosts and while understanding 92 zoonotic diseases remain vital, loss of microbial diversity can cause dysbiosis(Berg et al., 2020). 93 Dysbiosis is caused when the composition of microbial diversity drops and leads to the 94 emergence of pathogens which affects the immune system of the host. Thus any dynamic built 95 environment can face the threat of dysbiosis, especially urban landscapes which are constantly 96 shaped by anthropogenic activities. In fact metagenomic maps of urban microbiomes and 97 antimicrobial resistance markers revealed that most cities have a unique microbial 98 fingerprint(Danko et al., 2021). 99 In this complex mosaic of microbial diversity in urban systems, one crucial and perhaps 100 understudied domain are urban freshwater systems that provide a juxtaposition of ecology and 101 society. Historically habitats and settlements have come up around water bodies whether built or 102 natural such as rivers, lakes, reservoirs, ponds, coastal or inland regions(Cruz-Cano et al., 2025). 103 These continue to underpin urban livelihood, recreational activities, tourism and even play a role 104 in mitigating climate change. They also bear the burden of waste, pollution, anthropogenic 105 activities, toxic waste, fecal contamination and cultural activities. Marginalised communities 106 depend on them for drinking water as well making them directly relevant to public health in the 107 surrounding area(Dickin & Gabrielsson, 2023). This is vital at a time when nearly 4 billion 108 people face water scarcity at least once a year(Mekonnen & Hoekstra, 2016). Thus water 109 management practices become crucial for the health and well being of the population around 110 waterbodies. Urban planning tends to look at waterbodies as passive infrastructure elements 111 instead of thriving ecosystems. Determining the urban waterbody microbiome is not just an 112 exercise in curiosity but data that can assist urban governance and planning, stormwater design, 113 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 6 restoration, control pollution and surveil public health. 114 With the advent of sequencing methods there is no scarcity of microbiome data from specific 115 urban ecosystems, especially waterbodies surrounding a city(González et al., 2025). However, 116 we have a fragmented view of the microbial make-up often in the form of case studies and 117 geographically restricted datasets which prevent us from having a wider context of the ecological 118 patterns around the world. A broader view of the literature can help in looking at both short term 119 fluctuations and long term geographical or climatic trends arising from climate change, pollution 120 or anthropogenic activities. Evidence based urban water management and ecological theory can 121 be strengthened by checking microbial trends beyond mere identification or pathogen dynamics. 122 Fundamental processes such as nutrient cycling and AMR need coordinated comparative 123 approaches. 124 Keeping this in mind we synthesized insights from 90 studies across three different research 125 search engines to look at the effect urbanisation has on the microbiome of waterbodies in cities 126 across the globe. We have taken a comprehensive approach and shed light on the different 127 human activities observed with respect to the microbial communities observed and nutrient 128 cycling types in global urban freshwater bodies. Our goal was to identify microbes beyond their 129 pathogenic traits and also look at the trend of urbanisation versus the type of microbe identified 130 over time. 131 132

133 The primary questions addressed in this paper are:- 134 ● What evidence exists for the effect of urbanisation on urban freshwater body 135 microbiomes in terms of microbial diversity and functional shifts? 136 ● What are the critical gaps in the available literature and where else can the focus lie? 137 ● How can we look at microbial ecological patterns across geography in urban freshwater 138 bodies? 139 ● Should policy makers focus on microbial make-up of wetlands beyond a few identifying 140 organisms while restoring waterbodies in cities? 141 This review was conducted to address the objectives. It is following a systematic approach, 142 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 7 conforming to the PRISMA reporting standards to ensure comprehensive and unbiased coverage 143 of literature (Page et al., 2020). 144

145 This study combines systematic review methodology with systematic mapping to identify 146 patterns in microbial taxa reporting, functional traits, and anthropogenic drivers across urban 147 freshwater ecosystems. 148 We used three different bibliographic databases for this literature review: Web of Science 149 (WOS), Google Scholar (GS) and SCOPUS. Each search string was adapted to suit the database. 150 The search was conducted between January 2025 and March 2025. The number of records 151 retrieved with each string is shown in Table 1. Table 2 contains the rationale for each inclusion 152 and exclusion criteria. We only considered research articles and not reviews. 