Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria

This study investigated the feasibility of improving the stability of aerobic granules through selecting slow-growing nitrifying bacteria. For this purpose, four sequencing batch reactors were operated at different substrate N/COD ratios ranging from 5/100 to 30/100. Results showed that aerobic granules formed in all four reactors, and aerobic granulation was a gradual process evolving from the dispersed seed sludge to mature and stable granules, and the whole granulation process could be divided into three phases, i.e. acclimation phase, granulation followed by granule maturation. The observed growth rate and mean size of mature aerobic granules were found to decrease as the substrate N/COD ratio was increased, while nitrifying population was enriched markedly in aerobic granules developed at high substrate N/COD ratios. The enriched nitrifying population in aerobic granules was responsible for the observed low growth rate of aerobic granules. It seems certain that the substrate N/COD ratio is an important factor in selecting nitrifying bacteria in aerobic granules. Aerobic granules with low growth rates showed strong structure and good settleability in terms of specific gravity, SVI and cell hydrophobicity that further lead to high stability as compared to those having high growth rates. This study demonstrated that the selection of slow-growing nitrifying bacteria through controlling substrate N/COD ratio would be a useful strategy for improving the stability of aerobic granules.     

Optimum growth requirements of nitrifying consortia developed from treated sewage

The optimum growth requirements of two nitrifying consortia developed from treated sewage by enrichment technique were determined by a series of experiments. There was total inhibition of nitrification at above 2.75 g l(-1) NH4(+)- N and 2.5 g l(-1) NO2(-)-N and the ammonia oxidizing consortium preferred a pH at 8.5 and the nitrite oxidizing consortium a pH of 7.5 as the optima for nitrification. Optimum temperatures were between 20 degrees and 30 degrees C for both the groups. As the rate of airflow was increased from 1 to 7 l/min, the build-up of NO2(-)-N increased 10-fold and the consumption of NO2(-)-N increased by a factor of 28.8 implying that the ammonia oxidizing consortium in a bioreactor required three times more aeration than that for nitrite oxidizers for expressing their full nitrifying potential. These data directly contribute for developing a fermentation process for the mass production of nitrifiers as well as for designing bioreactors for nitrifying sewage.     

Formation and long-term stability of nitrifying granules in a sequencing batch reactor

The formation and long-term stability of nitrifying granules in a sequencing batch reactor was investigated in this study. The results showed that nitrifying granules with a size of 240 microm and SVI of 40 ml g(-1) were formed on day 21 at a settling time of 10 min. Maintaining settling time at 15 min from day 57 to 183 did not affect the physical characteristics of sludge and the fraction of suspended floc in the sludge. In addition, nitrifying granules could tolerate the fluctuations of nitrogen loading rate from 0.72 to 1.8 g l(-1)d(-1) during 2 months without the change of physical characteristics. However, it was observed that complete nitrification to nitrate and partial nitrification to nitrite by sludge converted each other corresponding to the change of the influent NH4+-N concentration. Thus, an appropriate method is needed to maintain a stable complete nitrification or partial nitrification under the conditions with changing influent NH4+-N concentrations and nitrogen loading rates.     

Growth kinetics of aerobic granules developed in sequencing batch reactors

AIMS: This paper attempts to develop a kinetic model to describe the growth of aerobic granules developed under different operation conditions. METHODS AND RESULTS: A series of experiments were conducted by using four-column sequencing batch reactors to study the formation of aerobic granules under different conditions, e.g. organic loading rates, hydrodynamic shear forces and substrate N/COD ratios. A simple kinetic model based on the Linear Phenomenological Equation was successfully derived to describe the growth of aerobic granules. It was found that the growth of aerobic granules in terms of equilibrium size and size-dependent growth rate were inversely related to shear force imposed to microbial community, while a high organic loading favoured the growth of aerobic granules, leading to a large size granule. The effect of substrate N/COD ratio on the growth kinetics of aerobic granules was realized through change in microbial populations, and enriched nitrifying population in aerobic granules developed at high substrate N/COD ratio resulted in a low overall growth rate of aerobic granules. CONCLUSIONS: The proposed model can provide good prediction for the growth of aerobic granules indicated by the correlation coefficient >0.95. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model proposed could offer a useful tool for studying the growth kinetics of cell-to-cell immobilization process. The study confirmed that the growth of aerobic granules and biofilms are subject to a similar kinetic pattern. This work would also be helpful for better understanding the mechanism of aerobic granulation.     

Microbial community structure in autotrophic nitrifying granules characterized by experimental and simulation analyses

This study evaluates the community structure in nitrifying granules (average diameter of 1600 μm) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 μm below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 μm, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH₄⁺, NO₂^–, NO₃^– and O₂ concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria–substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.     

