c-di-GMP对大肠杆菌生物膜调控的研究进展

大肠杆菌生物膜是由聚集于特定介质上的大肠杆菌菌体细胞相互黏附并分泌胞外基质聚合物(extracellular polymeric substances,EPS)而产生的一种结构复杂的膜状聚集物。感染宿主后的致病性大肠杆菌在形成生物膜后会极大地逃避免疫系统以及环境中各种有害因素对其的影响,对宿主造成持续甚至致命的伤害。环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)是广泛存在于细菌中的第二信使,在调节生物膜形成过程中起到至关重要的作用。基于此,本文对近些年来有关c-di-GMP对大肠杆菌生物膜形成过程中菌体的运动、黏附以及EPS产生机制的研究进行了综述,以期为从c-di-GMP角度抑制大肠杆菌生物膜提供依据和思路。     

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.     

Potential coupling effects of ammonia-oxidizing and anaerobic ammonium-oxidizing bacteria on completely autotrophic nitrogen removal over nitrite biofilm formation induced by the second messenger cyclic diguanylate

The objective of this study was to investigate the influence of extracellular polymeric substance (EPS) on the coupling effects between ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria for the completely autotrophic nitrogen removal over nitrite (CANON) biofilm formation in a moving bed biofilm reactor (MBBR). Analysis of the quantity of EPS and cyclic diguanylate (c-di-GMP) confirmed that the contents of polysaccharides and c-di-GMP were correlated in the AOB sludge, anammox sludge, and CANON biofilm. The anammox sludge secreted more EPS (especially polysaccharides) than AOB with a markedly higher c-di-GMP content, which could be used by the bacteria to regulate the synthesis of exopolysaccharides that are ultimately used as a fixation matrix, for the adhesion of biomass. Indeed, increased intracellular c-di-GMP concentrations in the anammox sludge enhanced the regulation of polysaccharides to promote the adhesion of AOB and formation of the CANON biofilm. Overall, the results of this study provide new comprehensive information regarding the coupling effects of AOB and anammox bacteria for the nitrogen removal process.

     

Effect of organic carbon shock loading on endogenous denitrification in sequential batch reactors

This work was focused on the performance evaluation of sequential batch reactors (SBR) treating sewage, through a process of endogenous biological denitrification. Different operational conditions were carried out, and the behaviour under the effects of organic shock loading was examined. Three laboratory scale reactors were operated simultaneously and fed with similar wastewater. The substratum was molasses and nitrate, as carbon and nitrogen sources, respectively. The three reactors were operated during different aeration periods (0, 15 and 30 min). Sudden changes (shock loading) in organic matter concentration were performed during the experiment. Thus, influent load was quickly increased threefold in relation to the original concentration. Results indicated that SBR reactors withstand adequately moderate shock loading. With regard to substratum degradation, nitrate elimination achieved was approximately 80%, while denitrification rate was approximately 0.87 mgg(-1)h(-1).     

Stability in a Denitrifying Fluidized Bed Reactor

This study evaluates changes in the microbial community structure and function of a pilot-scale denitrifying fluidized bed reactor during periods of constant operating conditions and periods of perturbation. The perturbations consisted of a shutdown period without feed, two disturbances in which biofilms were mechanically sheared from carrier particles, and a twofold step increase in feed nitrate concentration. In the absence of perturbations, nitrate removal was stable and consistently greater than 99%. The structure and dynamics of the microbial community were studied using cloning and sequencing techniques and terminal restriction fragment length polymorphism (T-RFLP) of the SSU rRNA gene. Under unperturbed operating conditions, stable function was accompanied by high constancy and low variability of community structure with the majority of terminal restriction fragments (T-RFs) appearing throughout operation at consistent relative abundances. Several of the consistently present T-RFs correlated with clone sequences closely related to Acidovorax (98% similarity), Dechloromonas (99% similarity), and Zoogloea (98% similarity), genera recently identified by molecular analyses of similar systems. Significant changes in community structure and function were not observed after the shutdown period. In contrast, following the increase in loading rate and the mechanical disturbances, new T-RFs appeared. After both mechanical disturbances, function and community structure recovered. However, function was much more resilient than community structure. The similarity of response to the mechanical disturbances despite differences in community structure and operating conditions suggests that flexible community structure and potentially the activity of minor members under nonperturbation conditions promotes system recovery.     