153 154 We used Google Scholar, Web of Science and SCOPUS to search for articles, develop the search 155 string as well as the inclusion-exclusion criteria. The final search string used for each database 156 can be found in Table 1. WOS, GS and SCOPUS were the databases used to obtain studies for 157 analyses. In the search string, we used terms synonymous with lakes, waterbodies, urbanisation 158 and microbiome. We also used Boolean operators such as “AND” and “OR”. We identified more 159 articles by going through the bibliographies of the studies included after primary screening. For 160 WOS and SCOPUS we retrieved all the results of each search string, for GS we retrieved results 161 from the first 10 pages for each search string. These were collated and duplicates were removed. 162 163 Databases Search Strings Records retrieved WOS TS=( urban blue space* OR lake* OR pond* OR river* OR stream* OR canal* OR waterway* OR urban wetland* OR stormwater pond* OR reservoir*) AND TS=(microbiome* OR microbiota* OR bacteria* OR fungi OR prokaryote* OR eukaryote* 1055 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 8 OR belowground biodivers* OR soil microbe* OR AMR AND TS=( urbanisation OR urbanisation OR urban development OR urban expansion OR urban growth OR land use change OR urban sprawl OR urban migration ) NOT TS=( marine OR ocean* OR hospital OR clinical OR human gut OR medical OR COVID OR aquaculture OR coastal ) SCOPUS (TITLE-ABS-KEY(("urban blue space*" OR lake* OR pond* OR river* OR stream* OR canal* OR waterway* OR "urban wetland*" OR "stormwater pond*" OR reservoir*) AND (microbiome* OR microbiota* OR bacteria OR fungi OR prokaryotes* OR eukaryote* OR "belowground biodiversity*" OR "soil microbe*" OR AMR) AND (urbanisation* OR "urban development" OR "urban expansion" OR "urban growth" OR "land use change" OR "urban sprawl" OR "urban migration")) AND NOT TITLE-ABS- KEY(marine OR ocean* OR hospital OR clinical OR "human gut" OR medical OR COVID* OR aquaculture OR coastal)) 100 Google Scholar (urban blue space OR lake OR pond OR river OR stream OR canal OR waterway OR urban wetland OR stormwater pond OR reservoir) 406 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 9 AND (microbiome OR microbiota OR bacteria OR fungi OR prokaryote OR eukaryote OR belowground biodiversity OR soil microbiome OR AMR) AND (urbanisation OR urbanisation OR urban development OR urban expansion OR urban growth OR land use change OR urban sprawl OR urban migration) NOT (marine OR ocean OR hospital OR clinical OR human gut OR medical OR COVID OR aquaculture OR coastal) Table 1: Search strings and number of records retrieved. 164 165 The scoping exercise revealed that studies revolving around the water microbiome and 166 urbanisation began around 2000 and so, we included research published from 2000 to 2025. We 167 did not place any restriction on geographic locations but, we only considered published studies 168 (i.e., no grey literature) and studies published in English as it was difficult to obtain reliable 169 translations. 170 We estimated the comprehensiveness of the search string by checking for overlap between the 171 three databases. We found a significant overlap between the three databases, suggesting that all 172 three databases covered important studies published during the established time frame. The 173 collated list of papers was divided into 5 groups such that every section was screened by two 174 authors. This screening was based on the title and abstract. The agreement coefficient for each 175 section was calculated. The agreement coefficient is a statistical measure used to quantify the 176 reliability between two raters using different methods. In this study, the method used is Cohen’s 177 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 10 kappa. The kappa value was calculated for each section and overall value (0.85) was obtained by 178 averaging those values. This was followed by full-text screening. 179 Inclusion Rationale Urban blue spaces Studies conducted in freshwater bodies, lakes, rivers, ponds Water body microbe/microbiome/microbiota/bact eria/fungi/prokaryotes/eukaryotes Studies involving microbes present in the water hence, can include bacteria, fungi, protists, some types algae, individual taxa and groups/community Relevant ecosystem Only aquatic freshwater Analytical modelling Correlative studies are included but analytical techniques used are also noted to understand the cause-effect relationship depicted Time Period 2000-2025 Language Studies published in English language or a reliable translation to English Functional traits Studies depicting functional traits such as antibiotic resistance, nitrogen cycling genes are also considered Exclusion Rationale Urban blue spaces Excluding spaces like coastal areas, bays, harbors, stormwater Heavily industrialized areas, areas that have been left behind (like landfills and so on) Excluding spaces that involved extensive industrialisation, because those sites will be far away from urban spaces and consist of heavy metals which may drastically alter the community. Non-urban Excluding studies involving non urban blue spaces present in rural areas or pristine areas. Ecosystems Excluding air-borne microbiomes, microbiomes on surfaces other than soil, endosphere, phyllosphere and wetlands. Soil microbiome Excluding studies involves the microfauna, meiofauna, macrofauna megafauna present in the soil. Reviews/speculative studies/Human health Excluding Article/literature reviews/meta-analysis and studies which focus on human health 180 Table 2: Different inclusion and exclusion criteria that were used to select the studies meant for mapping. The criteria includes 181 synonyms and the rationale behind each criteria. 