Diffusivity of oxygen in aerobic granules.

This work for the first time estimated apparent oxygen diffusivity (D(app)) of two types of aerobic granules, acetate-fed and phenol-fed, by probing the dissolved oxygen (DO) level at the granule center with a sudden change in the DO of the bulk liquid. With a high enough flow velocity across the granule to minimize the effects of external mass transfer resistance, the diffusivity coefficients of the two types of granules were estimated with reference to a one-dimensional diffusion model. The carbon source has a considerable effect on the granule diameter (d) and the oxygen diffusivity. The diffusivity coefficients were noted 1.24-2.28 x 10(-9) m2/s of 1.28-2.50 mm acetate-fed granules, and 2.50-7.65 x 10(-10) m2/s of 0.42-0.78 mm phenol-fed granules. Oxygen diffusivity declined with decreasing granule diameter, in particular, the diffusivity of acetate-fed granules is proportional to the size, whereas the diffusivity of phenol-fed granules is proportional to the square of granule diameter. The existence of large pores in granule, evidenced by FISH-CLSM imaging, was proposed to correspond to the noted size-dependent oxygen diffusivity. The phenol-fed granules exhibited a higher excellular polymer (ECP) content than the acetate-fed granules, hence yielding a lower oxygen diffusivity.

Startup of pilot-scale aerobic granular sludge reactor by stored granules.

The startup of a pilot-scale aerobic granular sludge reactor was investigated by seeding with 4-month stored aerobic granules. Two liters of granules were inoculated into the reactor (5.9% of reactor volume), which gave a biomass concentration of 1.03 g l(-1). Experimental results showed that seeding granules could be successfully maintained in the reactor. The microbial activity of seeding granules could be fully recovered to that of fresh granules after 2 days of operation, and new granules started to grow after day 5. Newly developed aerobic granules at stable period had similar size and morphology as seeding granules, and a biomass concentration of 6.0 g l(-1) was achieved in the reactor. The experiment demonstrated for the first time that stored aerobic granules could be used for easy and quick startup of aerobic granular sludge reactor.

Phosphate removal from the returned liquor of municipal wastewater treatment plant using iron-reducing bacteria.

AIM: The application of iron-reducing bacteria (IRB) to phosphate removal from returned liquor (liquid fraction after activated sludge digestion and anaerobic sludge dewatering) of municipal wastewater treatment plant (WWTP) was studied. METHODS AND RESULTS: An enrichment culture and two pure cultures of IRB, Stenotrophomonas maltophilia BK and Brachymonas denitrificans MK identified by 16S rRNA gene sequencing, were produced using returned liquor from a municipal WWTP as carbon and energy source, and iron hydroxide as oxidant. The final concentration of phosphate increased from 70 to 90 mg l(-1) in the control and decreased from 70 to 1 mg l(-1) in the experiment. The mass ratio of removed P to produced Fe(II) was 0.17 g P g(-1) Fe(II). The strain S. maltophilia BK showed the ability to reduce Fe(III) using such xenobiotics as diphenylamine, m-cresol, 2,4-dichlorphenol and p-phenylphenol as sole sources of carbon under anaerobic conditions. CONCLUSIONS: Bacterial reduction of ferric hydroxide enhanced the phosphate removal from the returned liquor. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the facultative anaerobes S. maltophilia BK and B. denitrificans MK to reduce Fe(III) was shown. These micro-organisms can be used for anaerobic removal of phosphate and xenobiotics by bacterial reduction of ferric ions.

Bacteriological examination of ballast water in Singapore Harbour by flow cytometry with FISH.

In this study the concentrations of total bacteria, enterobacteria, Vibrio spp., and E. coli have been compared for ballast water samples taken from ships in Singapore Harbour. The cell concentrations were enumerated using FISH and flow cytometry. The data were highly variable, reflecting the many influences upon ballast water as it is utilized in the shipping industry. The concentration of bacterial species was determined as a proportion of the total concentration of cells for the ballast water sampled. For the ballast water sampled these concentrations were 0.67-39.55% for eubacteria, 0-2.46% for enterobacteria, 0.18-35.82% for Vibrio spp., and 0-2.46% for E. coli. Using FISH and flow cytometry, an informative determination of the bacterial hazards of ship ballast water can be made.

