Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Decolorization of textile azo dyes by aerobic bacterial consortium

The decolorization potential of two bacterial consortia developed from a textile wastewater treatment plant showed that among the two mixed bacterial culture SKB-II was the most efficient in decolorizing individual as well as mixture of dyes. At 1.3 g L^?1 starch supplementation in the basal medium by the end of 120 h decolorization of 80–96% of four out of the six individual azo dyes Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC (10 mg L^?1) was noted. The culture exhibited good potential ability in decolorizing 50–60% of all the dyes (Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC) when present as a mixture at 10 mg L^?1. The consortium SKB-II consisted of five different bacterial types identified by 16S rDNA sequence alignment as Bacillus vallismortis, Bacillus pumilus, Bacillus cereus, Bacillus subtilis and Bacillus megaterium which were further tested to decolorize dyes. The efficient ability of this developed consortium SKB-II to decolorize individual dyes and textile effluent using packed bed reactors is being carried out.     

Functional consortium for hydrogen production from cellobiose: Concentration-to-extinction approach

A functional bacterial consortium that can effectively hydrolyze cellobiose and produce bio-hydrogen was isolated by a concentration-to-extinction approach. The sludge from a cattle feedlot manure composting plant was incubated with 2.5–20 g l^?1 cellobiose at 35 °C and pH 6.0. The microbial diversity of serially concentrated suspensions significantly decreased following increasing cellobiose concentration, finally leaving only two viable strains, Clostridium butyricum strain W4 and Enterococcus saccharolyticus strain. This consortium has a maximum specific hydrogen production rate of 2.19 mol H₂ mol hexose^?1 at 5 g l^?1 cellobiose. The metabolic pathways shifted from ethanol-type to acetate-butyrate type as cellobiose concentration increased from 2.5 to >7 g l^?1. The concentration-to-extinction approach is effective for isolating functional consortium from natural microflora. In this case the functional strains of interest are more tolerant to the increased loadings of substrates than the non-functional strains.     

Methanol vapor biofiltration coupled with continuous production of recombinant endochitinase Ech42 by Pichia Pastoris

Methanol biofiltration using methylotrophic microorganisms has been previously reported by various authors. In a previous study, a modified strain of Pichia pastoris was tested for the ability to produce endochitinase (Ech42) when coupled with methanol vapor biodegradation in batch tests. The next challenge was to validate the process in a continuous system. Thus, in the present study, a biofilter packed with perlite and inoculated with P. pastoris transformed with the plasmid pPIC-ech42 was used for methanol vapor biofiltration and the continuous production of recombinant endochitinase (Ech42) for 60 days. The maximum elimination capacity (EC) of methanol obtained was 1320 g m^?3 h^?1 at a loading rate of 1465 g m^?3 h^?1. The extracellular protein production rate in the leachate was 2360 μg h^?1 with a chitinase enzymatic activity of 123 U L^?1. The protein content on the biofilm samples was negligible, indicating the effectiveness of the overall process and of P. pastoris to excrete proteins. The carbon balance indicated that 81% of the consumed methanol was mineralized and 5.8% was incorporated into biomass. The results of this study and the economic balance underscore the promising application of linking methanol vapor biofiltration to the continuous production of recombinant proteins.     

Removal of Cu(II) ions by biosorption onto powdered waste sludge (PWS) prior to biological treatment in an activated sludge unit: A statistical design approach

Biological treatment of synthetic wastewater containing Cu(II) ions was realized in an activated sludge unit with pre-adsorption of Cu(II) onto powdered waste sludge (PWS). Box-Behnken experimental design method was used to investigate Cu(II), chemical oxygen demand (COD) and toxicity removal performance of the activated sludge unit under different operating conditions. The independent variables were the solids retention time (SRT, 5–30 d), hydraulic residence time (HRT, 5–25 h), feed Cu(II) concentration (0–50 mg L^?1) and PWS loading rate (0–4 g h^?1) while percent Cu(II), COD, toxicity (TOX) removals and the sludge volume index (SVI) were the objective functions. The data were correlated with a quadratic response function (R² = 0.99). Cu(II), COD and toxicity removals increased with increasing PWS loading rate and SRT while decreasing with the increasing feed Cu(II) concentration and HRT. Optimum conditions resulting in maximum Cu(II), COD, toxicity removals and SVI values were found to be SRT of 30 d, HRT 15 h, PWS loading rate 3 g h^?1 and feed Cu(II) concentration of less than 30 mg L^?1.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

A stable immobilized d-psicose 3-epimerase for the production of d-psicose in the presence of borate