182 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 11 183 The data extracted (Table 3) was: microbial communities, microbial indices, microbial functional 184 traits, sample information, site information, study site details, climatic conditions, techniques 185 used for microbial identification, type of urban water body, type of human activity observed or 186 studied, relationship between urbanisation and microbes, types of statistical tests, electronic 187 publishing date, journal the study is published in, authors origins and funding agencies involved. 188 The results were through graphs and the graphs were made using R and QGIS. 189 190 191 Data Points Sub data points Rationale Microbial data Microbial communities It is collected to obtain a thorough understanding of the microbial community present in the water sample. Microbial indices Microbial functional traits Sample information Number of samples It is collected to understand how well the study captures the natural variations. Number of replicates Type of Sample Water or sediment sample It reconfirms if the samples observed were water only. Site Information Geographical region It is collected to make note of the country, the type of water body studied in the paper. Type of Site Age of Site Study site details Month(s) It is collected to understand the climatic conditions during which sampling took place. Year(s) Climatic conditions .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 12 Seasons Techniques for microbial identification It is collected to understand how microbial identification took place. Type of urban space Type of human activity observed or studied It is collected as a proxy for urbanisation. Relationship between urbanisation/human activity and microbes It is collected to check if the paper derives a relationship between urbanisation and microbes if yes, which direction. Types of Statistical tests It is collected to check the methodological rigour of the paper and compare analytical approaches between studies. Electronic Publishing date Month Year Journal Paper is published in Electronic journal Author origins Number of institutions Countries involved Funding agencies involved It was collected to understand if these studies were backed by governmental agencies or private agencies. 192 Table 3: Each data point that was extracted along with subcategories for each. It takes into account the variability 193 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 1 3 observed while scoping. 194

195 Number of Studies 196 The search string resulted in 1,561 papers from three unique databases. After removing 197 duplicates, 1,059 papers were screened. 940 papers were excluded after title and abstract 198 screening from the review based on the eligibility criteria. Out of 123 papers, 17 papers could not199 be retrieved. 106 papers were screened for full text screening, out of which 16 rejected further 200 based on the exclusion criteria. Thus, 90 papers were included in the review. Fig 1 shows the 201 PRISMA (Page et al., 2020) chart for details of the screening process. 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 3 ot .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 1 4 223 224 225 226 227 228 229 230 231 232 233 Fig. 1 -PRISMA flow chart for systematic literature reviews. The flow chart shows the systematic approach taken to select the 234 studies for the review. It shows the number of studies selected, removed and reason of exclusion at each step. The agreement 235 coefficient was calculated after the primary screening (Page et al., 2012). 236 Geographic Distribution of Studies 237 There is an uneven geographic distribution of papers across the globe depicted in Fig 2. 238 Developed countries such as China, United States of America and Canada are over represented 239 in water microbial research. China contributed the highest number of studies (55.5%, n = 50) 240 followed by the United States of America (12.22%, n = 11, Fig 3). South Asia and Africa had 241 fewer studies on urban freshwater microbiomes, despite facing more water stress due to rapid 242 urbanisation and industrialization. 243 244 245 4 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 1 5 246 Fig 2 - The global distribution of studies on urban freshwater microbiomes. More than 50 studies have been conducted in the 247 People's Republic of China followed by the USA with 11 studies. 248 249 Study Sites 250 Among the selected papers, lakes and rivers are the most commonly studied sites (n=27 and 37) 251 respectively, showing proportionally higher attention. In contrast, the other types of sites such as 252 channels, estuary, catchment areas as depicted in Fig. 3 are studied much less frequently, 253 appearing in only one or two papers. Few studies among the examined publications include 254 water samples from nearby construction sites that show the direct impact of anthropogenic 255 activities on waterbodies and the microbiome inside it. 256 257 5 as .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 16 258 Fig 3: This figure shows the number of studies conducted in different types of sites. Freshwater sites also include Freshwater 259 springs. NA in the graph, is for the studies that did not mention the type of sites just written as water samples taken from surface 260 waters. Waste water includes influents and effluents of the wastewater system. And the Rivers also include the riverine system. 