Enrichment of microbial communities for hexavalent chromium removal using a biofilm reactor.

Given the toxicity and widespread occurrence of hexavalent chromium [Cr(VI)] in aquatic environments, we investigated the feasibility of a down-flow hanging sponge (DHS) biofilm reactor for the enrichment of microbial communities capable of Cr(VI) removal. In the present study, a laboratory-scale DHS reactor fed with a molasses-based medium containing Cr(VI) was operated for 112 days for the investigation. The enrichment of Cr(VI)-removing microbial communities was evaluated based on water quality and prokaryotic community analyses. Once the DHS reactor began to operate, high average volumetric Cr(VI) removal rates of 1.21-1.45 mg L-sponge-1 h-1 were confirmed under varying influent Cr(VI) concentrations (approximately 20-40 mg L-1). 16S rRNA gene amplicon sequencing analysis suggested the presence of phylogenetically diverse prokaryotic lineages, including phyla that contain well-known Cr(VI)-reducing bacteria (e.g., Bacteroidetes, Firmicutes, and Proteobacteria) in the polyurethane sponge media of the DHS reactor. Therefore, our findings indicate that DHS reactors have great potential for the enrichment of Cr(VI)-removing microbial communities.

Defining microbial community composition and seasonal variation in a sewage treatment plant in India using a down-flow hanging sponge reactor.

The characteristics of the microbial community in a practical-scale down-flow hanging sponge (DHS) reactor, high in organic matter and sulfate ion concentration, and the seasonal variation of the microbial community composition were investigated. Microorganisms related to sulfur oxidation and reduction (2-27%), as well as Leucobacter (7.50%), were abundant in the reactor. Anaerobic bacteria (27-38% in the first layer) were also in abundance and were found to contribute to the removal of organic matter from the sewage in the reactor. By comparing the Simpson index, the abundance-based coverage estimator (ACE) index, and the species composition of the microbial community across seasons (summer/dry, summer/rainy, autumn/dry, and winter/dry), the microbial community was found to change in composition only during the winter season. In addition to the estimation of seasonal variation, the difference in the microbial community composition along the axes of the DHS reactor was investigated for the first time. Although the abundance of each bacterial species differed along both axes of the reactor, the change of the community composition in the reactor was found to be greater along the vertical axis than the horizontal axis of the DHS reactor.

Development of downflow hanging sponge (DHS) reactor as post treatment of existing combined anaerobic tank treating natural rubber processing wastewater.

Conventional aerated tank technology is widely applied for post treatment of natural rubber processing wastewater in Southeast Asia; however, a long hydraulic retention time (HRT) is required and the effluent standards are exceeded. In this study, a downflow hanging sponge (DHS) reactor was installed as post treatment of anaerobic tank effluent in a natural rubber factory in South Vietnam and the process performance was evaluated. The DHS reactor demonstrated removal efficiencies of 64.2 ± 7.5% and 55.3 ± 19.2% for total chemical oxygen demand (COD) and total nitrogen, respectively, with an organic loading rate of 0.97 ± 0.03 kg-COD m-3 day-1 and a nitrogen loading rate of 0.57 ± 0.21 kg-N m-3 day-1. 16S rRNA gene sequencing analysis of the sludge retained in the DHS also corresponded to the result of reactor performance, and both nitrifying and denitrifying bacteria were detected in the sponge carrier. In addition, anammox bacteria was found in the retained sludge. The DHS reactor reduced the HRT of 30 days to 4.8 h compared with the existing algal tank. This result indicates that the DHS reactor could be an appropriate post treatment for the existing anaerobic tank for natural rubber processing wastewater treatment.

Distinct comammox Nitrospira in high-rate down-flow hanging sponge reactor treating municipal wastewater.

A down-flow hanging sponge (DHS) reactor is a trickling filter system used for wastewater treatment, which employs sponges to retain biomass. This study assessed the process performance of a compact DHS combined with a sedimentation tank with seven phases at varying hydraulic retention times (HRT) over 500 days. The BOD of the DHS effluent was maintained at 4.0 ±â€¯0.5 mg·L-1 for the shortest HRT 0.3 ±â€¯0.1 h. The nitrification efficiency was considerably impacted by the reduced HRT, with NH4+-N and NO3--N concentrations of 9.0 ±â€¯1.2 mgN·L-1 and 2.2 ±â€¯0.5 mgN·L-1, respectively. Nevertheless, the effluent complied with effluent discharge standards throughout the trial period. The number of comammox 16S rRNA gene copies ranged from 5.58 to 13.2 × 107 copies·mL-1, indicating that sponges biomass retained carrier can provide favorable conditions for comammox growth and could contribute to nitrification in the high-rate DHS reactor.

Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems.

To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.IMPORTANCECulture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

Characteristics of organic removal for supermarket wastewater treatment with an anaerobic baffled reactor and efficacy evaluation of changing HRT.

An anaerobic baffled reactor (ABR) is one of the useful wastewater treatment technologies, but the knowledge about its treatment performance for actual wastewater with load fluctuation is limited. The organic removal performance of an ABR for treating supermarket wastewater was evaluated. The ABR, which consisted of eight columns, was examined under four hydraulic retention time (HRT) conditions of 19.4, 12.9, 8.0, and 4.4 h. As a result, the unfiltered chemical oxygen demand (COD) removal efficiency was 80 (±8) % at an HRT of 19.4 h. When the HRT was shortened to 12.9 h, the average unfiltered COD removal efficiency decreased to 58 (±15) %. However, it showed buffering effect against high load inflow in the first column, indicating that it is useful as a pretreatment system under this condition. At an HRT of 4.4 h, the unfiltered COD removal efficiency decreased to 9%, indicating the system failed. The results of the microbial community structure analysis showed that the detection frequency of acidogenic bacteria decreased in proportion to the extension of residence time in the reactor. These results indicate that the ABR is useful for the treatment of supermarket wastewater with load fluctuations as a main treatment system at a HRT of 19.4 h and as a pretreatment system at a HRT of 12.9 h.

Identification and cultivation of anaerobic bacterial scavengers of dead cells.

The cycle of life and death and Earth's carbon cycle(s) are intimately linked, yet how bacterial cells, one of the largest pools of biomass on Earth, are recycled back into the carbon cycle remains enigmatic. In particular, no bacteria capable of scavenging dead cells in oxygen-depleted environments have been reported thus far. In this study, we discover the first anaerobes that scavenge dead cells and the two isolated strains use distinct strategies. Based on live-cell imaging, transmission electron microscopy, and hydrolytic enzyme assays, one strain (designated CYCD) relied on cell-to-cell contact and cell invagination for degrading dead food bacteria where as the other strain (MGCD) degraded dead food bacteria via excretion of lytic extracellular enzymes. Both strains could degrade dead cells of differing taxonomy (bacteria and archaea) and differing extents of cell damage, including those without artificially inflicted physical damage. In addition, both depended on symbiotic metabolic interactions for maximizing cell degradation, representing the first cultured syntrophic Bacteroidota. We collectively revealed multiple symbiotic bacterial decomposition routes of dead prokaryotic cells, providing novel insight into the last step of the carbon cycle.

Complete Genome Sequence of "Candidatus Hydrogeosomobacter endosymbioticus," an Intracellular Bacterial Symbiont of the Anaerobic Ciliate Scuticociliate GW7.

The bacterium "Candidatus Hydrogenosomobacter endosymbioticus" is an intracellular symbiont of anaerobic scuticociliate GW7, which is associated with hydrogenosome together with methanogenic archaea. Here, we report a complete genome sequence of the symbiont consisting of 827 kbp. Knowing this sequence would contribute to the understanding of the metabolic interactions and evolution of the tripartite symbiosis.

Symbiosis between Candidatus Patescibacteria and Archaea Discovered in Wastewater-Treating Bioreactors.