Maximal activity of the immobilized d-psicose 3-epimerase from Agrobacterium tumefaciens on Duolite A568 beads was achieved at pH 9.0 and 55 °C with borate, and at pH 8.5 and 50 °C without borate. The half-lives of the immobilized enzyme at 50 °C with and without borate were increased 4.2- and 128-fold compared to that of the free enzyme without borate, respectively. The immobilized enzyme with borate produced 441 g l^?1 psicose from 700 g l^?1 fructose at pH 9.0 and 55 °C, whereas 193 g l^?1 psicose was produced without borate at pH 8.5 and 50 °C after 120 min in a batch reaction. The immobilized enzyme in a packed-bed bioreactor without borate was produced continuously 325 g l^?1 psicose from 500 g l^?1 fructose at a dilution rate of 1.62 h^?1 over a 236 h period with productivity of 527 g l^?1 h^?1 while that without borate produced 146 g l^?1 psicose at 4.15 h^?1 over a 384-h period with productivity of 606 g l^?1 h^?1. The operational half-lives of the enzyme with and without borate in the bioreactor were 601 and 645 h, respectively. In the present study, psicose was produced stably with high productivity using the immobilized d-psicose 3-epimerase in the presence of borate.     

Detoxification of Indigo carmine using a combined treatment via a novel trimeric thermostable laccase and microbial consortium

For the first time, the investigation of Indigo carmine decolorization was done using an atypical Scytalidium thermophilum laccase. Crude and purified laccases required high temperatures and slight acidic pH to achieve maximum Indigo decolorization. Kinetic parameters (K_m and k_cat) of the homotrimeric laccase toward Indigo carmine were determined and laccase efficacy toward repeated dye decolorization process was studied. For the first time, 5 g l⁻¹ as initial Indigo carmine concentration were efficiently transformed up to 50% within 6 h of incubation using 0.1 U ml⁻¹ of laccase and without presence of any mediators. In this study, we showed that the atypical laccase transformed the indigoid dye structure, confirmed by the color changing from blue to red. This intermediate (red) was a subject to an efficient microbial consortium treatment monitored by measuring the decrease in optical density and the total organic carbon removal efficiencies. Toxicological studies via micro-toxicity test showed that the released enzymatic and adapted consortium degradation products were both non-toxic while the initial product was toxic.     

Silica-supported fluoroboric acid (HBF4–SiO2) catalyzed highly productive synthesis of thiomorpholides as activators of l-asparaginase as well as the antioxidant agent

An efficient solvent-free procedure for the synthesis of thiomorpholides in the presence of a catalytic amount of solid-supported fluoroboric acid (HBF₄–SiO₂) is described. The advantages of this method are high yields, short reaction times, ease of product isolation, low cost, and the catalyst can be recycled for a number of times without significant loss of activity. Three thiomorpholides possessing electron-donating group (4c, 4g, and 4h) were exhibiting excellent stimulatory activities against Erwinia carotovora l-asparaginase. The most potent activator, compound 4h displayed the following kinetic parameters, K_m = 75 μM and V_max = 1000 μmol mg^?1 min^?1 and K_A = 0.985 μM. Furthermore, these compounds (4g, 4h, 4c, 4f, 4a, and 4d) have also shown promising 2,2′-diphenyl-1-picrylhydrazyl (DPPH) reducing antioxidant activity (21–36%) at 1 mM concentration as compared to standard butylated hydroxyl anisole (72% at 1 mM).     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

Performance of a composite membrane bioreactor treating toluene vapors: Inocula selection,reactor performance and behavior under transient conditions

In this study, a membrane biofilm reactor performance for toluene as a model pollutant is presented. A composite membrane consisting of a porous polyacrylonitrile (PAN) support layer coated with a very thin (0.3 μm) dense polydimethylsiloxane (PDMS) top layer was used. Batch experiments were performed to select an appropriate inocula (slaughterhouse wastewater treatment sludge with a specific toluene consumption rate of 118 ± 23 μg g^?1 VSS L^?1) among the three available sources of inoculums. The maximum elimination capacity gas-side reactor volume based (EC)_v and membrane based (EC)_{m, max} obtained were 609 g m^?3 h^?1 and 1.2 g m^?2 h^?1 respectively, which is much higher than other membrane bioreactors. Further experiments involved the study of the membrane biofilm reactor flexibility when operational parameters as temperature, loading rate etc. were modified. In all cases, the membrane biofilm reactor showed a rapid adaptation and new steady-states were obtained within hours. Overall, the results illustrate that membrane bioreactors can potentially be a good option for treatment of air pollutants such as toluene.     