Biotechnology of intensive aerobic conversion of sewage sludge and food waste into fertilizer.

Biotechnology for intensive aerobic bioconversion of sewage sludge and food waste into fertilizer was developed. The wastes were treated in a closed reactor under controlled aeration, stirring, pH, and temperature at 60 degrees C, after addition of starter bacterial culture Bacillus thermoamylovorans. The biodegradation of sewage sludge was studied by decrease of volatile solids (VS), content of organic carbon and autofluorescence of coenzyme F420. The degradation of anaerobic biomass was faster than biodegradation of total organic matter. The best fertilizer was obtained when sewage sludge was thermally pre-treated, mixed with food waste, chalk, and artificial bulking agent. The content of volatile solid and the content of organic carbon decreased at 24.8% and 13.5% of total solids, respectively, during ten days of bioconversion. The fertilizer was a powder with moisture content of 5%. It was stable, and not toxic for the germination of plant seeds. Addition of 1.0 to 1.5% of this fertilizer to the subsoil increased the growth of different plants tested by 113 to 164%. The biotechnology can be applied in larger scale for the recycling of sewage sludge and food wastes in Singapore.

Iron-mediated removal of ammonium from strong nitrogenous wastewater from food processing.

The combination of microbial reduction and further microbial oxidation of iron was applied to the treatment of food-processing wastewater and recovery of ammonium. Fe2+ ions were formed by iron-reducing bacteria under anaerobic conditions. Ammonium was recovered by co-precipitation with negatively charged iron hydroxides produced during oxidation of Fe2+ by iron-oxidizing bacteria under microaerophilic conditions. The value-added by-product of this process can be used as a slowly released ammonium fertilizer.

Microbiological monitoring in the biodegradation of sewage sludge and food waste.

AIM: To study the microbiology of intensive, in-vessel biodegradation of a mixture of sewage sludge and vegetable food waste. METHODS AND RESULTS: The biodegradation was performed in a closed reactor with the addition of a starter culture of Bacillus thermoamylovorans SW25 under conditions of controlled aeration, stirring, pH and temperature (60 degrees C). The content of viable bacterial cells, determined by flow cytometry, increased from 5 x 108 g-1 of dry matter to 61 x 108 g-1 for 6 days of the process and then dropped to the initial value at the end of the process. The reductions of organic matter, 16S rRNA of methanogens and coenzyme F420 fluorescence during 10 days of the treatment were 67, 54 and 87% of the initial values, respectively. The biodegradability of the organic matter decreased during the 10 days of the treatment from 3.8 to 1.3 mg CO2 g-1 of organic matter per day. The treatment of sewage sludge and food waste at 60 degrees C did not remove enterobacteria, which are the agents of intestinal infections, from the material. The percentage of viable enterobacterial cells, determined by fluorescent in situ hybridization (FISH) with Enterobacteriaceae-specific oligonucleotide probe and flow cytometry, varied from 1 to 14% of the viable bacterial cells. CONCLUSIONS: The mixture of sewage sludge and food waste can be degraded by the aerobic thermophilic bacteria; the starter culture of Bacillus thermoamylovorans SW25 can be used to perform this process; and enterobacteria can survive under treatment of sewage sludge and food waste at 60 degrees C for 13 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that FISH with an oligonucleotide probe can be used to study not only the growth but also the degradation of biomass. Obtained results could be used to design the bioconversion of sewage sludge and food waste into organic fertilizer.

The effect of hydraulic retention time on the stability of aerobically grown microbial granules.

AIMS: The aim of this study is to evaluate the effect of hydraulic retention time (HRT) on the development of aerobically grown microbial granules. METHODS AND RESULTS: Five column-shaped sequential aerobic sludge blanket reactors (SASBRs) were seeded with aerobically grown microbial granules and operated in a cyclic mode at different HRTs. At the shortest HRT of 1 h, the strong hydraulic pressure triggered biomass washout and led to reactor failure. At the longest HRT of 24 h, which represented the weakest hydraulic selection in this study, aerobic granules were gradually substituted by bioflocs because of the lower frequency of volumetric exchange. Within the optimum range of HRTs from 2 to 12 h, however, aerobic granules became stabilized in the presence of adequate hydraulic selection in the reactors, with good mixed liquor volatile suspended solids (MLVSS) retention, high volumetric chemical oxygen demand (COD) removal, low sludge volume index (SVI) values, good effluent quality, low sludge production rate, stronger and more compact structures, high cell hydrophobicity and high ratios of extracellular polysaccharides (PS) to extracellular proteins (PN). CONCLUSIONS: HRTs between 2 and 12 h provided the hydraulic selection pressures favourable for the formation and maintenance of stable aerobic granules with good settleability and activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first systematic study on the effect of HRT on heterotrophic aerobic granules. The results of the investigation are useful in understanding how aerobic granules can be applied for wastewater treatment.