Each prokaryotic domain, Bacteria and Archaea, contains a large and diverse group of organisms characterized by their ultrasmall cell size and symbiotic lifestyles (potentially commensal, mutualistic, and parasitic relationships), namely, Candidatus Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) and DPANN archaea, respectively. Cultivation-based approaches have revealed that Ca. Patescibacteria and DPANN symbiotically interact with bacterial and archaeal partners and hosts, respectively, but that cross-domain symbiosis and parasitism have never been observed. By amending wastewater treatment sludge samples with methanogenic archaea, we observed increased abundances of Ca. Patescibacteria (Ca. Yanofskybacteria/UBA5738) and, using fluorescence in situ hybridization (FISH), discovered that nearly all of the Ca. Yanofskybacteria/UBA5738 cells were attached to Methanothrix (95.7 ± 2.1%) and that none of the cells were attached to other lineages, implying high host dependency and specificity. Methanothrix filaments (multicellular) with Ca. Yanofskybacteria/UBA5738 attached had significantly more cells with no or low detectable ribosomal activity (based on FISH fluorescence) and often showed deformations at the sites of attachment (based on transmission electron microscopy), suggesting that the interaction is parasitic. Metagenome-assisted metabolic reconstruction showed that Ca. Yanofskybacteria/UBA5738 lacks most of the biosynthetic pathways necessary for cell growth and universally conserves three unique gene arrays that contain multiple genes with signal peptides in the metagenome-assembled genomes of the Ca. Yanofskybacteria/UBA5738 lineage. The results shed light on a novel cross-domain symbiosis and inspire potential strategies for culturing CPR and DPANN. IMPORTANCE One highly diverse phylogenetic group of Bacteria, Ca. Patescibacteria, remains poorly understood, but, from the few cultured representatives and metagenomic investigations, they are thought to live symbiotically or parasitically with other bacteria or even with eukarya. We explored the possibility of symbiotic interactions with Archaea by amending wastewater treatment sludge samples that were rich in Ca. Patescibacteria and Archaea with an isolate archaeon that is closely related to a methanogen population abundant in situ (Methanothrix). This strategic cultivation successfully established enrichment cultures that were mainly comprised of Ca. Patescibacteria (family level lineage Ca. Yanofskybacteria/UBA5738) and Methanothrix, in which we found highly specific physical interactions between the two organisms. Microscopic observations based on transmission electron microscopy, target-specific fluorescence in situ hybridization, and metagenomic analyses showed evidence that the interaction is likely parasitic. The results show a novel cross-domain parasitism between Bacteria and Archaea and suggest that the amendment of host Archaea may be an effective approach in culturing novel Ca. Patescibacteria.

Novel Cross-domain Symbiosis between Candidatus Patescibacteria and Hydrogenotrophic Methanogenic Archaea Methanospirillum Discovered in a Methanogenic Ecosystem.

To identify novel cross-domain symbiosis between Candidatus Patescibacteria and Archaea, we performed fluorescence in situ hybridization (FISH) on enrichment cultures derived from methanogenic bioreactor sludge with the newly designed 32-520-1066 probe targeting the family-level uncultured clade 32-520/UBA5633 lineage in the class Ca. Paceibacteria. All FISH-detectable 32-520/UBA5633 cells were attached to Methanospirillum, indicating high host specificity. Transmission electron microscopy observations revealed 32-520/UBA5633-like cells that were specifically adherent to the plug structure of Methanospirillum-like rod-shaped cells. The metagenome-assembled genomes of 32-520/UBA5633 encoded unique gene clusters comprising pilin signal peptides and type IV pilins. These results provide novel insights into unseen symbiosis between Ca. Patescibacteria and Archaea.

Adsorption and biodegradation removal of methylene blue in a down-flow hanging filter reactor incorporating natural adsorbent.

This study was carried out to explore the importance of adsorption and biodegradation mechanisms for methylene blue (MB) removal by a novel natural adsorbent (purified coconut fibre; PCF) incorporated to a down-flow hanging fibre (DHF) reactor. An adsorption DHF (Ads-DHF) reactor demonstrated the adsorption removal mechanism, while a combined adsorption-biological DHF (Bio-DHF) reactor simulated the processes of both adsorption and biodegradation were investigated for the MB removal capability. PCF prepared from coconut fibre waste was applied as a media in the DHF reactors. The process performance and the removal mechanisms of the DHF reactors were evaluated for 62 days. The results showed that a total MB removal efficiency of 93 ± 7% was achieved for the Bio-DHF reactor and 36 ± 25% for the Ads-DHF reactor. The combined adsorption and biological degradation in the Bio-DHF reactor enhanced the removal efficiency and the life-time of the reactor compared with the performance of the adsorption process alone in the Ads-DHF reactor. Moreover, microbial community analysis revealed that microorganisms, commonly involved in the biodegradation of dyes, were predominant in the Bio-DHF reactor. The PCF media of the Bio-DHF reactor was essential to keep the dye degrading bacteria in the reactor. Therefore, it can be concluded that the Bio-DHF reactor is an appropriate treatment system for treating dyes wastewater. This research is significant and useful for environmental protection and reuse of biomass wastes.

Food selectivity of anaerobic protists and direct evidence for methane production using carbon from prey bacteria by endosymbiotic methanogen.