261 262 Microbial Functional Traits Studies 263 Nutrient cycling was the most frequently reported functional trait, appearing in 30.8% of 264 included studies(Fig. 4), contributing 30.80%, followed by AMR/pathogen-related traits at 265 20.09%. Stress-related traits, such as seasonal variation, biofilm formation, and temperature 266 tolerance, accounted for 14.73%. In contrast, physical traits and mobile genetic 267 elements/horizontal gene transfer were underrepresented. 268 The temporal look at the functional trait, Fig 4b, shows that studies on microbiome were limited 269 until 2010 but have grown exponentially since 2016, and all traits have been studied relatively 270 evenly across the years since. 271 272 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 1 7 273 274 Fig 4 a.The above figure shows the frequency of various microbial functional traits occurring among the selected papers. b. The 275 frequency of various functional traits being studied each year. 276 7 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 18 Anthropogenic Activities 277 The most studied anthropogenic activity across the studies were tourism and tourist residence 278 (21.6%, n = 106, Fig 5), followed by land use impact (20%, n = 100), sewage (16.2%, n = 81) 279 and discharge ( 12.8%, n = 64) as shown in Fig 5. Because studies often report multiple drivers, 280 percentages represent frequency of mention rather than mutually exclusive categories.Certain 281 drivers such as stormwater runoff and cultural practices appear underrepresented, suggesting 282 potential gaps in current research focus. 283 284 285 Fig 5.The above figure shows the frequency of various anthropogenic activities studied in the selected papers. Tourism and 286 residential land use were the most frequently studied anthropogenic drivers, followed by land-use change, sewage discharge, and 287 wastewater inputs. 288 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 19 Microbial Diversity 289 Many studies reported the presence of bacteria without further classification. The most 290 commonly reported bacterial groups were Proteobacteria, followed by Cyanobacteria and 291 Actinobacteria(Fig 6). Other frequently reported groups included Firmicutes, Chloroflexi, and 292 Bacteroidetes. Some of the least reported groups were Deferribacteres, Frankiales, Parcubacteria, 293 and Thaumarchaeota, as depicted in Fig. 6. 294 It represents the frequency of reporting across studies and does not necessarily reflect relative 295 abundance changes under urbanisation. The most frequently reported microbes were microbes 296 from the human gut, indicating a sewage contamination in the waterbodies. Further, many of the 297 reported microbes have proven to have harmful impacts on human health, such as Proteobacteria, 298 having evidence in metabolic disorders and inflammatory bowel disease and playing a role also 299 in lung diseases, such as asthma and chronic obstructive pulmonary disease (Rizzatti et al, 2017). 300 Cyanobacterial blooms result in the bioaccumulation of cyanotoxins that are associated with 301 renal failure, decreased platelets, increased leukocytes, and the development of hepatotoxicosis 302 (Villalobos et al, 2025). 303 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 20 304 Fig 6. The top 50 microbes classes found in the papers. Proteobacteria is a broad generalist bacterial phylum that is present in 305 almost all the papers studied. It was followed by Cyanobacteria, Actinobacteria, Bacteriodetes, Firmicutes and Chloroflexi. 306 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 21 Microbial Diversity across Anthropogenic Activities 307 The heatmap reflects co-reporting patterns of microbial taxa across studies examining different 308 anthropogenic drivers. Fig 7 does not represent quantitative abundance measurements within 309 individual waterbodies. Tourism, Discharge, Pollution, Wastewater, Land Use, and 310 Sewage)shows significantly higher microbial abundance and diversity. Proteobacteria and 311 Actinobacteria, generalists, show positive association with sewage, tourism and land use impact, 312 suggesting they play an important role in the formation of the particular microbial signature of a 313 water body. Agriculture, Deforestation, and Drainage promote the establishment of a few more 314 niche or specialist species due to the conditions provided. Activities like Sewage, Land Use, and 315 Pollution are clustered together because they all show high microbial abundance. On the other 316 hand, Agriculture and Deforestation are branched off alone because their microbial communities 317 are different. This could be due to the uneven distribution of papers across all anthropogenic 318 activities, but this still highlights how different anthropogenic stressors mold the microbial 319 signatures of various ecosystems. 320 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 22 321 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 23 Fig 7. The above heat map shows the presence of microbial communities between various anthropogenic activities. The bacterial 322 count is taken in logarithmic scale to show the variation between the count.Generalist taxa such as Proteobacteria, Cyanobacteria, 323 and Actinobacteria were consistently reported across studies examining multiple anthropogenic activities.The groupings show the 324 anthropogenic activities as well as microbial community composition that are the most identical to one another. 