Effective green treatment of sewage sludge from Fenton reactions: Utilizing MoS2 for sustainable resource recovery.

Effectively managing sewage sludge from Fenton reactions in an eco-friendly way is vital for Fenton technology's viability in pollution treatment. This study focuses on sewage sludge across various treatment stages, including generation, concentration, dehydration, and landfill, and employs chemical composite MoS2 to facilitate green resource utilization of all types of sludge. MoS2, with exposed Mo4+ and low-coordination sulfur, enhances iron cycling and creates an acidic microenvironment on the sludge surface. The MoS2-modified iron sludge exhibits outstanding (>95%) phenol and pollutant degradation in hydrogen peroxide and peroxymonosulfate-based Fenton systems, unlike unmodified sludge. This modified sludge maintains excellent Fenton activity in various water conditions and with multiple anions, allowing extended phenol degradation for over 14 d. Notably, the generated chemical oxygen demand (COD) in sludge modification process can be efficiently eliminated through the Fenton reaction, ensuring effluent COD compliance and enabling eco-friendly sewage sludge resource utilization.

Insights into the evolutionary and ecological adaption strategies of nirS- and nirK-type denitrifying communities.

Denitrification is a crucial process in the global nitrogen cycle, in which two functionally equivalent genes, nirS and nirK, catalyse the critical reaction and are usually used as marker genes. The nirK gene can function independently, whereas nirS requires additional genes to encode nitrite reductase and is more sensitive to environmental factors than nirK. However, the ecological differentiation mechanisms of those denitrifying microbial communities and their adaptation strategies to environmental stresses remain unclear. Here, we conducted metagenomic analysis for sediments and bioreactor samples from Lake Donghu, China. We found that nirS-type denitrifying communities had a significantly lower horizontal gene transfer frequency than that of nirK-type denitrifying communities, and nirS gene phylogeny was more congruent with taxonomy than that of nirK gene. Metabolic reconstruction of metagenome-assembled genomes further revealed that nirS-type denitrifying communities have robust metabolic systems for energy conservation, enabling them to survive under environmental stresses. Nevertheless, nirK-type denitrifying communities seemed to adapt to oxygen-limited environments with the ability to utilize various carbon and nitrogen compounds. Thus, this study provides novel insights into the ecological differentiation mechanism of nirS and nirK-type denitrifying communities, as well as the regulation of the global nitrogen cycle and greenhouse gas emissions.

Generational Specific Recruitment of Arsenic- and Antimony-Reducing Microorganisms in Plant Root-Associated Niches for Adapting to Metalloid-Metal Pollution.

The recruitment of microorganisms by plants can enhance their adaptability to environmental stressors, but how root-associated niches recruit specific microorganisms for adapting to metalloid-metal contamination is not well-understood. This study investigated the generational recruitment of microorganisms in different root niches of Vetiveria zizanioides (V. zizanioides) under arsenic (As) and antimony (Sb) stress. The V. zizanioides was cultivated in As- and Sb-cocontaminated mine soils (MS) and artificial pollution soils (PS) over two generations in controlled conditions. The root-associated microbial communities were analyzed through 16S rRNA, arsC, and aioA gene amplicon and metagenomics sequencing. V. zizanioides accumulated higher As(III) and Sb(III) in its endosphere in MS in the second generation, while its physiological indices in MS were better than those observed in PS. SourceTracker analysis revealed that V. zizanioides in MS recruited As(V)- and Sb(V)-reducing microorganisms (e.g., Sphingomonales and Rhodospirillaceae) into the rhizoplane and endosphere. Metagenomics analysis further confirmed that these recruited microorganisms carrying genes encoding arsenate reductases with diverse carbohydrate degradation abilities were enriched in the rhizoplane and endosphere, suggesting their potential to reduce As(V) and Sb(V) and to decompose root exudates (e.g., xylan and starch). These findings reveal that V. zizanioides selectively recruits As- and Sb-reducing microorganisms to mitigate As-Sb cocontamination during the generational growth, providing insights into novel strategies for enhancing phytoremediation of metalloid-metal contaminants.

Community coalescence under variable hydrochemical conditions of the Chesapeake Bay shaped bacterial diversity and functional traits.

