Typology and activity of the nitrifying bacteria underlying selected bioprocesses treating animal and municipal wastewater | |
| Author | Anceno, Alfredo J. |
| Call Number | AIT Diss. no.EV-09-01 |
| Subject(s) | Sewage--Purification--Nitrogen removal Sewage--Purification--Anaerobic treatment |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Environmental Technology and Management |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. EV-09-01 |
| Abstract | With a view of producing renewable energy and reduced emissions of greenhouse gases and problematic nitrogenous effluents from animal wastes in Brittany, the animal farming capital of France, efforts were pursued in CEMAGREF-Remles to employ animal wastes in biogas production with subsequent nitrogen removal (N-removal). A major challenge was to develop an operationally cost-effective bioprocess that selects nitrifying consortia performing nitritation-denitritation instead of the conventional nitrification-denitrification and sufficiently meets the organic substrate requirement for the N-removal treatment. The undertaking consisted of process engineering aspect, modelling and kinetics aspect and process microbiology aspect. This dissertation focuses on the process microbiology aspect and is divided into three parts. These included studies on the (i) sources of bias in the 16S rRNA end-point polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) which was heavily used in profiling the nitrifying bacteria, (ii) quantification/profiling of nitrogen transformation related genes/phylotypes and correlation with bioreactor performance during start-up and (iii) long-term operation, and (iv) development of an approach leading to the elucidation of underlying N-removal pathways in select animal and municipal N-removal treatment processes. Study approaches ad results are detailed as follows. First, the end-point bias in PCR-SSCP was elucidated from the perspective of DNA extraction method used, multitemplate assays employing proportional amounts of genomic DNA from select target bacteria, and multitemplate assays employing proportional amounts of cloned V3 region of 16S rDNA from select target bacteria. Across mixtures of a-, high-GC, B-, low-GC and y-proteobacterial type strains (~10⁸ each) as such and/or mixed with manure, compost and soil matrices (100 mg), the DNA extraction method based on enzymatic lysis resulted in best template yield and quality. However, DNA extraction methods based on mechanical lysis with silica-column binding during purification step efficiently eliminated PCR inhibitors, undesirable low-molecular weight DNA fragments and reflected the widest range of microfloral diversity among sample matrices tested and therefore used in subsequent experiments. Multitemplate assay results utilizing proportional genomic DNA (10-100 pg) from select a-, high-GC, B-, low-GC and y-proteobacterial type strains and different DNA polymerases (Plu, Paq, Taq, TaqH) suggested that end-point bias could be minimized by avoiding overamplification (20-30 cycles of amplification was sufficient), performing assays soonest following the DNA extraction and utilization of thermo cyclers with tightly sealed block heaters to prevent undesirable temperature gradients in air surrounding the reaction tubes. Similar assays performed on cloned templates (V3 region of 16S rDNA) suggested that higher ramping rates (>2.5°C/s) between thermocycling steps (denaturation-annealing-extension) effectively reduced variability among obtained SSCP profiles. It also demonstrated that reaction volumes and thermo cycling could be reduced up to 20 uL and about 25-30 cycles, respectively, for a cost-effective and speedy assay. Second, the impact of the two sequencing batch process configurations treating swine wastewater ---- namely biological N-removal (BNR) and anaerobic digestion coupled biological N-removal (AD-BNR) ----- on the evolution of target N-converting bacteria in BNR reactor during start-up was investigated using the optimized PCR-SSCP methodology above and clone library analysis. For influent having 4.2 ± 0.1 g/L total Kjeldahl nitrogen (TKN), 2.4 ± 0.1 g/L ammonium-nitrogen (NH₄ ⁺ -N), 52.1 ± 6.2 g O₂/L total chemical oxygen demand (CODTOT), and 17.4 ± 2.9 g O₂/L soluble chemical oxygen demand (CODsoL) subjected to a treatment cycle of 6-h non-aeration and 6-aeration (~6 mg 0₂/L), complete NH₄ ⁺-N removal was achieved in BNR while higher effluent N0₂ ⁻ concentration (up to 20.1 ± 3.3 mg/L) was obtained in AD-BNR eight weeks after start-up with "full-sludge" inoculation. The utilization of 2/3 influent organic substrate in the anaerobic processes of AD-BNR resulted in higher CODTOT (36.8 ± 1.2%) and lower TKN (61.8 ± 6.2%) removal therein as compared to BNR (32.6 ± 3.3% for CODTOT and 70.1 ± 3.1 % for TKN). Group-specific PCR-SSCP analysis revealed that BNR and AD-BNR appeared to select Nitrosospira-INitrospira-like and Nitrosomonas-INitrobacter-like ammonia-/nitrite-oxidizing pair of taxa, respectively, correlating with the known survival strategies of these taxa with the physico-chemical milieu in respective configurations; the mean NH₄ ⁺ concentration being notably higher in AD-BNR. Clone library analysis of N₂0 reductase gene fragment showed that denitrifier taxa related to Ensifer, Rhizobium, Sinorhizobium