Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.     

Effect of COD/N ratio on cultivation of aerobic granular sludge in a pilot-scale sequencing batch reactor

Aerobic granular sludge was successfully cultivated with the effluent of internal circulation reactor in a pilot-scale sequencing batch reactor (SBR). Soy protein wastewater was used as an external carbon source for altering the influent chemical oxygen demand/nitrogen (COD/N) ratios of SBR. Initially, the phenomenon of partial nitrification was observed and depressed by increasing the influent COD/N ratios from 3.32 to 7.24 mg/mg. After 90 days of aerobic granulation, the mixed liquor suspended solids concentration of the reactor increased from 2.80 to 7.02 g/L, while the sludge volumetric index decreased from 105.51 to 42.99 mL/g. The diameters of mature aerobic granules vary in the range of 1.2 to 2.0 mm. The reactor showed excellent removal performances for COD and $ {\text{NH}}_4^{ + }{\text{ - N}} $ after aerobic granulation, and average removal efficiencies were over 93% and 98%, respectively. The result of this study could provide further information on the development of aerobic granule-based system for full-scale applications.     

Calcium accumulation characterization in the aerobic granules cultivated in a continuous-flow airlift bioreactor

Limited work has been done on the accumulation characterization of Ca²⁺ in aerobic granules that are cultivated in a continuous-flow bioreactor. In this work, the contribution of Ca²⁺ to the biogranulation in a continuous flow airlift fluidized bed (CAFB) reactor has been studied. The spatial distribution and form of calcium in the granules were investigated by scanning electron microscopy-mapping, energy dispersive X-ray and X-ray diffraction (XRD). Calcium was located throughout the Ca-rich granules, rather than accumulating in the center of the granules of the sequencing batch reactor. Furthermore, CaCO₃ was detected as the main crystalline mineral form of the calcium. Calcium augmentation of the inflow promoted the accumulation of magnesium in the granules in the CAFB. The magnesium was presented as Ca₇Mg₂P₆O₂₄ according to XRD analyses.     

Characterization of aerobic granules by microbial density at different COD loading rates

Aerobic granules were cultivated under temporal alternating aerobic and anoxic conditions without the presence of a carrier material in a sequencing batch reactor (SBR) with a high column height/column diameter ratio. The reactor was operated for 6h per cycle (aerobic: 4.75 h, anoxic: 1.25 h). To determine a new parameter for the definition of aerobic granules, a protocol of 4,6-diamidino-2-phenylindole hydrochloride staining and fluorescence image processing was developed. The d(tm) analysis showed that the increase in the chemical oxygen demand (COD) loading rate promoted no more growth of the aerobic granules. It was inconsistent with the results of the analysis of the sludge volume index (SVI) value but matched well with the results of the COD and nitrogen removal of the SBR and the particle size distribution by LS-PSA. The optimum COD loading rate for aerobic granulation in the SBR was 2.52 kg/m(3)d. When d(tm) was correlated with the biomass concentration and the SVI value during the period of granule formation, d(tm) could be used as a more sensitive and accurate parameter for classifying aerobic granules and optimizing the operational conditions for aerobic granulation processes.     

Simulation of wastewater treatment by aerobic granules in a sequencing batch reactor based on cellular automata

In the present paper, aerobic granules were developed in a sequencing batch reactor (SBR) using synthetic wastewater, and 81 % of granular rate was obtained after 15-day cultivation. Aerobic granules have a 96 % BOD removal to the wastewater, and the reactor harbors a mount of biomass including bacteria, fungi and protozoa. In view of the complexity of kinetic behaviors of sludge and biological mechanisms of the granular SBR, a cellular automata model was established to simulate the process of wastewater treatment. The results indicate that the model not only visualized the complex adsorption and degradation process of aerobic granules, but also well described the BOD removal of wastewater and microbial growth in the reactor. Thus, CA model is suitable for simulation of synthetic wastewater treatment. This is the first report about dynamical and visual simulation of treatment process of synthetic wastewater in a granular SBR.     