Community coalescence related to bacterial mixing events regulates community characteristics and affects the health of estuary ecosystems. At present, bacterial coalescence and its driving factors are still unclear. The present study used a dataset from the Chesapeake Bay (2017) to address how bacterial community coalescence in response to variable hydrochemistry in estuarine ecosystems. We determined that variable hydrochemistry promoted the deterioration of water quality. Temperature, orthophosphate, dissolved oxygen, chlorophyll a, Secchi disk depth, and dissolved organic phosphorus were the key environmental factors driving community coalescence. Bacteria with high tolerance to environmental change were the primary taxa accumulated in community coalescence, and the significance of deterministic processes to communities was revealed. Community coalescence was significantly correlated with the pathways of metabolism and organismal systems, and promoted the co-occurrence of antibiotic resistance and virulence factor genes. Briefly, community coalescence under variable hydrochemical conditions shaped bacterial diversity and functional traits, to optimise strategies for energy acquisition and lay the foundation for alleviating environmental pressures. However, potential pathogenic bacteria in community coalescence may be harmful to human health and environmental safety. The present study provides a scientific reference for ecological management of estuaries.

Protozoa as Hotspots for Potential Pathogens in the Drinking Water of a Subtropical Megacity: Diversity, Treatment, and Health Risk.

Drinking water systems host a wide range of microorganisms essential for biosafety. However, one major group of waterborne pathogens, protozoa, is relatively neglected compared to bacteria and other microorganisms. Until now, little is known about the growth and fate of protozoa and their associated bacteria in drinking water systems. In this study, we aim to investigate how drinking water treatment affects the growth and fate of protozoa and their associated bacteria in a subtropical megacity. The results showed that viable protozoa were prevalent in the city's tap water, and amoebae were the major component of tap water protozoa. In addition, protozoan-associated bacteria contained many potential pathogens and were primarily enriched in amoeba hosts. Furthermore, this study showed that current drinking water disinfection methods have little effect on protozoa and their associated bacteria. Besides, ultrafiltration membranes unexpectedly served as an ideal growth surface for amoebae in drinking water systems, and they could significantly promote the growth of amoeba-associated bacteria. In conclusion, this study shows that viable protozoa and their associated bacteria are prevalent in tap water, which may present an emerging health risk in drinking water biosafety.

Synthetic phylogenetically diverse communities promote denitrification and stability.

Denitrification is an important process of the global nitrogen cycle as some of its intermediates are environmentally important or related to global warming. However, how the phylogenetic diversity of denitrifying communities affects their denitrification rates and temporal stability remains unclear. Here we selected denitrifiers based on their phylogenetic distance to construct two groups of synthetic denitrifying communities: one closely related (CR) group with all strains from the genus Shewanella and the other distantly related (DR) group with all constituents from different genera. All synthetic denitrifying communities (SDCs) were experimentally evolved for 200 generations. The results showed that high phylogenetic diversity followed by experimental evolution promoted the function and stability of synthetic denitrifying communities. Specifically, the productivity and denitrification rates were significantly (P < 0.05) higher with Paracocus denitrificans as the dominant species (since the 50th generation) in the DR community than those in the CR community. The DR community also showed significantly (t = 7.119, df = 10, P < 0.001) higher stability through overyielding and asynchrony of species fluctuations, and showed more complementarity than the CR group during the experimental evolution. This study has important implications for applying synthetic communities to remediate environmental problems and mitigate greenhouse gas emissions.

Soil Amoebae Affect Iron and Chromium Reduction through Preferential Predation between Two Metal-Reducing Bacteria.

Soil protists are essential but often overlooked in soil and could impact microbially driven element cycling in natural ecosystems. However, how protists influence heavy metal cycling in soil remains poorly understood. In this study, we used a model protist, Dictyostelium discoideum, to explore the effect of interactions between soil amoeba and metal-reducing bacteria on the reduction of soil Fe(III) and Cr(VI). We found that D. discoideum could preferentially prey on the Fe(III)-reducing bacterium Shewanella decolorationis S12 and significantly decrease its biomass. Surprisingly, this predation pressure also stimulated the activity of a single S. decolorationis S12 bacterium to reduce Fe(III) by enhancing the content of electron-transfer protein cyt c, intracellular ATP synthesis, and reactive oxygen species (e.g., H2O2). We also found that D. discoideum could not prey on the Cr(VI)-reducing bacterium Brevibacillus laterosporus. In contrast, B. laterosporus became edible to amoebae in the presence of S. decolorationis S12, and their Cr(VI) reduction ability decreased under amoeba predation pressure. This study provides direct evidence that protists can affect the Cr and Fe cycling via the elective predation pressure on the metal-reducing bacteria, broadening our horizons of predation of protists on soil metal cycling.