Bioprocess strategies for mass multiplication of and metabolite synthesis by plant growth promoting pseudomonads for agronomical applications

The exponential substrate feeding (open-loop) and automated feedback substrate feeding (closed loop) strategies were developed to obtain high cell densities of fluorescent pseudomonad strains R62 and R81 and enhanced production of antifungal compound 2,4-diacetylphloroglucinol (DAPG) from glycerol as a sole carbon source. The exponential feeding strategy resulted in increased glycerol accumulation during the fed-batch cultivation when the predetermined specific growth rate (μ) was set at 0.10 or 0.20 h^?1 (<μ_m = 0.29 h^?1). Automated feeding strategies using dissolved oxygen (DO) or pH as feedback signals resulted in minimal to zero accumulation of glycerol for both the strains. In case of DO-based feeding strategy, biomass productivity of 0.24 g/(L h) and 0.29 g/(L h) was obtained for R62 and R81, respectively. Using pH-based feeding strategy, biomass productivity could be increased to a maximum of 0.51 and 0.54 g/(L h), for the strains R62 and R81, respectively, whereas the DAPG concentration was enhanced to 298 mg/L for R62 and 342 mg/L for R81 strains. These yields of DAPG are thus far the highest reported from GRAS organisms.     

Optimization of Cd2+ removal by the cyanobacterium Synechocystis pevalekii using the response surface methodology

Response surface methodology (RSM) has been used to optimize the critical parameters responsible for higher Cd²⁺ removal by a unicellular cyanobacterium Synechocystis pevalekii. A three-level Box–Behnken factorial design was used to optimize pH, biomass and metal concentration for Cd²⁺ removal. A coefficient of determination (R²) value (0.99), model F-value (86.40) and its low p-value (F < 0.0001) along with lower value of coefficient of variation (5.61%) indicated the fitness of response surface quadratic model during the present study. At optimum pH (6.48), biomass concentration (0.25 mg protein ml^?1) and metal concentration (5 μg ml^?1) the model predicted 4.29 μg ml^?1 Cd²⁺ removal and experimentally, 4.27 μg ml^?1 Cd²⁺ removal was obtained.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

New integrated bioprocess for the continuous production,extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor

Recently, a bubbleless membrane bioreactor (BMBR) has been successfully developed for biosurfactant production by Bacillus subtilis [1]. In this study, for the first time, continuous culture were carried out for the production of surfactin in a BMBR, both with or without a coupled microfiltration membrane. Results from continuous culture showed that a significant part of biomass was immobilized onto the air/liquid membrane contactor. Immobilized biomass activity onto the air/liquid membrane contactor was monitored using a respirometric analysis. Kinetics of growth, surfactin and primary metabolites production were investigated. Planktonic biomass, immobilized biomass and surfactin production and productivity obtained in batch culture (3 L) of 1.5 days of culture were 4.5 g DW, 1.3 g DW, 1.8 g and 17.4 mg L^?1 h^?1, respectively. In continuous culture without total cell recycling (TCR), the planktonic biomass was leached, but immobilized biomass reached a steady state at an estimated 6.6 g DW. 11.5 g of surfactin was produced after 3 days of culture, this gave an average surfactin productivity of 54.7 mg L^?1 h^?1 for the continuous culture, which presented a surfactin productivity of 30 mg L^?1 h^?1 at the steady state. TCR was then investigated for the continuous production, extraction and purification of surfactin using a coupled ultrafiltration step. In continuous culture with TCR at a dilution rate of 0.1 h^?1, planktonic biomass, immobilized biomass, surfactin production and productivity reached 7.5 g DW, 5.5 g DW, 7.1 g and 41.6 mg L^?1 h^?1 respectively, after 2 days of culture. After this time, biomass and surfactin productions stopped. Increasing dilution rate to 0.2 h^?1 led to the resumption of biomass and surfactin production and these values reached 11.1 g DW, 10.5 g DW, 7.9 g and 110.1 mg L^?1 h^?1, respectively, after 3 days of culture. This study has therefore shown that with this new integrated bioprocess, it was possible to continuously extract and purify several grams of biosurfactant, with purity up to 95%.     

Treatment of high-strength wastewater in tropical vertical flow constructed wetlands planted with Typha angustifolia and Cyperus involucratus