Kinetic analysis on the two-step processes of AOB and NOB in aerobic nitrifying granules

Complete granulation of nitrifying sludge was achieved in a sequencing batch reactor. For the granular sludge, batch experiments were conducted to characterize the kinetic features of ammonia oxidizers (AOB) and nitrite oxidizers (NOB) in the granules using the respirometric method. A two-step nitrification model was established to determine the kinetic parameters of both AOB and NOB. In addition to nitrification reactions, the new model also took into account biomass maintenance and mass transfer through the granules. The yield coefficient, maximum specific growth rate, and affinity constant for ammonium for AOB were 0.21 g chemical oxygen demand (COD) g⁻¹ N, 0.09 h⁻¹, and 9.1 mg N L⁻¹, respectively, whereas the corresponding values for NOB were 0.05 g COD g⁻¹ N, 0.11 h⁻¹, and 4.85 mg N L⁻¹, respectively. The model developed in this study performed well in simulating the oxygen uptake rate and nitrogen conversion kinetics and in predicting the oxygen consumption of the AOB and NOB in aerobic granules.     

The role of loading rate, backwashing, water and air velocities in an up-flow nitrifying tertiary filter

The vertical distribution of nitrification performances in an up-flow biological aerated filter operated at tertiary nitrification stage is evaluated in this paper. Experimental data were collected from a semi-industrial pilot-plant under various operating conditions. The actual and the maximum nitrification rates were measured at different levels inside the up-flow biofilter. A nitrogen loading rate higher than 1.0 kg NH₄-N m⁻³_media d⁻¹ is necessary to obtain nitrification activity over all the height of the biofilter. The increase in water and air velocities from 6 to 10 m h⁻¹ and 10 to 20 m h⁻¹ has increased the nitrification rate by 80% and 20% respectively. Backwashing decreases the maximum nitrification rate in the media by only 3-14%. The nitrification rate measured at a level of 0.5 m above the bottom of the filter is four times higher than the applied daily average volumetric nitrogen loading rate up to 1.5 kg NH₄-N m⁻³_media d⁻¹. Finally, it is shown that 58% of the available nitrification activity is mobilized in steady-state conditions while up to 100% is used under inflow-rate increase.     

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by ¹³CO₂ after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the ¹³C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the ¹³C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that ¹³CO₂ assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.     

Effects of mechanical stress on Anammox granules in a sequencing batch reactor (SBR)

The effect of shear stress on Anammox process was studied in a sequencing batch reactor (SBR). The reactor was operated during 218 days under different stirring speeds (60-250 rpm) in order to expose the system to different shear conditions and to study the stability of the Anammox granules referred to their biological activity and size. The nitrogen loading rate (NLR) fed to the SBR was kept around 0.3g N(L day)(-1). The nitrite (limiting substrate) removal percentage was 98% during most of the operational period. The specific Anammox activity of the biomass was practically constant and around 0.4 g N(g VSSday)(-1) and the average feret diameter of the formed granules was 0.64 mm. Obtained results indicated that stirring speeds up to 180 rpm have no negative effect on the performance of the Anammox process, whereas Anammox activity decreased to 40% when a rotating speed of 250 rpm was tested and the average diameter decreased in 45%, the concentration of solids in the effluent increased to 0.2g TSSL(-1) and nitrite was accumulated in the reactor up to 60 mg NL(-1).     

Population dynamics of nitrifying bacteria in an aquatic ecosystem.

In a space environment such as Space Shuttle or Space Station, animal experiments with aquatic species in a closed system pose a crucial problem in maintaining their water quality for a long term. In nature, ammonia as an animal wastes is converted by nitrifying bacteria to nitrite or nitrate compounds, which usually become nitrogen sources for plants. Thus an application of the biological reactor with such bacteria attached on some filters has been suggested and experimentally studied for efficient waste managements of ammonia. Although some successful results were reported (Kozu et al. 1995, Nagaoka et al. 1998, Nakamura et al. 1997, 1998) in the space applications, purely empirical approaches have so far been taken to develop a biological filter having a stable nitrifying activity. In this study, we constructed a mathematical model to deal with the dynamics of the ammonia nitrifying processes in a biological reactor. The model describes population dynamics of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria cultivated on the same filter. We estimated parameters involved in the model using the experimental data. The result shows that these estimated parameters could be applied to general cases and that the two bacteria are in a symbiotic relationship; they can better perform when both coexist, as has been empirically recognized. Based on the model analysis, we discuss how to prepare a high performance biological filter.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

A comparative study on the formation and characterization of aerobic 4-chloroaniline-degrading granules in SBR and SABR