Evidence for Assimilatory Nitrate Reduction as a Previously Overlooked Pathway of Reactive Nitrogen Transformation in Estuarine Suspended Particulate Matter.

Suspended particulate matter (SPM) contributes to the loss of reactive nitrogen (Nr) in estuarine ecosystems. Although denitrification and anaerobic ammonium oxidation in SPM compensate for the current imbalance of global nitrogen (N) inputs and sinks, it is largely unclear whether other pathways for Nr transformation exist in SPM. Here, we combined stable isotope measurements with metagenomics and metatranscriptomics to verify the occurrence of dissimilatory nitrate reduction to ammonium (DNRA) in the SPM of the Pearl River Estuary (PRE). Surprisingly, the conventional functional genes of DNRA (nirBD) were abundant and highly expressed in SPM, which was inconsistent with a low potential rate. Through taxonomic and comparative genomic analyses, we demonstrated that nitrite reductase (NirBD) in conjunction with assimilatory nitrate reductase (NasA) performed assimilatory nitrate reduction (ANR) in SPM, and diverse alpha- and gamma-proteobacterial lineages were identified as key active heterotrophic ANR bacteria. Moreover, ANR was predicted to have a relative higher occurrence than denitrification and DNRA in a survey of Nr transformation pathways in SPM across the PRE spanning 65 km. Collectively, this study characterizes a previously overlooked pathway of Nr transformation mediated by heterotrophic ANR bacteria in SPM and has important implications for our understanding of N cycling in estuaries.

Complex Bilateral Interactions Determine the Fate of Polystyrene Micro- and Nanoplastics and Soil Protists: Implications from a Soil Amoeba.

Nano- and microplastics have become a serious global concern, threatening our living environments. Previous studies have shown that many organisms, including bacteria, animals, and plants, can be affected by microplastics. However, little is known about one ecologically important group of soil organisms, the protists. In this study, we investigated how polystyrene micro- and nanoplastics interacted with a soil amoeba Dictyostelium discoideum. The results showed that environmental concentrations of nano- and microplastics could negatively affect the soil amoeba's fitness and development. D. discoideum ingested both nano- and microplastics through phagocytosis but packed and excreted them during slug migration, which also promoted their biodegradation. Fourier transform infrared spectroscopy analyses revealed the formation of new oxygen-containing functional groups and the sign of possible oxidation of polystyrene. Also, nano- and microplastic exposure disrupted the nutrient and energy metabolisms of D. discoideum and affected the expression of key genes (e.g., cf45-1, dcsA, aprA, dymB, and gefB) related to morphogenesis and phagocytosis. In conclusion, our results show that nano- and microplastics have complex bilateral interactions with the soil amoeba, affecting each other's fate in the soil environment. This study provides new insights into how soil protists interact with nano- and microplastics in the soil ecosystem.

Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota.

The Ecology and Evolution of Amoeba-Bacterium Interactions.

Amoebae are protists that have complicated relationships with bacteria, covering the whole spectrum of symbiosis. Amoeba-bacterium interactions contribute to the study of predation, symbiosis, pathogenesis, and human health. Given the complexity of their relationships, it is necessary to understand the ecology and evolution of their interactions. In this paper, we provide an updated review of the current understanding of amoeba-bacterium interactions. We start by discussing the diversity of amoebae and their bacterial partners. We also define three types of ecological interactions between amoebae and bacteria and discuss their different outcomes. Finally, we focus on the implications of amoeba-bacterium interactions on human health, horizontal gene transfer, drinking water safety, and the evolution of symbiosis. In conclusion, amoeba-bacterium interactions are excellent model systems to investigate a wide range of scientific questions. Future studies should utilize advanced techniques to address research gaps, such as detecting hidden diversity, lack of amoeba genomes, and the impacts of amoeba predation on the microbiome.

