Community structure and function in a H2-based membrane biofilm reactor capable of bioreduction of selenate and chromate

Two different H₂-based, denitrifying membrane-biofilm reactors (MBfRs) initially reduced Se(VI) or Cr(VI) stably to Se⁰ or Cr(III). When the oxidized contaminants in the influent were switched, each new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been in its original MBfR. The precipitation of reduced selenium and chromium in the biofilm was verified by scanning electron microscopy and energy dispersive X-ray analysis. These results on selenate and chromate reduction are consistent with the interpretation that the H₂-based biofilm community had a high level of functional diversity. The communities’ structures were assessed by cloning analysis. Dechloromonas spp., a known perchlorate-reducing bacteria, dominated the clones from both reactors during selenate and chromate reductions, which suggests that it may have functional diversity capable of reducing selenate and chromate as secondary and dissimilatory acceptors.     

Simultaneous Bio-reduction of Nitrate,Perchlorate, Selenate,Chromate, Arsenate,and Dibromochloropropane Using a Hydrogen-based Membrane Biofilm Reactor

We tested the hypothesis that the H₂-based membrane biofilm reactor (MBfR) is capable of reducing multiple oxidized contaminants, a common situation for groundwater contamination. We conducted bench-scale experiments with three groundwater samples collected from California’s San Joaquin Valley and on two synthetic groundwaters containing selenate and chromate. The actual groundwater sources had nitrate levels exceeding 10 mg-N l⁻¹ and different combinations of anthropogenic perchlorate + chlorate, arsenate, and dibromochloropropane (DBCP). For all actual groundwaters, the MBfR reduced nitrate to less than 0.01 mg-N l⁻¹. Present in two groundwaters, perchlorate + chlorate was reduced to below the California Notification Level, 6 μg-ClO₄ l⁻¹. As(V) was substantially reduced to As(III) for two groundwaters samples, which had influent As(V) concentrations from 3 to 8.8 μg-As l⁻¹. DBCP, present in one groundwater at 1.4 μg l⁻¹, was reduced to below its detection limit of 0.01 μg l⁻¹, which is well below California’s 0.2 μg l⁻¹ MCL for DBCP. For the synthetic groundwaters, two MBfRs initially reduced Se(VI) or Cr(VI) stably to Se° or Cr(III). When we switched the influent oxidized contaminants, the new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been reduced in its original MBfR. These results support that the H₂-based MBfR can reduce multiple oxidized contaminants simultaneously.     

Autohydrogenotrophic Denitrifying Microbial Community in a Glass Beads Biofilm Reactor

A glass bead biofilm reactor was operated using H₂ as an electron donor to remove nitrate at 150 mg NO₃–N l⁻¹ to below detection level. The microbial community in the glass beads biofilm reactor was investigated by using denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. In DGGE analysis of the biofilm, five bands were dominant and indicated the presence of eight β-proteobacteria, one γ-proteobacteria and twelve clostridia. An unculturable Hydrogenophaga sp., which is a new genus of hydrogen-oxidizing bacterium was dominant in microbial community of the biofilm reactor.     

Limestone Corrosion by Neutrophilic Sulfur-Oxidizing Bacteria: A Coupled Microbe-Mineral System

Subsurface karst aquifers receiving sulfidic water can host complex chemolithotrophic microbial communities that are capable of dissolving limestone, forming new karstic habitat. Neutrophilic sulfur-oxidizing bacteria use reduced sulfur compounds as energy rich substrate, potentially producing sulfuric acid as a geochemically reactive byproduct. The physicochemical relationship between a biofilm forming on a limestone surface and the extent of microbial influence on dissolution rate, however, are unknown. We investigated the rate of Madison limestone dissolution by sulfur-oxidizers both in the field at Lower Kane Cave, WY (LKC), and in the laboratory using continuous flow culture reactors and microbial mat collected from LKC. In the field, a microbial consortium rapidly colonized limestone chips forming a thick biofilm, with deep etching of mineral surfaces underneath. In the laboratory we found that a microbial biofilm oxidizing thiosulfate on the limestone surface accelerated dissolution rate up to 7 times faster than the abiotic baseline rate. In contrast, experiments done with H₂S or a mixture of H₂S and thiosulfate had no effect on dissolution rate. We hypothesize that the laboratory mat community dominated by Thiothrix sp. oxidizes thiosulfate to sulfate and H⁺, while H₂S is partially oxidized to S°. When all sulfur substrate is withheld, the community oxidizes stored intracellular sulfur, briefly accelerating limestone dissolution even in the absence of external supplied substrate. Accelerated corrosion occurs only in the reactive micro-environment under the biofilm, disconnected from the bulk reactor solution. When experiments are repeated where the microbial population is separated from the limestone by a dialysis membrane barrier, measured pH drop is greater, but there is only slight enhancement of rate. This work confirms our working hypothesis that neutrophilic sulfur-oxidizers colonize and rapidly dissolve limestone surfaces, possibly to buffer the production of excess acidity.     