The Synergistic Effects of High Nitrate Concentrations on Sediment Bioreduction

Groundwaters at nuclear sites can be characterized by low pH and high nitrate concentrations (10–100 mM). These conditions are challenging for bioremediation, often inhibiting microbial Fe(III)-reduction which can limit radionuclide migration. Here, sediment microcosms representative of the UK Sellafield site were used to study the influence of variable pH and nitrate concentrations on microbially-mediated TEAP (terminal electron accepting processes) progression. The rate of reduction through the terminal electron accepting cascade NO^? ₃ > NO^? ₂ > Mn(IV)/Fe(III) > SO^2? ₄ at low pH (～5.5) was slower than that in bicarbonate buffered systems (pH ～ 7.0), but in the low pH systems, denitrification and associated pH buffering resulted in conditioning of the sediments for subsequent Fe(III) and sulfate-reduction. Under very high nitrate conditions (100 mM), bicarbonate buffering (pH ～ 7.0) was necessary for TEAP progression beyond denitrification and the reduction of 100 mM nitrate created alkaline conditions (pH 9.5). 16S rRNA gene analysis showed that close relatives of known nitrate reducers Bacillus niacini and Ochrobactrum grignonense dominated the microbial communities in this reduced sediment. In Fe(III)-reducing enrichment cultures from the 100 mM nitrate system, close relatives of the Fe(III)-reducing species Alkaliphilus crotonatoxidans and Serratia liquifaciens were observed. These results highlight that under certain conditions and contrary to expectations, denitrification may support bioreduction via pH conditioning for optimal metal reduction and radionuclide immobilization.     

Influence of microbial interactions and EPS/polysaccharide composition on nutrient removal activity in biofilms formed by strains found in wastewater treatment systems

The study of biofilm function, structure and microbial interactions might help to improve our understanding of biofilm wastewater treatment processes. However, few reports specifically address the influence of interactions within multispecies biofilms on microbial activity and biofilm composition. Thus, the relationship between biofilm formation, denitrification activity, phosphorus removal and the composition of extracellular polymeric substances (EPS), exopolysaccharides and the bacterial community was investigated using biofilms of denitrifying and phosphorus removing strains Comamonas denitrificans 110, Brachymonas denitrificans B79, Aeromonas hydrophila L6 and Acinetobacter calcoaceticus ATCC23055. Denitrification activity within the biofilms generally increased with the amount of biofilm while phosphorus removal depended on bacterial growth rate. Synergistic effects of co-growth on denitrification (B. denitrificans B79 and A. hydrophila L6) and phosphorus removal (C. denitrificans 110 with either A. calcoaceticus or A. hydrophila L6) were observed. B. denitrificans B79 was highly affected by interspecies interactions with respect to biofilm formation, denitrification activity and EPS composition, while C. denitrificans 110 remained largely unaffected. In some of the dual and quadruple strain biofilms new exopolysaccharide monomers were detected which were not present in the pure strain samples.     