The formation and characterization of the aerobic 4-chloroaniline-degrading granules in the three column-type sequencing batch reactors were investigated in this paper. The granular sludge was observed since 15 days after start-up in R2 and R3 which had the high ratio of height to diameter (H/D). Since then and within the subsequent 75 days, the granulation of aerobic sludge was apparently developed by the decreased settling time and gradually increased 4-chloroaniline (4-ClA) concentration to above 400 mg.L(-1) in R1 to R3. The aerobic granules tended to be mature in all reactors continuously operated with 4-ClA loading rates of around 800 g.m(-3).d(-1), and the removal efficiencies of chemical oxygen demand, total nitrogen, and 4-ClA were maintained above 93%, 70%, and 99.9%, respectively. Mature aerobic granules in R1 to R3 featured with the average diameter of 0.78, 1.68, and 1.25 mm, minimal settling velocity of 20.5, 70.1 and 66.6 m.h(-1), specific 4-ClA degradation rates of 0.14, 0.21, and 0.27 g.gVSS(-1).d(-1), and the ratio of proteins to polysaccharides of 8.2, 10.8, and 13.7 mg.mg(-1), respectively. This study demonstrates that the reactor with a high H/D ratio and internal circulation favors the granulation and stabilization of aerobic sludge.     

Characteristics and determinants of osmotic lysis in chromaffin granules

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.     

Degradation pathway, toxicity and kinetics of 2,4,6-trichlorophenol with different co-substrate by aerobic granules in SBR

The present study deals with cultivation of 2,4,6-trichlorophenol (TCP) degrading aerobic granules in two SBR systems based on glucose and acetate as co-substrate. Biodegradation of TCP containing wastewater starting from 10 to 360 mg L⁻¹ with more than 90% efficiency was achieved. Sludge volume index decreases as the operation proceeds to stabilize at 35 and 30 mL g⁻¹ while MLVSS increases from 4 to 6.5 and 6.2 g L⁻¹ for R1 (with glucose as co-substrate) and R2 (with sodium acetate as co-substrate), respectively. FTIR, GC and GC/MS spectral studies shows that the biodegradation occurred via chlorocatechol pathway and the cleavage may be at ortho-position. Haldane model for inhibitory substrate was applied to the system and it was observed that glucose fed granules have a high specific degradation rate and efficiency than acetate fed granules. Genotoxicity studies shows that effluent coming from SBRs was non-toxic.     

Characteristics of methanogenic granules grown on propionate in an upflow anaerobic sludge blanket (UASB) reactor

Granulation of a propionate-degrading consortia was performed with a mesophilic propionate-acclimatized sludge in an upflow anaerobic sludge blanket (UASB) reactor. The granules formed were relatively small, ranging mainly from 0.3 to 0.6 mm in diameter, but had an excellent sedimentation velocity due to a high specific gravity of 1.355 g/cm³ (ash content, 48.2%). The ash consisted mainly of calcium (30.2%), phosphorus (19.7%), and magnesium (3.95%) forming plate crystals in the granules. The populations of three bacterial trophic groups present in the granules, propionate-degraders, hydrogenotrophic and aceticlastic methanogens were 5.6 × 10⁸, 1.6 × 10¹⁰, and 2 × 10⁹ (in most probable number/g mixed-liquor volatile suspended solids [MLVSS]), respectively, while the specific utilization rates of propionate, hydrogen, and acetate of the granules were 9.4, 850, and 20.9 (mmol/g MLVSS·d), respectively. Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. Total granular sludge concentration retained in the UASB reactor during 178 d of operation was 80.0 g mixed-liquor suspended solids (MLSS)/l-reactor, corresponding to 41.4 g MLVSS/l-reactor, which realized a high-rate methanogenic fermentation of propionate of 85 g chemical oxygen demand (COD)/l-reactor·d.     

Characteristics of rapidly formed hydrogen-producing granules and biofilms

The physicochemical and microbiological characteristics of rapidly formed hydrogen-producing granules and biofilms were evaluated in the present study. Microbial species composition was examined using the 16S rDNA-based separation and sequencing techniques, and spatial distribution and internal structure of microbial components were evaluated by examining the confocal laser scanning microscope (CLSM) images. Phylogenetic analysis indicated that a pure culture of Clostridium pasteurianum-like bacterium (98% similarity) was found in microbial community of granules and biofilms. It is postulated that containing such a species favored the rapid immobilization of hydrogen-producing culture. Manure granules and biofilms secreted 24-35 mg extracellulous proteins and 142-175 mg extracellulous polysaccharides in each gram of culture (in VSS). Such a high productivity of extracellulous polymers (ECP), a bio-glue to facilitate cell-to-cell and/or cell-to-substratum interaction, may work as the driving forces for the immobilization of C. pasteurianum. As abundant proteins were noted in the granule cores, it can be derived that rapid formation of the hydrogen-producing granules could be due to the establishment of precursor protein-rich microbial nuclei.