Characterization and evaluation of aerobic granules in sequencing batch reactor

In order to investigate the aerobic granules cultured under alternating aerobic and anoxic conditions, a sequencing batch reactor (SBR) was operated without the presence of a carrier material. Nitrification and denitrification occurred alternately in the SBR operation, with an increased nitrification efficiency of up to 97% and a high chemical oxygen demand (COD) removal efficiency of up to 95%. It was observed that physical characteristics of granule play an important role in the performance of the SBR process. Light microscopy was used to observe the time dependent development of the granules in the SBR. Based on the microscopic observations, some floc-like sludges remained in the form of a mixture with granules for 30 days of operation. Even though various granule sizes had been formed in the reactor after 50 days, the granule sizes were primarily from 1 +/- 0.35 to 1.3 +/- 0.45 mm, rarely exceeding 2 mm. The granules were analyzed by a combination of microelectrodes and fluorescent in situ hybridization (FISH), which provides more detailed information on what happens inside the granules. Based on their results, ammonia oxidizing bacteria (AOB) existed primarily in the upper and middle layers of the granule. Assuming a first-order reaction for nitrification, most of the nitrification is likely to occur from the surface to 300 microm into the granular thickness.     

降解苯胺和氯苯胺类污染物好氧污泥颗粒化及微生物种群结构分析

以序批式气提生物反应器(SABR)为平台,研究了苯胺和氯苯胺类有毒有机废水处理过程好氧污泥颗粒化。结果表明,通过缩短污泥沉降时间、逐步提升目标污染物进水负荷,反应器连续运行3个月,最终在污泥沉降时间5min、COD负荷1.0~3.6kg/(m3.d)、苯胺和氯苯胺负荷1kg/(m3.d)条件下实现污泥颗粒化,COD、苯胺和氯苯胺去除率分别稳定在90%、99.9%以上;获得的成熟好氧颗粒粒径在0.45~2.5mm,SOUR稳定在150mgDO/(gVSS·h)以上,颗粒污泥EPS中PN含量为28.0±1.9mg/gVSS,PN/PS比值为6.5mg/mg,苯胺类比降解速率达0.18g/(g·d);应用PCR-DGGE分子指纹图谱技术分析了稳定运行的颗粒化反应器内好氧污泥微生物种群结构,结果表明好氧颗粒内主要细菌分属β-Proteobacteria、γ-Proteobacteria及Flavobacteria等类群,优势菌为Pseudomonas sp.、Flavobacterium sp.;与已获得的降解氯苯胺好氧颗粒相比,苯胺存在下培养获得的好氧颗粒污泥微生物菌群结构更为丰富。     

Aerobic granulation and nitrogen removal with the effluent of internal circulation reactor in start-up of a pilot-scale sequencing batch reactor

Aerobic granular sludge was successfully cultivated with the effluent of internal circulation (IC) reactor in a pilot-scale sequencing batch reactor (SBR) using activated sludge as seeding sludge. N removal was investigated in the start-up of aerobic granulation process. Initially, the phenomenon of partial nitrification was observed and nitrite accumulation rates (NO₂ ^?-N/NO _x ^? -N) were between 84.6 and 99.1?%. It was potentially caused by ammonium oxidizing bacteria (AOB) in the seeding activated sludge, high external environmental temperature (~32?°C) and free ammonia (FA) concentration. After 50?days’ running, the aerobic granules-based bioreactor demonstrated perfect performance in simultaneous removal of organic matter and ammonia nitrogen, and average removal efficiencies were maintained above 93 and 96?%, respectively. The maximum nitrogen removal efficiency of 83.1?% was achieved after the formation of aerobic granules. The average diameter of mature aerobic granular sludge mostly ranged from 0.5 to 1.0?mm. Furthermore, one typical cyclic test indicated that pH and DO profiles could be used as effective parameters for biological reactions occurring in the aerobic/anoxic process. The obtained results could provide further information on the cultivation of aerobic granular sludge with practical wastewater, especially with regard to nitrogen-rich industrial wastewater.     

Long-term impact of dissolved O(2) on the activity of anaerobic granules

The impact of influent dissolved O(2) on the characteristics of anaerobic granular sludge was investigated at various dissolved O(2) concentrations (0.5-8.1 ppm) in 1- and 5-L laboratory-scale upflow anaerobic sludge bed (UASB)-like anaerobic/aerobic coupled reactors with a synthetic wastewater (carbon sources containing 75% sucrose and 25% acetate). The rate of dissolved O(2) supplied to the coupled reactor was as high as 0.40 g O(2)/L(rx).d, and the anaerobic/aerobic coupled reactors maintained excellent methanogenic performances at a COD loading rate of 3 g COD/L(rx).d even after the reactors had been operated with dissolved O(2) for 3 months. The activities of granular sludge on various substrates (glucose, propionate, and hydrogen) were not impaired, and acetate activity was even improved over a short term. However, after 3 months of operation, slight declines on the acetoclastic activities of granules were observed in the coupled reactor receiving the recirculated fluid containing 8.1 ppm dissolved O(2).Methane yield in the anaerobic control reactor and anaerobic/aerobic coupled reactors revealed that a significant aerobic elimination (up to 30%) of substrate occurred in the coupled reactors, as expected. The presence of dissolved O(2) in the recirculated fluid resulted in the development of fluffy biolayers on the granule surface, which imposed a negative impact on the settleability of granular sludge and caused a slightly higher sludge washout. This research shows that the anaerobic/aerobic coupled reactor can be successfully operated under O(2)-limited conditions and is an ideal engineered ecosystem integrating oxic and anaerobic niches. (c) 1996 John Wiley & Sons, Inc.     