Anaerobic protists are major predators of prokaryotes in anaerobic ecosystems. However, little is known about the predation behavior of anaerobic protists because almost none have been cultured. In particular, these characteristics of anaerobic protists in the phyla Metamonada and Cercozoa have not been reported previously. In this study, we isolated three anaerobic protists, Cyclidium sp., Trichomitus sp., and Paracercomonas sp., from anaerobic granular sludge in an up-flow anaerobic sludge blanket reactor used to treat domestic sewage. Ingestion and digestion of food bacteria by anaerobic protists with or without endosymbiotic methanogens were demonstrated using tracer experiments with green fluorescent protein and a stable carbon isotope. These tracer experiments also demonstrated that Cyclidium sp. supplied CO2 and hydrogen to endosymbiotic methanogens. While Cyclidium sp. and Trichomitus sp. ingested both Gram-negative and -positive bacteria, Paracercomonas sp. could only take up Gram-negative bacteria. Archaeal cells such as Methanobacterium beijingense and Methanospirillum hungatei did not support the growth of these protists. Metabolite patterns of all three protists differed and were influenced by food bacterial species. These reported growth rates, ingestion rates, food selectivity, and metabolite patterns provide important insights into the ecological roles of these protists in anaerobic ecosystems.

Simple and reliable enumeration of Escherichia coli concentrations in wastewater samples by measuring ß-d-glucuronidase (GUS) activities via a microplate reader.

Monitoring of Escherichia coli concentrations at wastewater treatment plants (WWTPs) is important to ensure process performance and protect public health. However, conventional E. coli enumeration methods are complicated and time- and labor-consuming. Here, we report a novel simple and reliable method based on ß-d-glucuronidase (GUS) activity assay to enumerate E. coli concentrations in wastewater (WW) samples. An aliquot (20 µL) of the medium with fluorogenic enzyme substrate for E. coli and 180 µL of a WW sample were added to one well of a 96-well microplate. The microplate was placed in a microplate reader at 37 °C. To this end, the fluorescence intensity of a fluorogenic enzyme substrate for E. coli was measured every 10 min over 3 h to determine GUS activity. The linear increase in the fluorescence intensity representing the GUS activities showed a positive correlation with E. coli concentrations in wastewater samples. However, the correlation equations were specific to WWTPs, which could be due to the difference in the E. coli population structures among WWTPs. We observed that the wastewater matrix is not a limitation to measure the GUS activity, and a WWTP-specific correlation equation can be used as a calibration curve to estimate the E. coli concentrations in the samples collected from that site. A comparison of the results with those of culture-dependent Colilert method proved that the current method is simple and useful for the enumeration of E. coli concentrations in wastewater samples reliably.

Temporal variation of eukaryotic community structures in UASB reactor treating domestic sewage as revealed by 18S rRNA gene sequencing.

Eukaryotes are important components of ecosystems in wastewater treatment processes. However, little is known about eukaryotic community in anaerobic wastewater treatment systems. In this study, eukaryotic communities in an up flow anaerobic sludge blanket (UASB) reactor treating domestic sewage during two years of operation were investigated using V4 and V9 regions of 18S rRNA gene for amplicon sequencing. In addition, activated sludge and influent sewage samples were also analyzed and used as the references for aerobic eukaryotic community to characterize anaerobic eukaryotes. The amplicon sequence V4 and V9 libraries detected different taxonomic groups, especially from the UASB samples, suggesting that commonly used V4 and V9 primer pairs could produce a bias for eukaryotic communities analysis. Eukaryotic community structures in the UASB reactor were influenced by the immigration of eukaryotes via influent sewage but were clearly different from the influent sewage and activated sludge. Multivariate statistics indicated that protist genera Cyclidium, Platyophrya and Subulatomonas correlated with chemical oxygen demand and suspended solid concentration, and could be used as bioindicators of treatment performance. Uncultured eukaryotes groups were dominant in the UASB reactor, and their physiological roles need to be examined to understand their contributions to anaerobic processes in future studies.

Non-aerated single-stage nitrogen removal using a down-flow hanging sponge reactor as post-treatment for nitrogen-rich wastewater treatment.

A laboratory-scale experiment is conducted to remove nitrogen from nitrogen-rich wastewater using a down-flow hanging sponge (DHS) reactor. Effluent from an anaerobic-aerobic system for treating synthetic natural rubber wastewater, which still contains high levels of ammonia, was used as nitrogen-rich wastewater. Experimental period was divided into four phases based whether a carbon source was fed to the DHS reactor. The highest nitrogen removal efficiency (59.5 ±â€¯5.4%) was achieved during phase 4, when a sodium acetate solution was fed into bottom section of the DHS reactor. In the DHS reactor, the nitrification occurred in the upper and middle sections. Then, after adding the sodium acetate solution, denitrification occurred. The final chemical oxygen demand, ammonia, and total inorganic nitrogen concentrations in the DHS reactor effluent were 37 ±â€¯24 mg/L, 34 ±â€¯5 mgN/L, and 42 ±â€¯8 mgN/L, respectively. These concentrations were sufficient to meet the effluent standards of the Vietnamese natural rubber industry, which are the strictest in South-East Asia. The dominant bacteria in the sludge retained by the reactor's sponge media were the nitrifying bacteria Nitrosovibrio (0.2%) and Nitrospira (0.2-0.3%), the denitrifying bacteria Hylemonella (1.0-13.7%), Pseudoxanthomonas (1.2-2.1%), and Amaricoccus (2.4-3.5%), and the anammox bacterium Candidatus Brocadia (0.1-0.2%). Significant amounts of the nitrogen-fixing bacterium Xanthobacter (11.2-14.8%) and the rubber-degrading bacterium Gordonia (11.0-28.6%) were also found in the DHS reactor. These bacteria were thus considered to be the key microbes for nitrogen removal in a DHS reactor fed with a carbon source for denitrification.

