3.3 Experimental methods and design
3.3.5 Terminal Restriction Fragment Length Polymorphism
TRFLP is based on PCR amplification of a target gene. In the case of TRFLP, the amplification is performed with one or both the primers having their 5’ end labeled with a fluorescent molecule. Add 0.5 μl of restriction endonuclease enzyme, Hhal, and 2 μl of complimentary buffer into 15μl sample of positive PCR product. The restriction enzyme and complimentary buffer, Buffer C (R003 A), are Catalog No.
R6441 System Lot No. 221280 produced by Promega Corporation. The cut sites of the enzyme are 5’GCG^C3’ and 3’C^GCG5’. Then put it into thermocycler at 37℃
for two hours. The above procedures are called as digestion reaction. The labeled fragments cuted in digestioin reaction were sent to Nucleic Acid Analysis and
Synthesis Core Laboratory to analyze with ABI PRISM3100 Genetic Analyzer[43,44].
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Chapter 4
Result and Discussion
4.1 Profiles of pH and DO
Fig. 4 shows the profiles of pH and DO concentration in the SBR under various HRTs investigated. The pH profile was fairly constant over the HRTs, except the final 6 d of HRT, owing to the malfunction of the aerators on 3 d of HRT. The decrease in pH at any point of time was compensated by the addition of alkalinity to the reactor. If ammonium concentration increased in the effluent, the DO valve was adjusted in such a way that the excess ammonium undergoes partial nitrification. The DO concentration in the reactor was varying a lot in the initial days of operation, i.e. 9 d HRT. The activity of anammox bacteria and denitrifiers in the SNAD system relies on partial nitrification becuase the later supplies NO2--N to anammox and denitrification.
Moreover, anammox bacteria and denitrifiers prefer anoxic/anaerobic environment.
Therefore, there was some difficulty in controlling the air flow rate to the system in the initial days of SBR operation (0-19 d, Fig. 4). After this stage, the airflow was adjusted in such a way to maintain the DO of the reactor at a constant level. To measure the DO concentration precisely in the reactor DO was measured using the BOD bottle at the end of 3 d HRT. The DO concentration at HRT 6 d was close to 0.3-0.4 mg/L.
19 47 77 110 142 175 208 240 273 306 338 different HRTs. 3d a without aerator and water jacket problems, 3d b with aerator and water jacket problems.
4.2 Nitrogen and COD removals under various HRTs
At 9 d HRT, the SBR was operated with influent NH4+–N and COD concentrations of 200 mg/L and 100 mg/L, respectively, corresponding to the NLR of 22.2 g/m3-d and OLR of 11.1 g/m3-d. Table 6 compare the range of nitrogen loading rates used and total nitrogen removal under different nitrogen removal processes. As shown in Table 6, the loading rates of anammox and OLAND processes are lower than the present study, which evidences that autotrophic nitrogen removal can happen in lower loading rates also. Moreover, this is the first stage of SNAD seed sludge acclimation in the SBR; thus, the reactor was operated in moderate loading conditions to avoid substrate inhibition. The operating conditions of the SBR under various HRTs are shown in Table 6. The organic loading rate (OLR) and nitrogen loading rate (NLR) to the SBR under various HRTs were worked out, and are also shown in Table 5. However, the NH4+
-N and COD concentrations were kept constant under all HRTs and the ratio of influent COD/TN was maintained at a constant level (0.5).