Organics and nitrogen removal and sludge stability in aerobic granular sludge membrane bioreactor

Novel aerobic granular sludge membrane bioreactor (GMBR) was established by combining aerobic granular sludge technology with membrane bioreactor (MBR). GMBR showed good organics removal and simultaneous nitrification and denitrification (SND) performances for synthesized wastewater. When influent total organic carbon (TOC) was 56.8-132.6 mg/L, the TOC removal of GMBR was 84.7-91.9%. When influent ammonia nitrogen was 28.1-38.4 mg/L, the ammonia nitrogen removal was 85.4-99.7%, and the total nitrogen removal was 41.7-78.4%. Moreover, batch experiments of sludge with different particle size demonstrated that: (1) flocculent sludge under aerobic condition almost have no denitrification capacity, (2) SND capacity was caused by the granular sludge, and (3) the denitrification rate and total nitrogen removal efficiency were enhanced with the increased particle size. In addition, study on the sludge morphology stability in GMBR showed that, although some granular sludge larger than 0.9 mm disaggregated at the beginning of operation, the granular sludge was able to maintain the stability of its granular morphology, and at the end of operation, the amount of granular sludge (larger than 0.18 mm) stabilized in GMBR was more than 56-62% of the total sludge concentration. The partial disaggregation of large granules is closely associated with the change of operating mode from sequencing batch reactor (SBR) system to MBR system.     

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part II. The application of aerobic sequencing batch reactor (aerobic SBR) to produce high biomass of Dioszegia sp. TISTR 5792

This study aimed to investigate the efficiency of an aerobic sequencing batch reactor (aerobic SBR) in a nonsterile system using the application of an experimental design via central composite design (CCD). The acidic whey obtained from lactic acid fermentation by immobilized Lactobacillus plantarum sp. TISTR 2265 was fed into the bioreactor of the aerobic SBR in an appropriate ratio between acidic whey and cheese whey to produce an acidic environment below 4.5 and then was used to support the growth of Dioszegia sp. TISTR 5792 by inhibiting bacterial contamination. At the optimal condition for a high yield of biomass production, the system was run with a hydraulic retention time (HRT) of 4 days, a solid retention time (SRT) of 8.22 days, and an acidic whey concentration of 80% feeding. The chemical oxygen demand (COD) decreased from 25,230 mg/L to 6,928 mg/L, which represented a COD removal of 72.15%. The yield of biomass production and lactose utilization by Dioszegia sp. TISTR 5792 were 13.14 g/L and 33.36%, respectively, with a long run of up to 180 cycles and the pH values of effluent were rose up to 8.32 without any pH adjustment.     

Aerobic granular sludge formation for high strength agro-based wastewater treatment

The present study investigates the formation of aerobic granular sludge in sequencing batch reactor (SBR) fed with palm oil mill effluent (POME). Stable granules were observed in the reactor with diameters between 2.0 and 4.0 mm at a chemical oxygen demand (COD) loading rate of 2.5 kg COD m⁻³ d⁻¹. The biomass concentration was 7600 mg L⁻¹ while the sludge volume index (SVI) was 31.3 mL g SS⁻¹ indicating good biomass accumulation in the reactor and good settling properties of granular sludge, respectively. COD and ammonia removals were achieved at a maximum of 91.1% and 97.6%, respectively while color removal averaged at only 38%. This study provides insights on the development and the capabilities of aerobic granular sludge in POME treatment.     