The ability of vertical flow (VF) constructed wetland systems to treat high-strength (ca. 300 mg L^?1 of COD and ca. 300 mg L^?1 total-nitrogen) wastewater under tropical climatic conditions was studied during a 5-month period. Nine 0.8-m diameter experimental VF units (depth 0.6 m) were used: three units were planted with Typha angustifolia L., another three units were planted with Cyperus involucratus Rottb and three units were unplanted. Each set of units were operated at hydraulic loading rates (HLRs) of 20, 50 and 80 mm d^?1. Cyperus produced more shoots and biomass than the Typha, which was probably stressed because of lack of water. The high evapotranspirative water loss from the Cyperus systems resulted in higher effluent concentrations of COD and total-P, but the mass removal of COD did not differ significantly between planted and unplanted systems. Average mass removal rates of COD, TKN and total-P at a HLR of 80 mm d^?1 were 17.8, 15.4 and 0.69 g m^?2 d^?1. The first-order removal rate constants at a HLR of 80 mm d^?1 for COD, TKN and total-P were 49.8, 30.1 and 13.5 m year^?1, respectively, which is in the higher range of k-values reported in the literature. The oxygen transfer rates were ca. 80 g m^?2 d^?1 in the planted systems as opposed to ca. 60 g m^?2 d^?1 in the unplanted systems. The number of Nitrosomonas was two to three orders of magnitude higher in the planted systems compared to the unplanted systems. Planted systems thus had significantly higher removal rates of nitrogen and phosphorus, higher oxygen transfer rates, and higher quantities of ammonia-oxidizing bacteria. None of the systems did, however, fully nitrify the wastewater, even at low loading rates. The vertical filters did not provide sufficient contact time between the wastewater and the biofilm on the gravel medium of the filters probably because of the shallow bed depth (0.6 m) and the coarse texture of the gravel. It is concluded that vertical flow constructed wetland systems have a high capacity to treat high-strength wastewater in tropical climates. The gravel and sand matrix of the vertical filter must, however, be designed in a way so that the pulse-loaded wastewater can pass through the filter medium at a speed that will allow the water to drain before the next dose arrives whilst at the same time holding the water back long enough to allow sufficient contact with the biofilm on the filter medium.     

Catalytic removal of sulfide by an immobilized sulfide-oxidase bioreactor

A bioreactor packed with chitosan immobilized sulfide-oxidase from Streptomyces species LD048 was developed to treat a liquid stream of sulfide. The inoculation system was composed of glass with a 0.7 L working volume and enzyme activity of 2 mmol S g^?1 carrier. The sulfide removal efficiency was almost 100% when the volumetric loading was increased up to 3.9 mmol S L^?1 h^?1 at a space velocity of 18 h^?1. The maximal elimination capacity was 22.1 mmol S L^?1 h^?1 with a space velocity of 72 h^?1. When the aeration was increased from 0.05 to 0.1 L min^?1, the average removal efficiency improved from 81% to 94%. A removal efficiency of 90% was obtained after 15 days of operation with a load rate of 8.9 mmol S L^?1 h^?1 and a space velocity of 14.28 h^?1. An operational equation based on the ideal plug flow bioreactor and the Michaelis–Menten model predicted the performance of this bioreactor.     

Immune responses,ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254

The toxic effects of Aroclor 1254 (0.05, 0.5, 5 and 50 μg l^?1) on scallop (Chlamys farreri) immune system in vivo were studied. The results showed that Aroclor 1254 had significant toxic effect on the parameters tested in this paper (P < 0.05). The total number of haemocytes, the proportion of granulocytes, phagocytosis in all groups as well as the lysosomal membrane stability (LMS) in 5, 50 μg l^?1 and bacteriolytic activity 0.5, 5, 50 μg l^?1 treatments decreased significantly, while the proportion of hyalinocytes and the production of O₂^- in all treatments remarkably increased during the sampling time and tended to be stable gradually after 6–15 d. The bacteriolytic activity in 0.05 μg l^?1 treatments, LMS in 0.05, 0.5 μg l^?1 groups and the DNA damage (comet ratios and arbitrary values) in all treatments increased at the beginning of exposure and reached their peaks on day 1, day 1, day 6 and day 3, following that they all decreased gradually and became stable after 9–15 d. When the indices reached stability, except for DNA damage was higher than controls, the others were all significantly lower than those of controls (P < 0.05). Thus, Aroclor 1254 has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms. Also it supports the speculation that the PCBs pollution is one of the important reasons of the mass mortality of the C. farreri.     

Aerobic granulation in sequencing batch reactors with different reactor height/diameter ratios

Effects of reactor height/diameter ratios ranged from 24 to 4 corresponding to reactor settling velocities from 12 to 2 m h^?1 on aerobic granulation were investigated. It was found that granules appeared after 1-week operation and granule volume percentages exceeded 50% after 2–3 weeks in four reactors. In addition, similar granule fraction of 94–96% was found at steady state in all four reactors. Sludge volume index (SVI), average sludge size, biomass density and granule settling velocity at steady state were around 50 ml g^?1, 1800 μm, 53 g l^?1 and 40 m h^?1, respectively, in four reactors. Extracellular polymeric substances (EPS) and specific oxygen uptake rate (SOUR) were around 38 mg g^?1 VSS and 40 mg O₂ g^?1 VSS h^?1, respectively. Denaturing gradient gel electrophoresis (DGGE) fingerprint of sludge in four reactors showed the same microbial population shift during the start-up period and same microbial community structure during steady-state period. These results recommended strongly that reactor height/diameter ratio or reactor setting velocity in the used range in this study did not affect granule formation, physical characteristics, microbial community structure of granules and stable operation of granular sludge reactor. Reactor height/diameter ratio thus can be very flexible in the practice, which is important for the application of aerobic granule technology.