Characteristics of extracellular polymeric substances from sludge and biofilm in a simultaneous nitrification and denitrification system under high salinity stress

The composition and distribution of extracellular polymeric substance (EPS) both from suspended sludge and attached biofilm were investigated in a simultaneous nitrification and denitrification (SND) system with the increase of the salinity from 1.0 to 3.0 %. Fourier-transform infrared (FTIR) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy were used to examine proteins (PN), polysaccharides (PS) and humic substances (HS) present in EPS. High total nitrogen removal (above 83.9 %) via SND was obtained in the salinity range of 1.0–2.5 %. Total EPS in the sludge increased from 150.2 to 200.6 mg/gVSS with the increase of salinity from 1.0 to 3.0 %, whereas the corresponding values in the biofilm achieved the maximum of 288.6 mg/g VSS at 2.0 % salinity. Dominant composition of EPS was detected as HS in both sludge and biofilm, having the percentages of 50.6–68.6 and 41.1–69.9 % in total EPS, respectively. Both PN and PS contents in soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) of sludge and biofilm increased with the increased salinity. The FTIR spectrum and 3D-EEM fluorescence spectroscopy of S-EPS, LB-EPS and TB-EPS in the sludge and biofilm showed the changes of functional groups and conformations of the compositions in EPS with the increase of salinity. The results demonstrated that the characteristics of EPS varied from sludge to biofilm. The obtained results could provide a better understanding of the salinity effect on the EPS characteristics in a SND system.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

The effect of seasonality upon the development of lotic biofilms and microbial biostabilisation

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Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

Benthic organic carbon influences denitrification in streams with high nitrate concentration

1. Anthropogenic activities have increased reactive nitrogen availability, and now many streams carry large nitrate loads to coastal ecosystems. Denitrification is potentially an important nitrogen sink, but few studies have investigated the influence of benthic organic carbon on denitrification in nitrate‐rich streams. 2. Using the acetylene‐block assay, we measured denitrification rates associated with benthic substrata having different proportions of organic matter in agricultural streams in two states in the mid‐west of the U.S.A., Illinois and Michigan. 3. In Illinois, benthic organic matter varied little between seasons (5.9–7.0% of stream sediment), but nitrate concentrations were high in summer (>10 mg N L⁻¹) and low (<0.5 mg N L⁻¹) in autumn. Across all seasons and streams, the rate of denitrification ranged from 0.01 to 4.77 μg N g⁻¹ DM h⁻¹ and was positively related to stream‐water nitrate concentration. Within each stream, denitrification was positively related to benthic organic matter only when nitrate concentration exceeded published half‐saturation constants. 4. In Michigan, streams had high nitrate concentrations and diverse benthic substrata which varied from 0.7 to 72.7% organic matter. Denitrification rate ranged from 0.12 to 11.06 μg N g⁻¹ DM h⁻¹ and was positively related to the proportion of organic matter in each substratum. 5. Taken together, these results indicate that benthic organic carbon may play an important role in stream nitrogen cycling by stimulating denitrification when nitrate concentrations are high.     

Using modern low-oxygen marine ecosystems to understand the nitrogen cycle of the Paleo- and Mesoproterozoic oceans

During the productive Paleoproterozoic (2.4–1.8 Ga) and less productive Mesoproterozoic (1.8–1.0 Ga), the ocean was suboxic to anoxic and multicellular organisms had not yet evolved. Here, we link geologic information about the Proterozoic ocean to microbial processes in modern low-oxygen systems. High iron concentrations and rates of Fe cycling in the Proterozoic are the largest differences from modern oxygen-deficient zones. In anoxic waters, which composed most of the Paleoproterozoic and ~40% of the Mesoproterozoic ocean, nitrogen cycling dominated. Rates of N₂ production by denitrification and anammox were likely linked to sinking organic matter fluxes and in situ primary productivity under anoxic conditions. Additionally autotrophic denitrifiers could have used reduced iron or methane. 50% of the Mesoproterozoic ocean may have been suboxic, promoting nitrification and metal oxidation in the suboxic water and N₂O and N₂ production by partial and complete denitrification in anoxic zones in organic aggregates. Sulfidic conditions may have composed ~10% of the Mesoproterozoic ocean focused along continental margins. Due to low nitrate concentrations in offshore regions, anammox bacteria likely dominated N₂ production immediately above sulfidic zones, but in coastal regions, higher nitrate concentrations probably promoted complete S-oxidizing autotrophic denitrification at the sulfide interface.     

Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.     