Long-term effect of weak nitrogen limitation on polyhydroxyalkanoates production of propionate-fed activated sludge operated at long sludge retention time

A sequencing batch reactor (SBR) submitted to aerobic dynamic feeding condition was fed with weakly nitrogen-limited substrate (COD/N ratio of 60?mg/mg, propionate as the sole carbon source), and operated at 15?days sludge retention time (SRT). Then, the nitrogen-limited sludges at different cultivating periods (45th, 120th, 195th and 240th days) were harvested to conduct aerobic batch experiments of PHAs production under various nitrogen concentrations. Experimental results indicated that PHAs accumulating behavior of the propionate-fed sludge declined significantly after long-term cultivation, implying that weak nitrogen limitation and long SRT operation cannot achieve a stable PHAs producing performance of the sludge. Supplementing enough nitrogen was consequently a requisite to guarantee the stability of the propionate-fed PHAs accumulating culture. Besides, the sludge with higher PHAs content in SBR did not ensure a better PHAs producing capability in the batch PHAs production phase. Batch experiments also indicated that the propionate-fed sludge with 0.7?N?mmol/L exhibited a better PHAs producing capability than with other nitrogen concentrations.     

Biodegradation of tannery wastewater using sequencing batch reactor--respirometric assessment

This investigation proved that respirometry combined with sequencing batch reactor (SBR) could be an effective way for the removal of COD in tannery wastewater. Measurement of oxygen uptake rates (OUR) and corresponding COD uptake rates showed that a 12-h operating cycle was optimum for tannery wastewater. The removal of COD by degradation was stoichiometric with oxygen usage. A plot of OUR values provided a good indication of the biological activity in the reactor. A high OUR value corresponded to the feed period; at the end of the cycle, when the substrate was depleted, the OUR value was low. At a 12-h SBR cycle with a loading rate of 1.9-2.1 kgm(-3) d(-1), removal of 80-82% COD, 78-80% TKN and 83-99% NH(3)-N were achieved. These removal efficiencies were much higher than the conventional aerobic systems. A simple method of COD fractionation was performed from the OUR and COD uptake rate data of the SBR cycle. About 66-70% of the influent COD was found to be readily biodegradable, 10-14% was slowly degradable and 17-21% was non-biodegradable. The oxygen mass transfer coefficient, K(L)a (19 +/- 1.7 h(-1)) was derived from respirometry. It was observed that with the exception of high organic load at the initial feed the oxygen transfer capacity was in excess of the OUR, and aerobic condition was generally maintained. Simultaneous nitrification-denitrification was observed in the SBR during the feed period as proved by mass balance.     

Effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors

Copper (Cu(II)) and nickel (Ni(II)) are often encountered in wastewaters. This study investigated the individual toxic effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors (SBRs). The biochemical properties of aerobic granules were characterized by extracellular polymeric substances (EPS) content, dehydrogenase activity, microbial community biodiversity, and SBR performance. One SBR was used as a control system, while another two received respective concentration of Cu(II) and Ni(II) equal to 5 mg/L initially and increased to 15 mg/L on day 27. Results showed that the addition of Cu(II) drastically reduced the biomass concentration, bioactivity, and biodiversity of aerobic granules, and certainly deteriorated the treatment performance. The toxic effect of Ni(II) on the biodiversity of aerobic granules was milder and the aerobic granular system elevated the level of Ni(II) toxicity tolerance. Even at a concentration of 15 mg/L, Ni(II) still stimulated the biomass yield and bioactivity of aerobic granules to some extent. The elevated tolerance seemed to be owed to the concentration gradient developed within granules, increased biomass concentration, and promoted EPS production in aerobic granular systems.     

Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor

A novel bioreactor containing self-flocculated anaerobic granular sludge was developed for high-performance hydrogen production from sucrose-based synthetic wastewater. The reactor achieved an optimal volumetric hydrogen production rate of approximately 7.3 L/h/L (7,150 mmol/d/L) and a maximal hydrogen yield of 3.03 mol H2/mol sucrose when it was operated at a hydraulic retention time (HRT) of 0.5 h with an influent sucrose concentration of 20 g COD/L. The gas-phase hydrogen content and substrate conversion also exceeded 40 and 90%, respectively, under optimal conditions. Packing of a small quantity of carrier matrices on the bottom of the upflow reactor significantly stimulated sludge granulation that can be accomplished within 100 h. Among the four carriers examined, spherical activated carbon was the most effective inducer for granular sludge formation. The carrier-induced granular sludge bed (CIGSB) bioreactor was started up with a low HRT of 4-8 h (corresponding to an organic loading rate of 2.5-5 g COD/h/L) and enabled stable operations at an extremely low HRT (up to 0.5 h) without washout of biomass. The granular sludge was rapidly formed in CIGSB supported with activated carbon and reached a maximal concentration of 26 g/L at HRT = 0.5 h. The ability to maintain high biomass concentration at low HRT (i.e., high organic loading rate) highlights the key factor for the remarkable hydrogen production efficiency of the CIGSB processes.     