and the pathogenic Brucella, Bordatella, Alcaligenes were dominant in BNR but decreased in number with AD-BNR due to anaerobic inactivation during anaerobic digestion. Overall, notably higher denitrifier diversity was observed in AD-BNR than BNR due to continuous reinoculation. Third, long-term operation study involving three bioprocess configurations - namely BNR, AD-BNR with influent bypass to BNR (AD-BNRBP) and AD-BNRBP with recirculation between AD and BNR (AD-BNRBP+R) - utilizing the same influent and similar treatment cycle (nonaeration-aeration regime) with control of dissolved oxygen (DO) concentration was carried out in September 2007 to October 2008. Performance data was correlated to the numbers of N-transformation related functional genes as determined via a quantitative PCR (qPCR) and the identities of phylotypes harboring some of these senes as determined via asymmetric SSCP/T-RFLP analyses and clone library screening. ~tatistical analysis (ANOYA) revealed that effluent concentrations of N0₂ ⁻ -N (30.45±4.90 ng/L) and N0₃ ⁻ -N (53.67±1.60 mg/L) were both significantly high in AD-BNRBP (P-value < 0.001). In all configurations, the removal efficiency of CODToT (23-31 %) was not substantial as compared to CODSOL (72-76%), suggesting that soluble rather than legradable organic carbon was the major electron donor and energy source to the leterotrophic biomass during denitrification. The removal efficiencies for TKN (60-75%) and NH₄ ⁺ -N (92-100%) were higher in BNR and AD-BNRBP; all mean pairs being :ignificantly different at a = ~0.01. The specific removal rates for CODTOT (up to 0.05 g/g VSS/d), CODSOL (up to 0.04 g/g VSS/d), TKN (up to 0.006 g/g VSS/d) and NH₄ ⁺ -N (up to 0.006g/g VSS/d) are also significantly higher in BNR and AD-BNRBP (P-value = 0.001). However, nitritation rates both on the basis of biomass (1.7 g NO₂ ⁻ -N/g VSS/h) and volume (22 g N0₂ ⁻ -N/L/h) were significantly higher in AD-BNRBP+R (P-value = 0.001). These results suggest that, despite higher specific C-N removal rates in BNR and AD-NRBP, optimal nitritation was achieved by careful control of DO concentration (from 6 to 0.5 mg 0₂/L during uncontrolled and controlled aeration). The shifts of bioprocess onfiguration from BNR to AD-BNRBP+R has resulted in a relative decrease of biomass due to dilution during recirculation as seen from copy numbers of total 16S rRNA gene ragments (10.5 to 9.5 Log₁₀ copies/g YSS). Nevertheless, an increase in gene fragment copy numbers of NH₃ monooxygenase (amoA; 0.3 to 12% of 16S rRNA) and N₂O reductase (nosZ; 10 to 60% of l6S rRNA) and a decrease of N0₂ ⁻ oxidoreductase (nxrB; rom >5 to <0.5% of 16S rRNA) suggested that a shift of bioprocess configuration from BNR to AD-BNRBP+R has enriched the NH₃-oxidizing bacteria, suppressed the N0₂ ⁻ -xidizing bacteria and has installed nitritation-denitritation as the major N-removal pathway as originally sought. Also, while the survival or (co)dominance of certain hylotypes would have been a result of continuous reinoculation, results of asymmetric SCP/T-RFLP and clone library analyses suggested that a more complex community of nitrifiers were resilent to perturbations in process conditions and influent characteristics. In terms of richness in phylotypes retrieved from respective clone libraries (amoA, nxrB, IOSZ), functional gene based fingerprinting techniques employed here were superior to the 16S rRNA based approach by a factor of 2 to 5. Lastly, an approach of correlating the nitrification-denitrification activities in sludge with the quantitive data on relevant functional genes was extended to different sludge types representing several animal and municipal wastewater treatment approaches. Specific nitri(a)tation activities were measured by respirometry wherein NH₄ ⁺ or N0₂ ⁻ substrate was pulsed into endogenously respiring biomass (sludge) with oxygen uptake rates leasured until substrate depletion. Specific denitri(a)tation activities were determined by pulsing NP₂ ⁻ or NO₃ ⁻ (in the presence of excess acetate) into substrate-depleted sludge in a reaction chamber with oxygen-free headspace and thereafter N₂ gas produced was measured by alkali displacement method. Copy numbers of amoA and nxrB gene fragments (nitritation and nitratation markers) and those of napA, narG, nirK, nirS, norB and nosZ (denitrification cascade markers) in the original sludge were estimated by qPCR. Phylotypes harboring amoA, nxrB and nosZ were identified by asymmetric SSCP/T-RFLP and clone library analyses. The approach proved to be a uselful tool of elucidating patterns of association among the dominant/sub-dominant N-converting taxa and the prevailing N-removal pathway for a given bioprocess and wastewater type |
| Year | 2009 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. EV-09-01 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Energy and Climate Change (Former title: Department of Energy, Environment, and Climate Change (DEECC)) |
| Academic Program/FoS | Environmental Engineering and Management (EV) |
| Chairperson(s) | Shipin, Oleg V.; |
| Examination Committee(s) | Annachhatre, Ajit P. ;Rakshit. Sudip K.; |
| Scholarship Donor(s) | France ;Asian Institute of Technology Fellowship ;CEMAGREF International and European Affairs; |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2009 |