Microbial nitrogen removal of ammonia wastewater in poly (butylenes succinate)-based constructed wetland: effect of dissolved oxygen

Constructed wetland (CW) is popular in wastewater treatment for its prominent advantage of low construction and operation cost. However, the nitrogen removal in conventional CW is usually limited by the low dissolved oxygen (DO) and insufficient electron donor. This paper investigated the nitrogen removal performance and mechanisms in the poly (butylenes succinate)-based CW (PBS-CW) while treating ammonia wastewater under different DO levels. The average DO contents in limited-aeration and full-aeration phases were 1.68 mg L⁻¹ and 5.71 mg L⁻¹, respectively. Results indicated that, with the ammonia nitrogen loading rate of 25 g N m⁻³ day⁻¹, total nitrogen removal ratios in the PBS-CW under the limited-aeration and full-aeration phases were 72% and 99%, respectively. Combined analyses revealed that simultaneous nitrification and denitrification (SND) via nitrite/nitrate were the main microbial nitrogen removal pathways in the aeration phase of the PBS-CW (> 89%). The microbial carrier of biodegradable material was believed to play a significant role in prompting SND performance while dealing with low C/N wastewater. Due to the coexistence of micro-anaerobic zone and carbon supply inside the coated biofilm, the high DO level in the PBS-CW increased the abundance of the nitrifying bacteria (amoA and nxrA), denitrifying bacteria (narG, nirK, nirS, and nosZ), and even anammox bacteria (anammox 16s rRNA). These features are beneficial to many microbial processes which require the simultaneous aerobic, anoxic, and anaerobic environment.

     

Influences of extracellular polymeric substances (EPS) on the characteristics of activated sludge under non-steady-state conditions

Laboratory experiments were carried out on activated sludge (AS) to investigate the correlations between the content of extracellular polymeric substances (EPS) and the performance of biosolids–water separation, including sludge flocculation, sedimentation, compression, and dewatering, under non-steady-state conditions. On three stabilized AS reactors changes were made in sludge retention time (SRT), substrate composition, and loading rate, respectively, to bring about unstable operation to the reactors. A two-step heating method was used to extract from the sludge the easily extractable EPS, or loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS), respectively. The experimental results demonstrate dynamic changes in sludge characteristic and EPS production under the non-steady-state conditions. During the early phase of transition after a change was imposed, the sludge became generally worse in flocculation, compressibility, and dewaterability. With the acclimatization of the biomass to the new process conditions, biosolids–water separation showed a general trend of improvement. Changes in AS process condition also resulted in considerable variations in EPS production. The change of the LB-EPS content appeared to be more significant than that of the TB-EPS. Throughout the non-steady-state operation, the sludge flocculating behavior, settleability, compressibility, and dewaterability had a positive correlation with the LB-EPS content; however, no correlation could be found between these properties and the TB-EPS content. The results suggest that although EPS is essential to biofloc formation, excessive EPS in the form of LB-EPS would weaken cell attachment and deteriorate the AS floc structure, resulting in poor biosolids–water separation.     

