Granulation And Microbial Molecular Ecology Of Aerobic Sludge For Degrading Aniline And Chloroaniline

Aerobic biological technology has been extensively applied for treatment of domestic and industrial wastewater, and plays an important role in the mineralization of organic compounds and removal of nutrient. However, currently applied processes such as SBR and oxidition ditch, are sensitive to the fluctuation in organic loading and environmental conditions, and uneffectively degrade compounds due to the decrease of functional bacteria. In order to retain functional bacteria and obtain successful biodegradation of non-/slowly- degradable pollutants in bioreactors, the granulation and microbial mechanism of aerobic sludge in SBR and sequencing airlift bioreactor (SABR) for high-rate degradation of organic wastewater containing aniline (AN) and chloroanilines (ClAs) were studied in this thesis, and the main results are as follows:1. The granulation of aerobic sludge for degrading AN was developed in SABR using activated sludge as inoculum. High hydraulic shear force and short settling time were chosen as two major selection pressures for enrichment of biomass with excellent settleability, and high AN degradation activity were accomplished by stepwise increase of AN loading. Operated at AN loading rate of 1.2 kg·m^-3·d^-1, the granular SABR reached a steady state with biomass concentration of 4.4±0.1 g·L^-1. Mature aerobic granules had the average diameter of 1.2 mm, minimal settling velocity of 35 m·h^-1 and specific AN degradation rate of 0.84 g·gVSS^-1·d^-1. Results showed that aerobic granules were successfully developed in SABR fed with AN as sole carbon and energy source.Aerobic granules were also cultivated with 4-chloroaniline (4-ClA) loading rate of 0.8 kg·m^-3·d^-1 in three column-type SBRs which had different geometrical configurations. The granular sludge was observed after 15 days of cultivation in R2 and R3 which had higher height to diameter (H/D) ratio, and mature granules formed in the reactors featured with average diameter of 1.68 and 1.25 mm, minimal settling velocity of 70.1 and 66.6 m·h^-1, and specific 4-ClA degradation rates of 0.21 and 0.27 g·gVSS^-1·d^-1, respectively. The granule strength, SOUR and specific 4-ClA degradation rate were all the highest in R3. Results demonstrated that a rapid aerobic sludge granulation could be achieved in the reactor with higher H/D ratio and internal-circulation.Based on the successful cultivation of aerobic granules for degrading 4-ClA or AN, the granulation of aerobic sludge for degrading ClAs and AN was investigated. Operated at COD loading rate of 3 kg·m^-3·d^-1 and ClAs/AN loading rate of 0.8 kg·m^-3·d^-1, two SABRs reached steady states after 120 days, as evidenced by constant COD and ClAs/AN removal efficiencies of above 90% and 99.9%, respectively. Mature granules had diameter of 0.9～2.5 mm, minimal settling velocity of above 60 m·h^-1 and specific ClAs/AN degradation rate of 0.18 g·gVSS^-1·d^-1. Aerobic granules had smooth outer surface with a compact and regular appearance, and were consisted of diverse microbial morphotypes including bacilus and coccus, embedded in extracellular polymeric substances (EPS). Compared to ClAs-degrading aerobic granules, the performance and stability were better in aniline and chloroaniline-degrading aerobic granules.2. The hydraulic selection pressures (e.g., shear force, settling time and hydraulic retention time) and microbial selection pressures (e.g., substrate composition, substrate loading rate and feast-famine regimes in bioreactor) are decisive parameters in the formation of aerobic granules. According to the results obtained, the bioreactor operated under conditions of superficial gas velocity higher than 2.4 cm·s^-1, settling time shorter than 10 min, ClAs loading rate at 0.6～0.8 kg·m^-3·d^-1, hydraulic retention time (HRT) at 11.4～17.2 h, anaerobic before aerobic period and removing fresh sludge, favored the granulation of aerobic sludge. Additionally, C-starvation could enhance the granule strength and surface hydrophobicity, and C- or K-starvation would lead the degradation of EPS and weaken the structural integrity of granule. The microbial activity of aerobic granules would be severely inhibited by C-, N- and P-starvation, and C- or Fe-starvation was proved to inhibit the growth of microorganism. This study is expected to be helpful in treating industrial wastewater in which nutrient deficiency is commonly encountered.The stabilities of three reactors which had different geometrical configuration were quantitatively compared. Results showed that the reactors with higher H/D had better stability when undergoing the ClAs shock load. The characteristics of hydraulic shear rate in three reactors were studied, and the overall shear rate of R3 was higher than those of both R2 and R1. Results demonstrated that the reactor with higher shear rate favored the formation and stability of aerobic granules.The relationships between granular characteristic indexes and factors affecting aerobic granulation were investigated using gray-system analysis method. Results indicated that the grey relation grade between settling velocity (SV) and total microbial density (d_tm) was 0.828, and those of SV-SVI and granular size (D)-d_tm were 0.804 and 0.779, respectively. According to the relationships between granular characteristic indexes and operation parameters, hydrodynamic shear force (HSF), sludge settling time (SST) and OLR