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Table 5. The ranges of loading rate under different nitrogen removal processes Nitrogen Removal
Process
Requirement of O2/COD
Nitrogen loading (Kg N m-3 reactor d-1)
Total nitrogen removal (%)
Application status
Common reactor configuration
Reference
Conventional High/Yes 0.3-9 95 Full-scale Activated sludge [23,24]
SND Low/No 1-3.5 100 Lab-scale SBR [43]
ANAMMOX None/No 0.003-20 87 Full/Lab scale FBR,SBR [6,7,12,36]
SHARON Low/No 0.5-1.5 90 Full-scale Activated sludge [2,3]
CANON Low/No 0.04-3 75 Lab-scale SBR, UASB [4,3,5]
OLAND Low/No 0.001-0.1 85 Lab-scale SBR, RBC [5,37]
In this study Yes/Yes 0.022-0.066 95 Lab-scale SBR [9,10,12]
Table 6. Characteristics of synthetic wastewater before and after treatment
aVFR increased by 3 times, and without aerator and water jacket problems
bVFR increased by 3 times, and with aerator and water jacket problems
cTN is the sum of NH4
+-N, NO2
- -N and NO3
- -N;
dVFR represents volumetric flow rate and the increases, i.e.1.5, 2 and 3 times, based on 9 d HRT.
32
The influent and effluent profiles of nitrogenous matter and organics are shown in Fig.
4 and 5, respectively. In the first 40 d of operation, a consistent NH4+–N removal (more than 90%) was observed and small quantities of NO2--N and NO3--N accumulation were found in the SBR. However, SBR displayed a very poor COD removal efficiency (less than 65%) during this period. In the subsequent days (40-65 d), the removal efficiencies increased gradually and have shown a stable NH4+–N and COD removal efficiencies of 96% and 87%, respectively.
0 30 60 90 120 150 180 210 240 270 300 330
Fig.7. Performance of the concentration of nitrogen compounds and removal efficiency of ammonium and total nitrogen in the SBR at different HRTs. 3d a without
aerator and water jacket problems, 3db with aerator and water jacket problems.
0 30 60 90 120 150 180 210 240 270 300 330
Fig. 8. Performance of the concentration of COD and removal efficiency of COD in the SBR at different HRTs. 3d a without aerator and water jacket problems, 3db with aerator and water jacket problems.
In order to find the effect of loading rate on the SNAD process, the NLR and OLR were progressively increased by decreasing the HRT from 9 d to 4.5 d, and operated for 47 d (Table 5). Despite the higher influent NLR and OLR, a stable conversion of NH4+–N, without accumulation of NO2
--N/NO3
--N was observed in the SBR. The increased NLR (44 g/m3-d) and OLR (22 g/m3-d), decreased the COD removal efficiency of the SBR from 87% to 78%, whereas the NH4+–N removal efficiency was maintained in the same level, i.e. 95%. This reveals that the increase NLR and OLR have no significant effect on the SNAD system. Table 5 shows the steady-state concentrations of NH4+–N, NO2
--N, NO3--N and COD under various HRTs.
Following to the steady-state condition at 4.5 d HRT, the reactor NLR and OLR were further increased to 66 g/m3-d and 33 g/m3-d, respectively, also the HRT was decreased to 3 d. The decrease in the HRT to 3 d has decreased the NH4+–N and COD
34
removals in the system. An increasing trend in the effluent NH4+–N concentration can be noticed in Fig. 5. This indicates that the increases in NLR and OLR (at 3 d HRT) have produced slight inhibition/toxicity to the partial nitrifiers; as a result, insufficient NO2
Unexpectedly, aerator and water jacket were went out-of-order under this recovery stage, which drastically decreased the reactor performance. During this stage, the DO in the SBR has went down to below 0.2 mg/L, pH drop down to less than 6 and the temperature decreased by 5 to 8C. It can be noticed in Table 5 that only 52% of the NH4+–N was removed in the reactor, and interestingly, around 86% of the COD was removed in the reactor. Under this situation, it is hypothesized that Anammox bacteria might be inactive and the NO2
--N produced as a result of partial nitrification could have been utilized only by denitrifiers. In order not to increase further loading under these circumstances, the reactor NLR and OLR were decreased to 33 g/m3-d and 16 g/m3-d. The reactor started to recover when the HRT was increased from 3 to 6 days.
The effluent concentration of NH4+
-N was decreased from 94 mg/L to 25 mg/L, also by slightly adjusting the DO and pH value back to optimal condition, the removal efficiencies of NH4+–N and TN has come back to 75% and 67%, respectively. These observations and hypothesis indicate that high DO concentrations (>2 mg/L) could result complete nitrification in the SNAD system, whereas low DO concentration (<0.5 mg/L) could reduce the rate of nitrification and overall performance of the reactor. Moreover, these data reveal that SNAD process is more resistant to substrate
shock loading compared to sudden change in aeration rate and temperature.