Sugar and organic acid availability modulate soil diazotroph community assembly and species co-occurrence patterns on the Tibetan Plateau.

Metabolites can mediate species interactions and the assembly of microbial communities. However, how these chemicals relate to the assembly processes and co-occurrence patterns of diazotrophic assemblages in root-associated soils remains largely unknown. Here, we examined the diversity and assembly of diazotrophic communities and further deciphered their links with metabolites on Tibetan Plateau. We found that the distribution of sugars and organic acids in the root-associated soils was significantly correlated with the richness of diazotrophs. The presence of these two soil metabolites explains the variability in diazotrophic community compositions. The differential concentrations of these metabolites were significantly linked with the distinctive abundances of diazotrophic taxa in same land types dominated by different plants or dissimilar soils by same plants. The assembly of diazotrophic communities is subject to deterministic ecological processes, which are widely modulated by the variety and amount of sugars and organic acids. Organic acids, for instance, 3-(4-hydroxyphenyl)propionic acid and citric acid, were effective predictors of the characteristics of diazotrophic assemblages across desert habitats. Diazotrophic co-occurrence networks tended to be more complex and connected within different land types covered by the same plant species. The concentrations of multiple sugars and organic acids were coupled significantly with the distribution of keystone species, such as Azotobacter, Azospirillum, Bradyrhizobium, and Mesorhizobium, in the co-occurrence network. These findings provide new insights into the assembly mechanisms of root-associated diazotrophic communities across the desert ecosystems of the Tibetan Plateau.Key points• Soil metabolites were significantly linked to the diversity of diazotrophic community.• Soil metabolites determined the assembly of diazotrophic community.• Sugars and organic acids were coupled mainly with keystone species in networks.

Responses of ammonia-oxidizing microorganisms to biochar and compost amendments of heavy metals-polluted soil.

Heavy metal pollution affects soil ecological function. Biochar and compost can effectively remediate heavy metals and increase soil nutrients. The effects and mechanisms of biochar and compost amendments on soil nitrogen cycle function in heavy-metal contaminated soils are not fully understood. This study examined how biochar, compost, and their integrated use affected ammonia-oxidizing microorganisms in heavy metal polluted soil. Quantitative PCR was used to determine the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB). Ammonia monooxygenase (AMO) activity was evaluated by the enzyme-linked immunosorbent assay. Results showed that compost rather than biochar improved nitrogen conversion in soil. Biochar, compost, or their integrated application significantly reduced the effective Zn and Cd speciation. Adding compost obviously increased As and Cu effective speciation, bacterial 16S rRNA abundance, and AMO activity. AOB, stimulated by compost addition, was significantly more abundant than AOA throughout remediation. Correlation analysis showed that AOB abundance positively correlated with NO3--N (r = 0.830, P < 0.01), and that AMO activity had significant correlation with EC (r = -0.908, P < 0.01) and water-soluble carbon (r = -0.868, P < 0.01). Those seem to be the most vital factors affecting AOB community and their function in heavy metal-polluted soil remediated by biochar and compost.

Defects on CoS2-x : Tuning Redox Reactions for Sustainable Degradation of Organic Pollutants.

It is important to develop self-producing reactive oxygen species (ROSs) systems and maintain the continuous and effective degradation of organic pollutants. Herein, for the first time, a system of ultrasound-treated CoS2-x mixed with Fe2+ is constructed to sustainably release singlet oxygen (1 O2 ) for the effective degradation of various organic pollutants, including dyes, phenols, and antibiotics. Ultrasonic treatment produces defects on the surface of CoS2 which promote the production of ROSs and the circulation of Fe3+ /Fe2+ . With the help of Co4+ /Co3+ exposed on the surface of CoS2-x , the directional conversion of superoxide radical (. O2- ) to 1 O2 is realized. The CoS2-x /Fe2+ system can degrade organic pollutants efficiently for up to 30 days, which is significantly better than the currently recognized CuPx system (<3 days). Therefore, CoS2-x provides a new choice for the long-term remediation of organic pollutants in controlling large area river pollution.

Constructing an Acidic Microenvironment by MoS2 in Heterogeneous Fenton Reaction for Pollutant Control.