were considered as the key controlling factors in aerobic sludge granulation.EPS plays crucial role in the formation and stability of aerobic granules. The values of proteins (PN) and ratio of proteins to polysaccharides (PN/PS) in EPS extracted from aerobic granules markedly increased along with sludge granulation. The hydrophobicity and surface charge were significantly positively correlated with PN content and PN/PS ratio. The increase of shear force and decrease of sludge settling time (less than 10 min) could stimulate the production of PN, and favor the stability of aerobic granules.We proposed a model for aerobic sludge granulation consisting of the following steps: Under an appropriate combination of hydraulic and microbial selection pressures, excessive EPS is secreted, and influences the hydrophilicity and surface charge of cells, then favors the adhesion of microorganisms. Formed microbial aggregates ulteriorly transfer to "microbial nuclei" via creating a network of filamentous bacteria, EPS and cations. Multi-microorganisms tend to be regularly distributed and form steady three-dimensional structure of microbial granules by hydrodynamic shear forces. Great aerobic granules tend to disaggregate because of the mass transfer resistance of oxygen and substrate, then fragments from disaggregated granules could act as "secondary nuclei" to accelerate the formation of mature aerobic granules.3. The degradation kinetics of ClAs and AN by aerobic granules followed the Haldane model. The order of maximal specific ClAs/AN degradation rates (V_max) followed: AN>4-ClA>2-ClA>3-ClA>3,4-DClA, and the order of inhibition constant (K_i) followed: AN>3-ClA>4-ClA>2-ClA>3,4-DClA. The biodegradation of ClAs and AN has significantly positive correlations with space and thermodynamics parameters of the compounds. The optimal granule size-range was suggested as 1.25-2.0 mm in diameter, which had higher V_max and K_i. Results showed the granulation of aerobic sludge could improve the performances of bioreactor for high-rate degradation of toxic organic compounds, however it is important to control the size of granular sludge.The intermediates of 4-ClA metabolized by aerobic granules were analyzed and identified by enzyme assay, HPLC and LC/MS methods. The results showed that the intermediates included 4-chlorocatechol, 5-chloro-4-acyl-crotonic acid, 2-hydroxy-5-chloromuconic semialdehyde and 4-hydroxy-5-chloro-2-ketovaleric acid, which are the intermediates of a typical meta-cleavage pathway. In addition, aerobic granules could produce the induced catechol 2,3-dioxygenase when they grow on 4-ClA or its intermediates of meta-cleavage pathway such as 4-chlorocatechol, 2-hydroxy-5-chloromuconic semialdehyde. It was concluded that aerobic granules could degrade 4-ClA through meta-cleavage pathway.The specific EPS and polyhydroxybutyrate (PHB) production rates interrelated with each other and both had an exponential correlation with specific cell growth rate. Therefore, PHB and EPS might be regarded as products of the secondary metabolisms by microorganisms and be used as secondary substrates for microorganisms under nutritional or hydraulic stress of aerobic starvation period.4. Microbial communities in aerobic sludge shifted obviously with the increased ClAs and AN loadings and granulation of aerobic sludge. DGGE analysis illustrated that aerobic granules in R2 and R3 showed more abundant DGGE bands and the cumunity structures were the most similar in steady state. Phylogenetic analysis indicated thatβ-,γ-Proteobacteria, Planctomycetales, Flavobacteriales, Clostridiales and Acidobacteria were the dominant classes in the 4-ClA-degrading aerobic granules, and the predominant functional bacteria were closely related to Thauera sp., thus these bacteria are considered as the 4-ClA-degrading functional bacteria. An obvious shift of microbial communities was also observed in the aerobic sludge with AN as sole carbon and energy source. Proteobacteria, Flavobacteriales, Cytophagales and Bacteroidete were the predominant bacteria in aerobic granules for degrading AN, and the predominant functional bacteria were closely related to Thauera spp., Pseudomonas putida sp. and Acinetobacter sp. The predominant functional bacteria of aerobic granules for degrading ClAs and AN were closely related to Xenophilus spp., Pseudomonas sp., Pseudomonas putida sp. and Flavobacterium columna, and mostly belong toβ-Proteobacteria,γ-Proteobacteria and Flavobacteria. Compared to ClAs-degrading aerobic granules, the population diversity was higher in the AN and ClAs-degrading aerobic granules.The dynamics of the total bacteria,β-Proteobacteria, ammonia oxidizer (AOB) and Thauera spp. abundant in 4-ClA-degrading aerobic sludge were analyzed by RTQ-PCR method, and notable increases in copy numbers of 16S rRNA gene ofβ-Proteobacteria, AOB and Thauera spp. were found during granulation of aerobic sludge (each reached 21.55%, 10.79% and 61.28% of total bacteria), suggesting that Thauera spp. were gradually developed and highly enriched with the increase of ClAs loading and granulation of aerobic sludge. Increases of copies of 16S rRNA gene ofβ-Proteobacteria, AOB and Thauera spp. were also observed in AN-degrading aerobic sludge, and Thauera sp reached 23.47% of total bacteria at AN loading of 1.44 kg·m^-3·d^-1. Results demonstrated that aerobic sludge granulation may play an important role in the enrichment and retaining of toxic organic pollutants-degrading microorganisms.