Causes and control of filamentous growth in aerobic granular sludge sequencing batch reactors

Poor long-term stability of aerobic granules developed in sequencing batch reactors (SBRs) remains a limitation to widespread use of aerobic granulation in treating wastewater. Filamentous growth has been commonly reported in aerobic granular sludge SBR. This review attempts to address the instability problem of aerobic granular sludge SBR from the perspective of filamentous growth in the system. The possible causes of filamentous growth are identified, including long retention times of solids, low substrate concentration in the liquid phase, high substrate gradient within the granule, dissolved oxygen deficiency in the granule, nutrient deficiency inside granule, temperature shift and flow patterns. Because of cyclic operation of aerobic granular sludge SBR and peculiarities of aerobic granules, various stresses can be present simultaneously and can result in progressive development of filamentous growth in aerobic granular sludge SBR. Overgrowth of filamentous bacteria under stress conditions appears to be a major cause of instability of aerobic granular sludge SBR. Specific recommendations are made for controlling filamentous growth.     

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part I. Optimal condition for lactic acid fermentation using a microaerobic sequencing batch reactor (microaerobic SBR) with immobilized Lactobacillus plantarum TISTR 2265 and microbial communities

Cheese whey contains a high organic content and causes serious problems if it is released into the environment when untreated. This study aimed to investigate the optimum condition of lactic acid production using the microaerobic sequencing batch reactor (microaerobic SBR) in a nonsterile system. The high production of lactic acid was achieved by immobilized Lactobacillus plantarum TISTR 2265 to generate an acidic pH condition below 4.5 and then to support single-cell protein (SCP) production in the second aerobic sequencing batch reactor (aerobic SBR). A hydraulic retention time (HRT) of 4 days and a whey concentration of 80% feeding gave a high lactic acid yield of 12.58 g/L, chemical oxygen demand (COD) removal of 62.38%, and lactose utilization of 61.54%. The microbial communities in the nonsterile system were dominated by members of lactic acid bacteria, and it was shown that the inoculum remained in the system up to 330 days.     

Investigation on the formation and kinetics of glucose-fed aerobic granular sludge

Aerobic granular sludge was cultivated in a glass sequencing batch reactor (SBR) with glucose synthetic wastewater. The spherical shaped granules were observed on 4th day with the mean diameter of 0.1 mm. With the increase of chemical oxygen demand (COD) concentration of the influent, aerobic granules grew matured, the size of which ranged from 1.2 to 1.9 mm. The aerobic granular sludge could sustain high organic loading rate (about 4.0 g COD L⁻¹ d⁻¹), with good settling ability (settling velocity 36 m/h) and high biomass concentration (MLSS 6.7 ±0.2 g/L). Experimental data indicated that the substrate utilization and biomass growth kinetics followed Monod's kinetics model approximately. The corresponding kinetic coefficients of maximum specific substrate utilization rate (k), half velocity coefficient (K_s), growth yield coefficient (Y) and decay coefficient (K_d) were 13.2 d⁻¹, 275.8 mg/L, 0.183–0.250 mg MLSS/mg COD and 0.023–0.075 d⁻¹, respectively, which made aerobic granules have short setup period, high rate of substrate utilization and little surplus sludge.     

Respirometric Activities of Heterotrophic and Nitrifying Populations in Aerobic Granules Developed at Different Substrate N/COD Ratios

Aerobic granules were successfully developed at substrate N/COD ratios ranging from 5/100 to 30/100 by weight. By measuring respective respirometric activities of heterotrophic, ammonia-oxidizing, and nitrite-oxidizing bacteria, it was found that the relative abundance of nitrifying bacteria over heterotrophs in aerobic granules was closely related to the substrate N/COD ratios. Results further showed that the populations of both ammonia and nitrite oxidizers were significantly enriched with the increase of the substrate N/COD ratio, while a decreasing trend of heterotrophic population was observed in the aerobic granules. These seem to indicate that high substrate N/COD ratio favors the selection of nitrifying bacteria in the aerobic granules, while the relative activity of nitrifying population against heterotrophic population evolved until a balance between two populations was reached in the aerobic granular sludge community. Moreover, cell elemental composition was correlated with the shift in microbial populations, e.g., the enriched nitrifying population in the aerobic granules resulted in a high cell nitrogen content normalized to cell carbon content. This study provides a good insight into microbial interaction in aerobic granules.