Removal of micro-particles by microbial granules used for aerobic wastewater treatment.

Microbial granules with a diameter from 0.4 mm to 3.0 mm have been produced by fast sedimentation and retention of microbial aggregates in sequencing batch airlift reactors used for model wastewater treatment. The wastewater was with or without addition of calcium salt. The granules were able not only to degrade organic matter but to remove nano- and micro-particles from wastewater due to microchannels and pores in the matrix of the granules. To detect the removal of 0.1 microm, 0.6 pm, 4.2 microm fluorescent microspheres, and cells of Escherichia coli, stained by permeable nucleic acid stain SYTO9, the granules were incubated with these particles. The rate of particle removal and their accumulation in the granules was measured by a Fluoview300 confocal laser scanning microscope (CLSM) (Olympus, Japan); a FACSCalibur flow cytometer (Becton Dickinson, CA, USA), and a fluorescence spectrometer LS-50B (Perkin-Elmer, UK). The release or removal of biological and non-biological particles was analyzed by a flow cytometer after DNA staining. Total number of the particles bigger than 0.1 microm in the reactors was approximately 4 x 10(7) per ml, and 23% of these particles were bacterial cells. The 0.1 microm and 4.2. microm microbeads were accumulated within 250 microm in the upper layer of the microbial granule but externally added cells of Escherichia coli penetrated to the depth of approximately 800 microm in the granules without calcium addition. Microbial granules contained also attached ciliates but accumulation of the particles in protozoan cells was smaller than in the granule matrix. Kinetics of particle sorption was revealed by flow cytometry and fluorescence spectrometry. Almost half of the stained cells of E. coli can be removed by the granules for one hour. The ability of the microbial granules to remove the particles can enhance their function in aerobic treatment of wastewater.

Functional analysis of microbial community in phenol-degrading aerobic granules cultivated in SBR.

Phenol-degrading aerobic granules were cultivated in a sequencing batch reactor with an influent phenol concentration of 500 mg l(-1). Eight strains were isolated from aerobic granules to characterize the functional redundancy of the microbial community in the granules. The specific oxygen utilization kinetics show the eight strains possessed different phenol-degrading activities, with half-saturation constants (Ks) ranging from 0.4 to 70.5 mg phenol l(-1). Two isolates belonging to dominant populations expressed differing functions. The first strain was linked to the function of phenol degradation as this strain has the highest phenol-degrading ability among all isolates, while the second strain was linked to the maintenance of the granule structure because of its strong self-flocculation activity. This study could be used to exploit the granule-based system for treating high-strength wastewaters.

Changes in structure, activity and metabolism of aerobic granules as a microbial response to high phenol loading.

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m(-3) day(-1). The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m(-3) day(-1), and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m(-3) day(-1). Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m(-3) day(-1), and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.

The effect of organic loading rate on the aerobic granulation: the development of shear force theory.