Electron-acceptor loadings affect chloroform dechlorination in a hydrogen-based membrane biofilm reactor

Chloroform (CF) can undergo reductive dechlorination to dichloromethane, chloromethane, and methane. However, competition for hydrogen (H₂), the electron-donor substrate, may cause poor dechlorination when multiple electron acceptors are present. Common acceptors in anaerobic environments are nitrate (NO₃⁻), sulfate (SO₄²⁻), and bicarbonate (HCO₃⁻). We evaluated CF dechlorination in the presence of HCO₃⁻ at 1.56 e⁻ Eq/m²-day, then NO₃⁻ at 0.04–0.15 e⁻ Eq/m²-day, and finally NO₃⁻ (0.04 e⁻ Eq/m²-day) along with SO₄²⁻ at 0.33 e⁻ Eq/m²-day in an H₂-based membrane biofilm reactor (MBfR). When the biofilm was initiated with CF-dechlorination conditions (no NO₃⁻ or SO₄²⁻), it yielded a CF flux of 0.14 e⁻ Eq/m²-day and acetate production via homoacetogenesis up to 0.26 e⁻ eq/m²-day. Subsequent addition of NO₃⁻ at 0.05 e⁻ Eq/m²-day maintained full CF dechlorination and homoacetogenesis, but NO₃⁻ input at 0.15 e⁻ Eq/m²-day caused CF to remain in the reactor's effluent and led to negligible acetate production. The addition of SO₄²⁻ did not affect CF reduction, but SO₄²⁻ reduction significantly altered the microbial community by introducing sulfate-reducing Desulfovibrio and more sulfur-oxidizing Arcobacter. Dechloromonas appeared to carry out CF dechlorination and denitrification, whereas Acetobacterium (homoacetogen) may have been involved with hydrolytic dechlorination. Modifications to the electron acceptors fed to the MBfR caused the microbial community to undergo changes in structure that reflected changes in the removal fluxes.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

A biofilm model to understand the onset of sulfate reduction in denitrifying membrane biofilm reactors

This work presents a multispecies biofilm model that describes the co‐existence of nitrate‐ and sulfate‐reducing bacteria in the H₂‐based membrane biofilm reactor (MBfR). The new model adapts the framework of a biofilm model for simultaneous nitrate and perchlorate removal by considering the unique metabolic and physiological characteristics of autotrophic sulfate‐reducing bacteria that use H₂ as their electron donor. To evaluate the model, the simulated effluent H₂, UAP (substrate‐utilization‐associated products), and BAP (biomass‐associated products) concentrations are compared to experimental results, and the simulated biomass distributions are compared to real‐time quantitative polymerase chain reaction (qPCR) data in the experiments for parameter optimization. Model outputs and experimental results match for all major trends and explain when sulfate reduction does or does not occur in parallel with denitrification. The onset of sulfate reduction occurs only when the nitrate concentration at the fiber's outer surface is low enough so that the growth rate of the denitrifying bacteria is equal to that of the sulfate‐reducing bacteria. An example shows how to use the model to design an MBfR that achieves satisfactory nitrate reduction, but suppresses sulfate reduction. Biotechnol. Bioeng. 2013; 110: 763–772. © 2012 Wiley Periodicals, Inc.     

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH₄-based MBfR (<1.0 g N/m²-day) is about one order of magnitude smaller than that in the H₂-based MBfR (1.1–6.7 g N/m²-day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH₄-based MBfR is limited to <1.7 g N/m²-day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K₂) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (k_max2), with k_max2 being more important. Model simulations show that increasing k_max2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (D_m) could bring the nitrate-removal flux to ≥4.0 g N/m²-day, which is close to the nitrate-removal flux for the H₂-based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when D_m and k_max2 increase together.     