Although Fenton or Fenton-like reactions have been widely used in the environment, biology, life science, and other fields, the sharp decrease in their activity under macroneutral conditions is still a large problem. This study reports a MoS2 cocatalytic heterogeneous Fenton (CoFe2 O4 /MoS2 ) system capable of sustainably degrading organic pollutants, such as phenol, in a macroneutral buffer solution. An acidic microenvironment in the slipping plane of CoFe2 O4 is successfully constructed by chemically bonding with MoS2 . This microenvironment is not affected by the surrounding pH, which ensures the stable circulation of Fe3+ /Fe2+ on the surface of CoFe2 O4 /MoS2 under neutral or even alkaline conditions. Additionally, CoFe2 O4 /MoS2 always exposes "fresh" active sites for the decomposition of H2 O2 and the generation of 1 O2 , effectively inhibiting the production of iron sludge and enhancing the remediation of organic pollutants, even in actual wastewater. This work not only experimentally verifies the existence of an acidic microenvironment on the surface of heterogeneous catalysts for the first time, but also eliminates the pH limitation of the Fenton reaction for pollutant remediation, thereby expanding the applicability of Fenton technology.

Keystone taxa of water microbiome respond to environmental quality and predict water contamination.

The human activity introduces strong environmental stresses, and results in great spatiotemporal heterogeneity for the environment. Although the effects of environmental factors on the microbial diversity and succession have been widely studied, knowledge about how keystone taxa respond to environmental stresses remains poorly understood. We examined bacterial and archaeal communities from 45 wetland ponds covering a wide range of waters in Hangzhou. We found that shifts in bacterial and archaeal communities were strongly correlated with water pollution as indicated by the comprehensive water quality identification (CWQI). The SEGMENTED analysis suggested that there were non-linear responses of microbial communities and keystone taxa to the water pollution gradient. Moreover, these significant tipping points (e.g., CWQI > 4.0) would afford a warning line for urban wetland management. Notably, keystone taxa of bacterial communities could be used to successfully (~88.9% accuracy) predict water contamination levels. This study provides new insights into the potential for keystone bacterial taxa to predict water contamination.

Differential distribution of and similar biochemical responses to different species of arsenic and antimony in Vetiveria zizanioides.

Vetiver grass (Vetiveria zizanioides L. Nash) has a great application potential to the phytoremediation of heavy metals pollution. However, few studies explored the bioavailability and distribution of different speciations of As and Sb in V. zizanioides. This study aimed to clarify the allocation and accumulation of two inorganic species arsenic (As(III) and As(V)) and antimony (Sb(III) and Sb(V)) in V. zizanioides, to understand the self-defense mechanisms of V. zizanioides to these metal(loids) elements. Thus, an experiment was conducted under greenhouse conditions to identify distribution of As and Sb in plant roots and shoots. Antioxidant enzymes (superoxide dismutase, SOD) and changes of subcellular structures were tested to evaluate metal(loids) tolerance capacities of V. zizanioides. This study demonstrated that V. zizanioides had higher capacity to accumulate Sb than As. For Sb absorption, Sb(III) content is significantly higher than Sb(V) in tissues of V. zizanioides under all concentration levels, despite the oxidation of Sb(III) on the nutrient solution surface. Additional Sb was mainly accumulated in plant roots due to Sb immobilization by transforming it into precipitates. As was more easily transferred to aerial tissues and had low accumulation rates, probably due to its restricted uptake rather than restricted transport. In many cases, two inorganic species of As and Sb showed almost same biotoxicity to V. zizanioides estimated from its biomass, SOD activity, and MDA content as well as functional groups. In summary, the results of this study provide new insights into understanding allocation, accumulation and phytotoxicity effects of arsenic and antimony in V. zizanioides. Schematic diagram of distribution of and biochemical responses to As(III), As(V), Sb(III), and Sb(V) in tissue of V. zizanioides.

The Impact of Anthropogenic Disturbance on Bacterioplankton Communities During the Construction of Donghu Tunnel (Wuhan, China).