Ureolytic Prokaryotes in Soil: Community Abundance and Diversity.

Although the turnover of urea is a crucial process in nitrogen transformation in soil, limited information is currently available on the abundance and diversity of ureolytic prokaryotes. The abundance and diversity of the soil 16S rRNA gene and ureC (encoding a urease catalytic subunit) were examined in seven soil types using quantitative PCR and amplicon sequencing with Illumina MiSeq. The amplicon sequencing of ureC revealed that the ureolytic community was composed of phylogenetically varied prokaryotes, and we detected 363 to 1,685 species-level ureC operational taxonomic units (OTUs) per soil sample, whereas 5,984 OTUs were site-specific OTUs found in only one of the seven soil types.

A nitrogen removal system to limit water exchange for recirculating freshwater aquarium using DHS-USB reactor.

This study proposes a biological nitrogen removal system for freshwater aquaria consisting of a down-flow hanging sponge (DHS) and an up-flow sludge blanket (USB). DHS-USB systems can perform nitrification and denitrification simultaneously, reducing ammonia (NH3) and nitrate (NO3-) toxicity in the water. The performance of the system was evaluated using on-site fresh water aquaria at ambient temperature (23-34°C) over 192 days. NH3 and nitrite (NO2-) were maintained at a detection limit of 0.01 mg N L-1 and NO3- was maintained below 10 mg N L-1, despite limited water exchange. The 16S rRNA gene of microorganisms from the sludge retained in the bioreactors was sequenced to identify the microbial communities present. Microbial community analysis revealed that ammonia oxidizing archaea (AOA), Ca. Nitrososphaera and Nitrosopumilus, played an important role in nitrification in the DHS reactor, while denitrifying bacteria Thauera played an important role in denitrification in the USB reactor. The proposed DHS-USB system is a promising technological advancement in the development of lower maintenance aquaria.

Performance evaluation of the pilot scale upflow anaerobic sludge blanket - Downflow hanging sponge system for natural rubber processing wastewater treatment in South Vietnam.

A pilot-scale upflow anaerobic sludge blanket (UASB)-downflow hanging sponge system (DHS) combined with an anaerobic baffled reactor (ABR) and a settling tank (ST) was installed in a natural rubber processing factory in South Vietnam and its process performance was evaluated for 267days. The UASB reactor achieved a total removal efficiency of 55.6±16.6% for chemical oxygen demand (COD) and 77.8±10.3% for biochemical oxygen demand (BOD) with an organic loading rate of 1.7±0.6kg-COD·m-3·day-1. The final effluent of the proposed system had 140±64mg·L-1 of total COD, 31±12mg·L-1 of total BOD, and 58±24mg-N·L-1 of total nitrogen. The system could significantly reduce 92% of greenhouse gas emissions and 80% of hydraulic retention times compared with current treatment systems.

Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge.

Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

Microbial community analysis using MiSeq sequencing in a novel configuration fluidized bed reactor for effective denitrification.

A novel configured fluidized bed reactor (FBR) with granular rubber as the fluidized media was operated without internal recirculation to achieve denitrification. This FBR could operate under a low hydraulic retention time (HRT) of 50min due to the low rubber media density and absence of recirculation. Synthetic nitrate-rich wastewater with a fixed nitrate (NO3--N) concentration and varying COD concentrations was fed into the FBR. The nitrate removal profile showed a rapid nitrate reduction at the bottom of the reactor with a high performance under the low HRT. Different microbial communities were identified using Illumina Miseq sequencing. The dominant microorganisms belonged to the Beta- and Gamma-proteobacteria classes and played important roles in nitrate reduction. Acidovorax was abundant at low COD: NO3--N ratios, while Rhizobium and Zoogloea were dominant at high COD: NO3--N ratios. The COD: NO3--N ratio strongly influenced the composition of the microbial community including the dominant species.