325 326 327 Effect of urbanisation across studies on microbial diversity 328 Urbanisation was consistently found to be a driver of microbial community restructuring, and 329 this was evident both taxonomically and functionally (Table 4 and 5). Alpha diversity (28 330 studies) was found to be inconsistent and therefore not a reliable measure of urbanisation. 331 Diversity was found to be maintained or even enhanced under nutrient enrichment, even where 332 changes to the microbial community were evident (26 studies). These changes were found to be 333 more indicative of the effects of urbanisation. Proteobacteria was dominant (33 studies), and this 334 was enhanced, especially in wastewater and nutrient-rich environments. This was due to the 335 ability of this group to thrive under disturbed conditions. Conversely, coliforms and E. coli (18 336 studies) were found to increase significantly in urbanized environments, especially where there 337 was a sewage input. This was a reliable indicator of urbanisation. Pathogens and virulence 338 factors (11 studies) were also found to be enriched.The functional response was found to be 339 closely associated with the urban drivers. The antibiotic resistance genes (ARGs) were found to 340 be widespread and enriched, especially in wastewater-influenced systems, suggesting that urban 341 waters harbor antimicrobial resistance. The nitrogen cycling genes and associated 342 microorganisms were found to be consistently altered, suggesting the enhanced nutrient 343 transformation potential under eutrophic conditions. Similarly, the carbon metabolism was found 344 to be enriched in response to organic pollution and DOM, suggesting enhanced microbial carbon 345 transformation potential. Overall, the functional potential was found to be enriched in response 346 to urban drivers, including pathways associated with respiration, nutrient cycling, and stress 347 response. 348 349 Urban Driver Microbial Response Evidence From Studies Ecological Implication .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 24 Wastewater discharge Increased coliforms, pathogens, and ARGs Many urban rivers and canals show elevated fecal indicators downstream Indicates direct human contamination and public health risk Nutrient enrichment Increased nitrogen and phosphorus cycling microbes Denitrification genes and nutrient- transforming taxa enriched Drives eutrophication and altered biogeochemical cycling Organic pollution / DOM inputs Increased respiration and carbon metabolism pathways Urban sites show higher organic carbon oxidation and DOM bioavailability Accelerates microbial carbon processing Land-use change and impervious surfaces Altered microbial composition and sometimes reduced diversity Urban streams show shifts toward disturbance-tolerant taxa Indicates environmental filtering and habitat modification Antibiotics and pollutants Enrichment of ARGs and defense pathways ARGs and antibiotic resistance phenotypes common in urban waters Promotes antimicrobial resistance reservoirs Hydrological alteration Community homogenization and bloom-associated microbes Urban wetlands and lakes show bloom- specialized microbial communities Alters ecosystem stability and microbial food webs Table 4. Different types of urban drivers explored across the 90 studies that alter microbial communities, increasing pathogens, 350 ARGs, and nutrient-cycling taxa, with implications for ecosystem function and public health. 351 352 Indicator Direction of Change Number of Studies Reporting Key Observations Across Studies Ecological Interpretation Alpha diversity (Shannon, Chao1, richness) Mixed 28 Some urban waters show lower diversity due to stress and pollution, while Diversity alone is not a consistent indicator of urban impact; .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 25 nutrient enrichment and runoff can increase diversity. Many studies show stable diversity but major compositional shifts. community composition is more sensitive. Proteobacteria abundance Frequently dominant 33 Proteobacteria commonly remain dominant across sites and often increase in disturbed waters, wastewater-influenced systems, and nutrient-rich environments. Highly adaptable taxa associated with organic pollution, nutrient enrichment, and disturbance tolerance. Antibiotic resistance genes (ARGs) Increased or detected frequently 19 ARGs and resistance phenotypes detected in sediments, water, and biofilms; some studies show strong ARG–mobile genetic element correlations. Urban waters may act as reservoirs and transmission pathways for antimicrobial resistance. Coliforms / E. coli / fecal indicators Strong increase in urban sites 18 Highest levels downstream of sewage outflows, wastewater discharge, and highly urbanized catchments. Reliable indicators of anthropogenic contamination and sewage influence. Pathogens / virulence factors Often enriched 11 Opportunistic pathogens and virulence genes frequently detected in urban rivers, ponds, and canals. Urban aquatic systems may act as pathogen reservoirs with potential public health implications. Nitrogen cycling genes Altered (often more denitrification or N- processing) 15 Changes in genes such as narG, nirS, amoA, and enrichment of nitrogen- transforming bacteria observed along urban Nutrient enrichment and runoff alter biogeochemical cycling and ecosystem functioning. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 26 gradients. Carbon metabolism / DOM processing Altered 13 urbanisation changes DOM composition, respiration rates, and microbial carbon metabolism pathways. Urban runoff and organic inputs reshape carbon processing and microbial energy pathways. Community composition Strong shifts or homogenization 26 Urban gradients drive major restructuring of microbial assemblages, even where alpha diversity remains stable. Environmental filtering and pollution select for disturbance-tolerant microbial taxa. Functional potential / metabolic pathways Frequently altered 17 Enrichment of metabolic pathways related to respiration, nutrient processing, and stress response. Indicates functional reorganization of microbial ecosystems under urban pressure. Table 5 Urbanisation alters microbial diversity, composition, and function, with consistent increases in fecal indicators, ARGs, 353 and disturbance-tolerant taxa, while community composition and functional potential shift more reliably than diversity alone. 354 Correlation of Microbial Diversity across anthropogenic activities 355 Fig 8 highlights how different anthropogenic stressors mold the microbial signatures of various 356 ecosystems. The correlation matrix shows the statistical relationship between the microbiome of 357 various anthropogenic stressors. Spearman's Correlation was used to calculate the correlation 358 coefficients. 359 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 27 The strongest positive correlation was found between Heavy Metal and Human Disturbance. 360 This suggests that patterns of human distribution are strongly correlated with levels of heavy 361 metal contamination in urban waterbodies, and are associated with shifts in the native 362 microbiome. Agriculture and wastewater with its high positive correlation indicate that the 363 farming structure shares the same basin for wastewater discharge and wastewater. Drainage has 364 the most independent microbiome composition, not dependent on the other anthropogenic 365 activities listed. The correlation matrix has more positive correlations due to the higher 366 abundance of generalists such as Proteobacteria and Actinobacteria, which drives homogeneity 367 across sites. The correlation analysis reflects similarities in reported microbial taxa across studies 368 investigating different anthropogenic drivers and should not be interpreted as within-site 369 ecological interactions. 370 371 Fig 8. The correlation plot between the microbial community found across various anthropogenic activities. The Spearman’s 372 coefficients are given between the microbial communities most reported in presence of anthropogenic activity. The strongest 373 correlation was found in agriculture with wastewater and population, heavy metal with human disturbance, wastewater, pollution 374 stressor and population.Higher positive correlation indicates presence of similar microbial communities in both the anthropogenic 375 activity sites. The correlation coefficients between all the activities show higher positive correlation due to presence of generalist 376 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 28 species such as Proteobacteria.This reflects similarity in taxa reported across studies rather than direct ecological interactions 377 within individual waterbodies. 378 379 Principal Component Analysis 380 In Fig 9 Dim1 represents the "total human impact or gradient of urban pollution". Samples on the 381 far right are highly contaminated; samples on the left are relatively "clean”. Dim1 has a high 382 explanatory power of 55.5% tells us that microbial diversity across waterbodies is driven by the 383 Human Impact gradient. Dim2 represents the secondary variation distinguishing between urban 384 and population-related impacts and industrial impacts. Sewage, Pollution, Wastewater, and 385 Tourism/Residence are packaged together and directed towards the right, which indicates that 386 they are correlated and drive similar microbial changes in waterbodies. Heavy Metal and Human 387 Disturbance point downward, suggesting they create a distinct environmental niche that differs 388 from “Sewage”. Drainage and Deforestation point in opposing directions from the main cluster 389 indication; they have a unique effect on the aquatic environment that is not completely 390 exhaustive with the urban pollution. The longer the arrow represents more importance of the 391 stressors in shaping the microbial communities of the waterbodies. Proteobacteria, 392 Cyanobacteria, and Actinobacteria are highly "enriched" by urban waste. They likely thrive in 393 high-nutrient (eutrophic) conditions provided by sewage and tourism-related runoff. The 394 microbes in the central area are not strongly associated with these specific stressors. 395 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 2 9 396 Fig 9. - PCA analysis revealed clustering of anthropogenic drivers such as sewage discharge, land-use change, and pollution 397 stressors, indicating similarity in microbial taxa reported across these disturbance categories.PC1 = disturbance intensity, PC2 = 398 functional variation. 399 400 9 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 30 Relative Stressor Composition 401 The Relative Stressor Composition, Fig 10, reveals that the most dominant taxa are heavily 402 partitioned by anthropogenic pressures. Proteobacteria, Cyanobacteria, and Actinobacteria show 403 larger sections for sewage, tourism and wastewater. High percentages of these blocks suggest 404 these microbes are robust indicators of urban runoff. This shows the microbial community 405 composition in urban waterbodies is not random but is specifically composed by the type of 406 environmental stressors present. 407 408 Fig 10. Relative Stressor composition of top 20 microbes.Proteobacteria, Cyanobacteria and Actinobacteria are comprised of 409 multiple stressors with various relative weightage. But some like Ciliophora, Armatimonadetes and Nitrospirae have fewer 410 stressors indicating they are present only in certain ecosystem conditions. 411