Bacterioplankton are both primary producers and primary consumers in aquatic ecosystems, which were commonly investigated to reflect environmental changes, evaluate primary productivity, and assess biogeochemical cycles. However, there is relatively less understanding of their responses to anthropogenic disturbances such as constructions of dams/tunnels/roads that may significantly affect the aquatic ecosystem. To fill such gap, this study focused on the bacterioplankton communities' diversity and turnover during a tunnel construction across an urban lake (Lake Donghu, Wuhan, China), and five batches of samples were collected within 2 months according to the tunnel construction progress. Results indicated that both resources and predator factors contributed significant to the variations of bacterioplankton communities, but the closed area and open areas showed different diversity patterns due to the impacts of tunnel construction. Briefly, the phytoplankton, TN, and TP in water were still significantly correlated with the bacterioplankton composition and diversity like that in normal conditions. Additionally, the organic matter, TN, and NH4-N in sediments also showed clear effects on the bacterioplankton. However, the predator effects on the bacterioplankton in the closed-off construction area mainly derived from large zooplankton (i.e., cladocerans), while small zooplankton such as protozoa and rotifers are only responsible for weak predator effects on the bacterioplankton in the open areas. Further analysis about the ecological driving forces indicated that the bacterioplankton communities' turnover during the tunnel construction was mainly governed by the homogeneous selection due to similar environments within the closed area or the open areas at two different stages. This finding suggests that bacterioplankton communities can quickly adapt to the environmental modifications resulting from tunnel construction activities. This study can also give references to enhance our understanding on bacterioplankton communities' response to ecological and environmental changes due to intensification of construction and urbanization in and around lake ecosystems.

Intestinal bacterial signatures of white feces syndrome in shrimp.

Increasing evidence suggests that the intestinal microbiota is closely correlated with the host's health status. Thus, a serious disturbance that disrupts the stability of the intestinal microecosystem could cause host disease. Shrimps are one of the most important products among fishery trading commodities. However, digestive system diseases, such as white feces syndrome (WFS), frequently occur in shrimp culture and have led to enormous economic losses across the world. The WFS occurrences are unclear. Here, we compared intestinal bacterial communities of WFS shrimp and healthy shrimp. Intestinal bacterial communities of WFS shrimp exhibited less diversity but were more heterogeneous than those of healthy shrimp. The intestinal bacterial communities were significantly different between WFS shrimp and healthy shrimp; compared with healthy shrimp, in WFS shrimp, Candidatus Bacilloplasma and Phascolarctobacterium were overrepresented, whereas Paracoccus and Lactococcus were underrepresented. PICRUSt functional predictions indicated that the relative abundances of genes involved in energy metabolism and genetic information processing were significantly greater in WFS shrimp. Collectively, we found that the composition and predicted functions of the intestinal bacterial community were markedly shifted by WFS. Significant increases in Candidatus Bacilloplasma and Phascolarctobacterium and decreases in Paracoccus and Lactococcus may contribute to WFS in shrimp.

Necessary Sequencing Depth and Clustering Method to Obtain Relatively Stable Diversity Patterns in Studying Fish Gut Microbiota.

The 16S rRNA gene is one of the most commonly used molecular markers for estimating bacterial diversity during the past decades. However, there is no consistency about the sequencing depth (from thousand to millions of sequences per sample), and the clustering methods used to generate OTUs may also be different among studies. These inconsistent premises make effective comparisons among studies difficult or unreliable. This study aims to examine the necessary sequencing depth and clustering method that would be needed to ensure a stable diversity patterns for studying fish gut microbiota. A total number of 42 samples dataset of Siniperca chuatsi (carnivorous fish) gut microbiota were used to test how the sequencing depth and clustering may affect the alpha and beta diversity patterns of fish intestinal microbiota. Interestingly, we found that the sequencing depth (resampling 1000-11,000 per sample) and the clustering methods (UPARSE and UCLUST) did not bias the estimates of the diversity patterns during the fish development from larva to adult. Although we should acknowledge that a suitable sequencing depth may differ case by case, our finding indicates that a shallow sequencing such as 1000 sequences per sample may be also enough to reflect the general diversity patterns of fish gut microbiota. However, we have shown in the present study that strict pre-processing of the original sequences is required to ensure reliable results. This study provides evidences to help making a strong scientific choice of the sequencing depth and clustering method for future studies on fish gut microbiota patterns, but at the same time reducing as much as possible the costs related to the analysis.