The effect of organic loading rate (OLR) on aerobic granulation was studied by adopting three column-shaped, sequential aerobic sludge blanket reactors (SASBR). The reactors had been fed with laboratory prepared, synthetic dextrose-nutrient broth substrate. Experimental results showed clearly that the formation, characteristics and stability of aerobic granules had a close relationship with the strength of OLR applied. Aerobic granules appeared firstly under the OLR of 4 kg COD x (m3 x day)(-1). The system stabilization was demonstrated by its little-changed amount and morphology of granules. The characteristics of the stabilized granules were: 5.4 mm in mean diameter, 1.29 in roundness, 118 mg O2 x (mg VSS x hr)(-1) in SPOUR. The respective biomass SVI was 50 mL x (g MLVSS)(-1) and the averaged COD removal rate was 95%. Under the OLR of 8 kg COD x (m3 x day)(-1), granules appeared two days later than those for 4 kg COD x (m3 x day)(-1) and they always coexisted with flocs. The formed granule bed was not as compact as that under 4 kg COD x (m3 x day)(-1). There were no granules formed under the OLR of 1 kg COD x (m3 x day)(-1). Instead, flocs with rather loose structure dominated reactor mixed-liquor. The respective SVI's were 65 and 138 mL x (g MLVSS)(-1) under OLR of 8 and 1 kg COD x (m3 x day)(-1). It was proposed that the growth and maintenance of aerobic granules follow the shear force balance theory. Under the OLR of 4 kg COD x (m3 x day)(-1), a balance was reached between the aeration shear force and organic loading rate. Under this favored condition aerobic granules formed quickly and, became stabilized with the experimental parameters remained unchanged.

Monitoring of microbial diversity by fluorescence in situ hybridization and fluorescence spectrometry.

The goal of the research was the development of a simple method to quantify microbial groups in environmental samples. Fluorescence intensity was measured in the sample before and after whole cell fluorescence in situ hybridization with rRNA-targeted, fluorochrome-labeled oligonucleotide probes. To determine specific and non-specific binding of different oligonucleotide probes the following approaches have been used: (1) incubation of the sample with probes at two different temperatures; (2) hybridization of labeled probe in the presence of unlabeled probe; (3) incubation of the sample with labeled specific probe or labeled nonsense probe. Specific binding (hybridization) of the probe was calculated as the difference between total binding and non-specific binding of the probe. Specific binding was 40-50% of total binding in the environmental samples tested. The ratio of the specific binding of different probes may be used to quantify the ratio of different microbial groups in the environmental samples. This quantification is suitable for the microbiological monitoring of microbial aggregates because it is a simple technique and the results can be measured by a portable fluorometer.

Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment.

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.

Size-effect on the physical characteristics of the aerobic granule in a SBR.

Owing to a fast growth rate, aerobic granules display a wide range of sizes, approximately 0.3-5.0 mm in diameter. As the diameter increases, the aerobic granule undergoes serial morphological and physical changes that could cause problems to the reactor operation, a phenomenon which, however, has not been fully studied hitherto. In this study, aerobic granules from a sequencing batch reactor were mechanically separated into various size-categories in order to investigate their physical properties, including sludge volumetric index (SVI), settling velocity (sv), specific surface hydrophobicity, granule strength, total solids, percentage volatile solids and other structural properties. Also, the live and dead biomass distribution was examined under a confocal laser scanning microscope after treatment with nucleic acid viability stains. Regardless of size, the biomass (both live and dead) was densest in the outer layer of the granule, which was about 600+/-50 microm thick. The live cells appeared only in the peripheral zone, while dead biomass spread into the inner zone. The biomass distribution pattern justified the changing physical properties of the granules as they grew bigger. As size increased, the sv, granule total density and biomass density increased but not in parallel with the size increment, while the granule strength, specific surface hydrophobicity and SVI decreased. Nonetheless, beyond a threshold size (4.0 mm diameter), the granules presented peculiar values in those properties, deviating from the initial trends. This was due to both inner and outer structural changes. The physical properties associated significantly with the size factor, for which the correlation coefficients were above 0.67. In view of biological viability and physical properties, the operational size-range suggested for optimal performance and economically effective aerobic SBR granular sludge is a diameter of 1.0-3.0 mm.

Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments.

AIMS: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading bacterial strains from oil-contaminated tropical marine sediments. METHODS AND RESULTS: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon and energy. Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while Micrococcus sp. str. MN-006 can also grow on benzene. Staphylococcus sp. str. MN-005 can only degrade naphthalene and was not able to use the other aromatic hydrocarbons tested. Strain MN-003 possessed the highest maximal specific growth rate with naphthalene as sole carbon source. An enrichment culture fed with naphthalene as sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of incubation. The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial community. Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l-1 of naphthalene. CONCLUSIONS: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-contaminated tropical marine sediments. Gram-positive bacteria might play an important role in naphthalene degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments. Among the three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and outgrew the other two isolates in competition experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in the development of bioremediation schemes for oil-contaminated marine environments. Strain MN-003 could potentially be exploited in such schemes.