412 One major limitation of this study is the lack of rural urban comparisons in the studies that we 413 picked up based on our search strings, which would have made the study stronger and more 414 comprehensive. We were also forced to rely on reporting frequency of different microbes which 415 may have resulted in over dominance of certain species across studies; while useful for detecting 416 major trends, this method does not take into account effect size, magnitude of change, or 417 significance, and therefore certain taxa may be emphasized while others, though showing small 418 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 31 changes, may not receive adequate representation. With the timeline we selected for this review 419 there is considerable heterogeneity between sequencing platforms, depth of sequencing, and 420 bioinformatic pipelines, such as different regions of 16S rRNA, and these differences make 421 studies less comparable to each other, which could lead to biases in community composition, 422 diversity, and taxonomic resolution. Additionally, with most of the studies reported from China 423 and North America our data gets skewed due to limited representation from the Global South, 424 and this bias constrains the generalizability of findings, particularly given that urbanisation 425 patterns, infrastructure, and environmental conditions differ substantially across regions. Finally, 426 we chose papers which were written in English, which may have resulted in language bias, as 427 studies conducted in other languages, especially in countries experiencing rapid urbanisation, 428 may have been excluded, leading to an incomplete global overview due to the lack of local 429 studies. 430

431 This is a comprehensive systematic review of the current global literature available on the 432 influence of urbanisation on microbial communities in freshwater bodies. From 90 selected 433 papers we identified critical gaps and biases that hinder a holistic understanding of these 434 interactions. Notably, there is a lack of research from economically developing regions, Africa, 435 and Southeast Asia (Fig. 2), limiting our ability to generalise global findings or look for 436 geographical patterns in these areas. This is particularly crucial given that the predicted AMR 437 burden in these regions is expected to be highest by 2050 especially affecting Africa and South 438 Asian countries(Naghavi et al., 2024). In this context, the functional and taxonomic patterns 439 identified in previous research become a key factor in understanding the ecological aspects of 440 this risk. 441 A high prevalence of functional traits related to nutrient cycling and AMR/pathogens were 442 identified, the co-occurrence of which suggests that urban freshwater systems are shaped by 443 resource enrichment and may serve as petri dishes for resistance proliferation behaviour. The 444 reporting frequency of Proteobacteria,Cyanobacteria, Actinobacteria, and Firmicutes adapt well 445 to nutrient-rich and organically loaded environments. This also mirrors well to what research 446 studies have reported over the last 25 years. Additionally, these phyla are ubiquitous in areas of 447 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 32 high anthropogenic pressure where freshwater are often at the receiving end of sewage, untreated 448 wastewater, runoff, and pollutants thus selecting for generalist microbial groups capable of rapid 449 growth and metabolic flexibility. This is consistent with studies from Germany that show 450 (Daga/i2 Quisbert et al., 2021) how urbanisation poses long-term health risks in waterbodies. 451 Similarly, in Numberger et al. (2022), Actinobacteriota and Gammaproteobacteria had ASVs 452 unique to urban waters in freshwater bodies, including Acidovorax (Burkholderiales), 453 Flavobacterium (Bacteroidota), and Pseudomonas (Gammaproteobacteria). Mohanta and Goel 454 (2014) found that multiple drug-resistant bacteria were highest in samples from rapidly 455 urbanising areas. Urbanisation thus acts as an ecological filter, with similar microbial patterns 456 emerging globally in climatically distinct regions, selecting for phyla that rapidly grow in areas 457 affected by sewage and tolerant of disturbances such as pollution thereby favouring opportunistic 458 pathogens. 