Perspectives and predictions on the microbial ecology of the hyporheic zone

1. Studies of hyporheic microbial ecology have suggested an important role for hyporheic microbial processes in stream ecosystem functioning. Using evidence from microbial communities in other aquatic habitats, some predictions are made concerning the diversity of microbial types and microbial processes likely to occur in the hyporheic zone, and the relative importance of these various types to the hyporheic ecosystem. 2. It is predicted that the biofilm growth form of interstitial micro-organisms will create a variety of microniches, allowing coexistence of a great diversity of microbial types, and promoting the activity of some otherwise poor competitors. It is further predicted that the confluence of reduced groundwaters and aerobic surface waters will favour chemolithotrophic processes in the hyporheic zone, but that these will contribute significantly to hyporheic production only if surface water is very low in dissolved organic carbon, or the groundwater is extremely reduced, such as by the influence of riparian wetlands. A variety of anaerobic respiratory pathways, such as nitrate, ferric ion, sulphate and even methanogenic respiration will be employed in the hyporheic zone, with biofilm dynamics permitting these to occur even in aerobic sediments. Anaerobic pathways may account for a significant proportion of total hyporheic organic matter mineralization. 3. The role of fungi in hyporheic dynamics is, as yet, almost completely unstudied. However, it is expected that they will be important in breaking down buried particulate organic matter (POM), which may account for a large proportion of total stream POM. 4. Physicochemical conditions in hyporheic sediments appear to be highly heterogeneous, and this heterogeneity may be very important in the cycling of certain nutrients, especially nitrogen, which involves a series of steps requiring different conditions. 5. Various new techniques are now available by which biofilm dynamics and in situ microbial processes may be measured. Studies are recommended of intact microbial communities both at the microscale of the biofilm and at the scale of the heterogeneities occurring in hyporheic sediments. Studies are needed that measure actual rates of microbial processes under in situ conditions.     

Characterization of microbial community and kinetics for spent sulfidic caustic applied autotrophic denitrification

Spent sulfidic caustic was applied to sulfur utilizing autotrophic denitrification as the simultaneous source of electron donor and alkalinity. The two experiment set-up of upflow anoxic hybrid growth reactor (UAHGR) and upflow anoxic suspended growth reactor (UASGR) was adopted and nitrate removals were similar in both reactors. Approximately 90% of the initial nitrate was denitrified at nitrate loading rate of 0.15∼0.40 kgNO₃ ⁻/m³·d. The experimental stoichiometric ratio of sulfate production to nitrate removal was ranged from 1.5 to 2.1 mgSO₄ ²⁻/mgNO₃ ⁻. During the operation period, denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments for the sludge sample of both reactors showed the change of microbial communities. Thiobacillus denitrificans-like microorganism occupied 28.5% (18 clones) of the 63 clones by cloning the PCR products from the sludge sample of UAHGR. Acidovorax avenae, which can reduce nitrate to nitrogen gas while oxidizing phenol (heterotrophic denitrifier), was also found in 7 clones (11.1%). Although an organic carbon source was not added to the medium, a microorganism (Kaistella koreensis) capable of oxidizing organic compounds was found in 7 clones (11.1%). Therefore, the microbial community of spent sulfidic caustic applied autotrophic denitrification process well corresponds to the substrate components of spent sulfidic caustic. Through the batch cultivation of microorganisms in UAHGR, the microbial kinetic coefficients of spent sulfidic caustic applied autotrophic denitrification were estimated to be μ _max = 0.097 h⁻¹, k _d = 0.0021 h⁻¹, K _s = 200 mgNO₃ ⁻/L, and Y = 0.31 mgMLVSS/mgNO₃ ⁻.     

Interpreting the effect of increasing COD loading rates on the performance of a pre-anoxic MBBR system: implications on the attached and suspended biomass dynamics and nitrification–denitrification activity

A pre-anoxic MBBR system was subjected to increasing organic loading rates up to 18 gCOD/(m² day). At 3 gCOD/(m² day), most of the incoming organic matter was removed via denitrification. However, at higher loads, anoxic COD removal became limited by the nitrite/nitrate supply from the aerobic reactor, which assumed an important role in this conversion. Despite the application of low dissolved oxygen (DO) levels (<2 mg/L) in this tank, nitrification was observed to be nearly complete until 8 gCOD/(m² day). As the organic input was increased, the maximum specific nitrifying activity gradually declined. Activity tests suggested that an oxygen-limited environment was established in the biofilm. At lower loads [3–8 gCOD/(m² day)], the nitrification product obtained was affected by the DO concentration, whereas from 16 to 21 gCOD/(m² day), nitrite/nitrate profiles were likely associated with microbial stratification in the biofilm. The results also indicated that the role of the suspended biomass in the overall nitrification and denitrification can be very significant in high loaded MBBRs and should not be neglected, even at low HRTs.