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane

Hydrogenotrophic methanogens can use gaseous substrates, such as H₂ and CO₂, in CH₄ production. H₂ gas is used to reduce CO₂. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH₄ production from CO₂ and H₂. CO₂ and H₂ were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5–5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH₄ production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH₄ production process. The results show that acidic operation of a CH₄ production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants

Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange (IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity (3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms in the three MBfRs and compared to those of their inocula. When H₂ was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In Situ Analysis of a Silver Nanoparticle-Precipitating Shewanella Biofilm by Surface Enhanced Confocal Raman Microscopy

Shewanella oneidensis MR-1 is an electroactive bacterium, capable of reducing extracellular insoluble electron acceptors, making it important for both nutrient cycling in nature and microbial electrochemical technologies, such as microbial fuel cells and microbial electrosynthesis. When allowed to anaerobically colonize an Ag/AgCl solid interface, S. oneidensis has precipitated silver nanoparticles (AgNp), thus providing the means for a surface enhanced confocal Raman microscopy (SECRaM) investigation of its biofilm. The result is the in-situ chemical mapping of the biofilm as it developed over time, where the distribution of cytochromes, reduced and oxidized flavins, polysaccharides and phosphate in the undisturbed biofilm is monitored. Utilizing AgNp bio-produced by the bacteria colonizing the Ag/AgCl interface, we could perform SECRaM while avoiding the use of a patterned or roughened support or the introduction of noble metal salts and reducing agents. This new method will allow a spatially and temporally resolved chemical investigation not only of Shewanella biofilms at an insoluble electron acceptor, but also of other noble metal nanoparticle-precipitating bacteria in laboratory cultures or in complex microbial communities in their natural habitats.     

The significance of the initiation process parameters and reactor design for maximizing the efficiency of microbial fuel cells

Microbial fuel cells (MFCs) can be used for electricity generation via bioconversion of wastewater and organic waste substrates. MFCs also hold potential for production of certain chemicals, such as H₂ and H₂O₂. The studies of electricity generation in MFCs have mainly focused on the microbial community formation, substrate effect on the anode reaction, and the cathode’s catalytic properties. To improve the performance of MFCs, the initiation process requires more investigation because of its significant effect on the anodic biofilm formation. This review explores the factors which affect the initiation process, including inoculum, substrate, and reactor configuration. The key messages are that optimal performance of MFCs for electricity production requires (1) understanding of the electrogenic bacterial biofilm formation, (2) proper substrates at the initiation stage, (3) focus on operational conditions affecting initial biofilm formation, and (4) attention to the reactor configuration.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Hydrogenotrophic denitrification and perchlorate reduction in ion exchange brines using membrane biofilm reactors

Halophilic (salt loving), hydrogenotrophic (H₂ oxidizing) denitrifying bacteria were investigated for treatment of nitrate <$>({\rm NO}_3^ ‐ )<$> and perchlorate <$>({\rm ClO}_4^ ‐ )<$> contaminated groundwater and ion exchange (IX) brines. Hydrogenotrophic denitrifying bacteria were enriched from a denitrifying wastewater seed under both halophilc and non‐halophilc conditions. The cultures were inoculated into bench‐scale membrane biofilm reactors (MBfRs) with an “outside in” configuration, with contaminated water supplied to the lumen of the membranes and H₂ supplied to the shell. Abiotic mass transfer tests showed that H₂ mass transfer coefficients were lower in brines than in tap water at highest Reynolds number, possibly due to increased transport of salts and decreased H₂ solubility at the membrane/liquid interface. An average <$>{\rm NO}_3^ ‐ <$> removal efficiency of 93% was observed for the MBfR operated in continuous flow mode with synthetic contaminated groundwater. Removal efficiencies of 30% for <$>{\rm NO}_3^ ‐ <$> and 42% for <$>{\rm ClO}_4^ ‐ <$> were observed for the MBfR operated with synthetic IX brine in batch operating mode with a reaction time of 53 h. Phylogenetic analysis focused on the active microbial community and revealed that halotolerant, <$>{\rm NO}_3^ ‐ <$> ‐reducing bacteria of the bacterial classes Gamma‐Proteobacteria and Sphingobacteria were the metabolically dominant members within the stabilized biofilm. This study shows that, despite decreased H₂ transfer under high salt conditions, hydrogenotrophic biological reduction may be successfully used for the treatment of <$>{\rm NO}_3^ ‐ <$> and <$>{\rm ClO}_4^ ‐ <$> in a MBfR. Biotechnol. Bioeng. 2009; 104: 483–491 © 2009 Wiley Periodicals, Inc.     