Despite of stable conversion of ammonium, nitrate accumulation was detected in effluent from the end of HRT 3 d. Nitrate production was related to a possible response of different electron acceptors such as sulfate which supplied from (NH4)2SO4 in the medium instead of nitrite in SNAD process. In many researches, except for nitrite, nitrate and propionate, there might be some other electron acceptors for ammonium oxidation and sulfate is considered to be a suitable selection for its strong oxidization capacity. Polanco et al. (2001) showed the possibility of removing ammonium and sulfate simultaneously. They postulated that the nitrite formation and subsequent Anammox process were responsible for nitrogen removal according to the following equations (Eq. (13), (14), (15) and (16))[44]:
3SO42-+4NH4+→3S2-+4NO2-+4H2O+8H+ (13) 3S2-+2NO2-+8H+→N2+3S+4H2O (14) 2NO2-+2NH4+→2N2+4H2O (15) SO42-+2NH4+→N2+S+4H2O (16)
After disturbance during HRT 3 d, the reactor was in unsteady state, the end product of combining sulfate with ammonium might also produce nitrate as well. On the other hand, the long period acclimation of SNAD system may result to accumulation of sulfide which can be toxic to microorganisms. The sludge might be covered by sulfur which could limit the sufficient contact among reactants. The Anammox activity might also affect by some middle medium, such as nitrite, H2S and sulfur causing
36
nitrate accumulation. With decreasing Anammox activity, further works need to focus on reduction of the released sulfureted hydrogen and collection of sulfur from reaction.
4.3 Model based evaluation of SNAD
The consumption of nitrogen compounds in partial nitrification, Anammox and denitrification are modeled using the stoichiometric equations and the experimental data. Generally, the presence of organic carbon is inhibitory to anammox bacteria. For example, the presence of methanol is found to have irreversible inhibition at concentration as low as 0.5 mM. However, a recent study indicated that anammox bacteria were successful in the oxidation of propionate, and the presence of glucose, acetate, formate and alanine had no effect on the anammox process[45]. The free energy of denitrification using typical organic carbon is shown in Table 7 Moreover, anammox bacteria can be competitive with heterotrophic denitrifiers for the utilization of organic matter, i.e. propionate. But, the rate of propionate utilization by anammox bacteria was 0.6 mM/mg of protein/d, which is far less than the utilization rate by denitrifiers in real-time wastewater systems.
Table 7. Free energy of typical organic carbon with different electron donor in denitrificaiton [46-48].
Denitrification
(organic carbon)
Stoichiometric equation Free energy
(kJ/mol)
Acetate with nitrite NO2
-+0.375CH3COO-+H+ 0.5N2+0.375CO2+0.375HCO3
-+0.875H2O
-360
Methanol NO2
Acetate with nitrate NO3
-+0.625CH3COO-+H+
The following stoichiometic relationships are used for modeling: (i) the molar ratio of NH4+-N: NO2--N in partial nitrification is 1:1, (ii) the stoichiometric consumption
--N is used for consuming 1.74 mg/L COD in denitrification. The TN removal in partial nitrification with Anammox and denitrification under all the HRTs based on the stoichiometric modeling are shown in Table 8. Moreover, the detailed modeling concept and the outcomes for 3 d HRT based on the average influent and effluent data are shown in Fig. 9.
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Fig.9. Model based evaluation of the SNAD system.