459 Above mentioned biases aside, due to the limited number of studies from the Global South, the 460 methodology of different studies is also varied. Some studies have relied on single-season, short-461 term sampling or screening, as compared to systematic sampling over different seasons (Wang et 462 al 2018, Zhao et al. 2022). It is difficult to infer the effect of seasonal variations on microbiome 463 diversity. Certain studies have also derived their data from 16s amplicon sequencing, while few 464 others from metagenomic analysis. There is a significant gap in the availability of transcriptomic, 465 proteomic, and metabolomic data. Although some research has examined samples across urban 466 gradients, most studies are restricted to a limited number of sampling sites, making broader 467 ecological inferences challenging. 468 The consistent reporting of similar bacterial phyla across diverse geographic regions and 469 anthropogenic disturbances suggests increasing microbial homogenisation in urban freshwater 470 ecosystems globally.China, which has the most number of studies on urban water microbiomes, 471 is suffering from waterbody loss (Xiao et al., 2022). This is mainly due to urbanisation, where 472 waterbodies play an important role in providing drinking water. China being one of the world’s 473 leading producers and consumers for antibiotics, is a potential centre for development and 474 dissemination of ARGs (Zhang et al., 2023). As a precautionary measure, they have invested a 475 lot of funding towards harmonious development between resources and environments, in the 476 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 33 form of ‘Sponge city programs’ and ‘The Administrative Measures of the Urban Blue 477 Line’(Xiao et al., 2022). 478 Similarly studies on freshwater microbiomes need to be expanded to the economically 479 developing countries as these studies reveal the presence of human health risks, impacts of 480 pollution and help in the long term monitoring of the ecosystem health (McLellan et al., 2015). 481 The global understanding of urban freshwater microbiomes is currently geographically skewed, 482 limiting predictive power and policy relevance in regions expected to face the highest AMR 483 burden by 2050. 484 As a finite resource it is vital to maintain the microbial biodiversity of urban waterbodies to 485 sustain people. This also brings up questions around equity as marginalised communities depend 486 more on freshwater bodies for survival. In a (Satterthwaite et al. 2022) Bengaluru case study, the 487 most rapidly urbanising city in India, it was discovered that lower income communities closer to 488 the lake faced higher risk of water related fecal contamination. Community distribution of 489 microbes, if conducted in countries of South America and South Asia, can help us estimate the 490 disease vulnerability of communities that depend on such wetlands for survival. Public health 491 welfare programs and education campaigns around the microbial make-up of an ecosystem like 492 waterbodies especially with AMR on the rise can create some much needed awareness around 493 these sites. 494 As Maria Magdalena Warter, points out “Just like our gut, freshwater ecosystems need a 495 functioning microbiome. Bacteria and other microorganisms form the basis of food chains and 496 metabolic processes, as well as the self-purification capacity of waterbodies"(Leibniz Institute of 497 Freshwater Ecology and Inland Fisheries (IGB), 2025). Thus there is certainly a need for a better 498 wastewater management system that protects the natural urban waterbodies and prevents further 499 entry of AMR. Across Table 4 and 5 the findings were consistent with the expected effects of 500 specific urban drivers. Antibiotics and pollutants are associated with enriched resistance traits, 501 and hydrological changes were associated with homogenization and bloom-forming 502 microorganisms. 503 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 34 Urban freshwater systems increasingly reflect wastewater-associated microbial signatures, 504 indicating that anthropogenic inputs contribute to a partial convergence between natural and 505 human-associated microbiomes(Numberger et al., 2022). One can infer from this study that 506 ARGs and fecal indicators may serve as more reliable monitoring targets than diversity metrics 507 alone. These patterns reinforce a One Health perspective, where environmental microbial 508 dynamics, ecosystem functioning, and human health risks are closely interconnected through 509 shared exposure pathways. Policies around better wastewater and runoff management along with 510 pathogen indicators can help mitigate AMR and associated risks. Future research should focus on 511 using expansive research methods and a multi-omics approach, aside from developing standard 512 methodologies and geographic representation, especially in rapidly urbanising areas. These 513 research methods are going to be crucial in developing futuristic frameworks that link microbial 514 ecology with urban planning and management. 515

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Characterization of microbial communities in seven 600 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 1, 2026. ; https://doi.org/10.64898/2026.03.31.715732doi: bioRxiv preprint 37 wetlands with different anthropogenic burden using next generation sequencing in 601 Bogotá, Colombia. Sci. Rep. 13, 16973. https://doi.org/10.1038/s41598-023-42970-w 602 ● Cai, S., Zhao, J., Sheng, E., Fan, L., Shen, Z., Li, Y., 2025. Similar but different assembly 603 processes of bacterial and micro-eukaryotic communities in an urban river. Sci. 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