The impact of medicinal brines on microbial biofilm formation on inhalation equipment surfaces

Abstract

Materials such as polyvinyl chloride, polypropylene, and polyethylene are used for the construction of medical equipment, including inhalation equipment. Inhalation equipment, because of the wet conditions and good oxygenation, constitutes a perfect environment for microbial biofilm formation. Biofilms may affect microbiological cleanliness of inhalation facilities and installations and promote the development of pathogenic bacteria. Microbial biofilms can form even in saline environments. Therefore, the aim of this study was to evaluate the effect of medicinal brines on microbial biofilm formation on the surfaces of inhalation equipment. The study confirmed the high risk of biofilm formation on surfaces used in inhalation equipment. Isolated microorganisms belonged to potential pathogens of the respiratory system, which can pose a health threat to hospital patients. The introduction of additional contaminants increased the amount of bacterial biofilm. On the other hand, the presence of brines significantly limited the amount of biofilm, thus eliminating the risk of infections.     

Integrating microbial ecology in bioprocess understanding: the case of gas biofiltration

Biofilters are packed-bed bioreactors where contaminants, once transferred from the gas phase to the biofilm, are oxidized by diverse and complex communities of attached microorganisms. Over the last decade, more and more studies aimed at opening the back box of biofiltration by unraveling the biodiversity-ecosystem function relationship. In this review, we report the insights provided by the microbial ecology approach in biofilters and we emphasize the parallels existing with other engineered ecosystems used for wastewater treatment, as they all constitute relevant model ecosystems to explore ecological issues. We considered three characteristic ecological indicators: the density, the diversity, and the structure of the microbial community. Special attention was paid to the temporal and spatial dynamics of each indicator, insofar as it can disclose the potential relationship, or absence of relation, with any operating or functional parameter. We also focused on the impact of disturbance regime on the microbial community structure, in terms of resistance, resilience, and memory. This literature review led to mitigated conclusions in terms of biodiversity–ecosystem function relationship. Depending on the environmental system itself and the way it is investigated, the spatial and temporal dynamics of the microbial community can be either correlated (e.g., spatial stratification) or uncoupled (e.g., temporal instability) to the ecosystem function. This lack of generality shows the limits of current 16S approach in complex ecosystems, where a functional approach may be more suitable.     

O2 versus N2O respiration in a continuous microbial enrichment

Despite its ecological importance, essential aspects of microbial N₂O reduction—such as the effect of O₂ availability on the N₂O sink capacity of a community—remain unclear. We studied N₂O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N₂O and O₂ can occur, (ii) the mechanism governing the competition for N₂O and O₂, and (iii) how the N₂O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N₂O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O₂. When supplied simultaneously, N₂O reduction to N₂ was only detected when the O₂ concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N₂O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture’s affinity constant (K_S) for O₂ was found to be two orders of magnitude lower than the value for N₂O, explaining the preferential use of O₂ over N₂O under most environmentally relevant conditions.

     

Formation of nesquehonite and other minerals as a consequence of biofilm dehydration

A microbial biofilm community was established over 971 days within gravel in an aquarium so as to model biofouling of an aquifer. When the water was allowed to evaporate slowly, white crystalline deposits, containing several carbonate and sulphate minerals including nesquehonite (MgCO₃.3H2O), were seen at the highest points on the surface of the biofouled gravel. No such deposits occurred in regions lacking biofilms. These crystals appeared to originate from evaporation of dissolved salts which had migrated through the biofilm. Surfaceadherent microbial biofilms may conceivably provide a conduit for solute transport in porous media such as soils and aquifers.     

Backwash intensity and frequency impact the microbial community structure and function in a fixed-bed biofilm reactor

Linkages among bioreactor operation and performance and microbial community structure were investigated for a fixed-bed biofilm system designed to remove perchlorate from drinking water. Perchlorate removal was monitored to evaluate reactor performance during and after the frequency and intensity of the backwash procedure were changed, while the microbial community structure was studied using clone libraries and quantitative PCR targeting the 16S rRNA gene. When backwash frequency was increased from once per month to once per day, perchlorate removal initially deteriorated and then recovered, and the relative abundance of perchlorate-reducing bacteria (PRB) initially increased and then decreased. This apparent discrepancy suggested that bacterial populations other than PRB played an indirect role in perchlorate removal, likely by consuming dissolved oxygen, a competing electron acceptor. When backwash intensity was increased, the reactor gradually lost its ability to remove perchlorate, and concurrently the relative abundance of PRB decreased. The results indicated that changes in reactor operation had a profound impact on reactor performance through altering the microbial community structure. Backwashing is an important yet poorly characterized procedure when operating fixed-bed biofilm reactors. Compared to backwash intensity, changes in backwash frequency exerted less disturbance on the microbial community in the current study. If this finding can be confirmed in future work, backwash frequency may serve as the primary parameter when optimizing backwash procedures.