A culture-independent approach for studying microbial diversity in aerobic granules.

This study reports the use of ribosomal-based molecular techniques to study the microbial diversity in aerobic granules. Aerobic granules at different growth stages (young, mature and old) were obtained from a laboratory scale sequential aerobic sludge blanket (SASB) bioreactor fed with glucose as the main source of carbon and energy. Deoxyribonucleic acid (DNA) was extracted from the young, mature and old granules. The polymerase chain reaction (PCR) was used to amplify the Eubacterial 16S ribosomal DNA (rDNA) and three clone libraries were constructed, corresponding to each of the three growth stages. The microbial diversity in each clone library was assessed by amplified ribosomal DNA restriction analysis (ARDRA). The results reveal that there was considerable diversity in each clone library and there were variations in microbial diversity among the three different clone libraries. This suggests a shift in the composition of the microbial communities. Microorganisms associated with 5 restriction fragment length polymorphism (RFLP) types (A, B, C, D and E) appear to play an important role in the development of aerobic granules.

Isolation of naphthalene-degrading bacteria from tropical marine sediments.

Oil pollution is a major environmental concern in many countries, and this has led to a concerted effort in studying the feasibility of using oil-degrading bacteria for bioremediation. Although many oil-degrading bacteria have been isolated from different environments, environmental conditions can impose a selection pressure on the types of bacteria that can reside in a particular environment. This study reports the successful isolation of two indigenous naphthalene-degrading bacteria from oil-contaminated tropical marine sediments by enrichment culture. Strains MN-005 and MN-006 were characterized using an extensive range of biochemical tests. The 16S ribosomal deoxyribonucleic acid (rDNA) sequence analysis was also performed for the two strains. Their naphthalene degradation capabilities were determined using gas chromatography and DAPI counting of bacterial cells. Strains MN-005 and MN-006 are phenotypically and phylogenetically different from each other, and belong to the genera Staphylococcus and Micrococcus, respectively. Strains MN-005 and MN-006 had maximal specific growth rates (micro(max)) of 0.082 +/- 0.008 and 0.30 +/- 0.02 per hour, respectively, and half-saturation constants (K(s)) of 0.79 +/- 0.10 and 2.52 +/- 0.32 mg per litre, respectively. These physiological and growth studies are useful in assessing the potential of these indigenous isolates for in situ or ex situ naphthalene pollutant bioremediation in tropical marine environments.

Aggregation of immobilized activated sludge cells into aerobically grown microbial granules for the aerobic biodegradation of phenol.

AIMS: The aim of this study is to evaluate the utility of aerobically grown microbial granules for the biological treatment of phenol-containing wastewater. METHODS AND RESULTS: A column-type sequential aerobic sludge blanket reactor was inoculated with activated sludge and fed with phenol as the sole carbon source, at a rate of 1.5 g phenol l-1 d-1. Aerobically grown microbial granules first appeared on day 9 of reactor operation and quickly grew to displace the seed flocs as the dominant form of biomass in the reactor. These granules were compact and regular in appearance, and consisted of bacterial rods and cocci and fungi embedded in an extracellular polymeric matrix. The granules had a mean size of 0.52 mm, a sludge volume index of 40 ml g-1 and a specific oxygen utilization rate of 110 mg oxygen g VSS-1 h-1 (VSS stands for volatile suspended solids). Specific phenol degradation rates increased with phenol concentration from 0 to 500 mg phenol l-1, peaked at 1.4 g phenol g VSS-1 d-1, and declined with further increases in phenol concentration as substrate inhibition effects became important. CONCLUSIONS: Aerobically grown microbial granules were successfully cultivated in a reactor maintained at a loading rate of 1.5 g phenol l-1 d-1. The granules exhibited a high tolerance towards phenol. Significant rates of phenol degradation were attained at phenol concentrations as high as 2 g l-1. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the ability of aerobically grown microbial granules to degrade phenol. These granules appear to represent an excellent immobilization strategy for microorganisms to biologically remove phenol and other toxic chemicals in high-strength industrial wastewaters.

Presence of anaerobic bacteroides in aerobically grown microbial granules.

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 micro m were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 micro m below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.