Table 8 indicates that around 85-87% of the TN removal is by the combination of Anammox and partial nitrification. The NO3
--N produced in Anammox process is utilized in denitrification along with COD, which is responsible for a TN removal of 7-9%. These observations indicate that under steady-state condition all three processes in the SBR, i.e. partial nitrification, Anammox and denitrification, synchronize each other and establish a firm relationship within the reactor irrespective of the NLR and OLR. However, the shock in the operating DO, pH and temperature of the SNAD system greatly affected the relationship of these processes. This can be evidenced from the poor NH4+-N removal efficiency of the system (52%). However, the overall TN removal efficiency of the SNAD system was maintained around 50.7%
owing to the consumption of NO2--N and/or NO3--N in denitrification. The stoichiometric modeling results also indicate that the decrease in the HRT of the system (from 9 to 3 d) could facilitate the increase in the production of Anammox bacteria (from 0.067 to 0.357 g/d). This approach could be useful to enrich the slow growing Anammox bacteria in the real-time conditions. However, a very high volumetric flow rate (VFRs) could wash out the Anammox bacteria from the system.
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Table 8. Performance of the SBR under various HRTs
HRT (d)
TN removal (%) Biomass
produced (g/d)
Sensitivity Index (SI)c*
Partial nitrification
+ anammox denitrification NH4 NO2- NO3- COD
9 85.7% 8.7% 0.067 - {9} - {2.1} - {1} - {14}
4.5 87.3% 7.8% 0.259 0.4（13） 0.1（2.3） 0.8（1.8） 0.9（27）
3a 85.5% 7.3% 0.357 1.3（21） 0.7（3.6） 2.6（3.6） 1.8（39）
3b 41.9% 8.7% 0.305 14（135） 0.1（2.2） 0.6（1.6） 2.6（50）
6 32.2% 8.8% 0.197 14（133） 2.5（7.5） 91（92） 1.5（35）
aVFR increased by 3 times, and without aerator and water jacket problems
bVFR increased by 3 times, and with aerator and water jacket problems
cSensitivity index based on the species concentration at 9 d HRT
* The values within ―{}‖ and ―()‖ indicates average and maximum concentrations in mg/L, respectively
Alternatively, the sensitivity of the SNAD system to the change in VFR was evaluated based on sensitivity index (SI) as shown in Eq. (17) [49].
(17)
where, Omax is the maximum concentration of substrate in the effluent at 4.5, 3 and 6 d HRTs (mg/L), and Os is the average concentration of substrate in the effluent at 9 d HRT (mg/L). The values of SI for all nitrogen species and COD are shown in Table 8.
The SI values indicate that the SNAD process is not greatly affected by the change in VFR of the system compared to the shock in the operating DO, pH and temperature conditions. Under the shocking DO, pH and temperature conditions, the SI values increased by 14 and 2.6 times for NH4+-N and COD, respectively. As indicated before, the Anammox bacteria might be inactive under the shocking condition and the NO2
--N produced as a result of partial nitrification could have been utilized only by denitrifiers.
This reveals that the SNAD system has the capability of acting as shortcut nitrification-denitrification (SND), i.e. NH4+-N is oxidized to NO2--N in nitritation, and subsequently, the NO2
--N is reduced to N2 gas. However, the removal efficiency of the SND system (under shocking condition) is far less than the efficiency observed in the SNAD system.
4.4 Comparison between full-scale SNAD system with lab-scale SNAD system
Landfill is the most common methods of organized waste disposal and remained so in many places around the world. A large number of adverse impacts may occur from landfill operations. One of the impacts is from landfill leachate which contained
42
organic and inorganic matters characterized by high concentration of nitrogen compounds generated during decomposition of waste in the landfill. Leachate has the specific meaning of having dissolved or entrained environmentally harmful substances which may then enter the environment. In older landfills and those with no membrane between the waste and the underlying, leachate is free to egress the waste directly into the groundwater. The most common method of handling collected leachate is on-site treatment.
The full scale SNAD system is applied in landfill leachate treatment plant [10]. The aeration tank is treating an average leachate flow of 304m3 d−1 with a sludge retention time between 12 and 18 d. Similarly, the full-scale SNAD system was evaluated by the model and sensitivity index describe in previous section. Table 9 shows the result of full-scale SNAD system, it indicated that the nitrogen removal mainly by partial nitrification and Anammox. In 2010, annual precipitation amounts vary from less than 332 mm/month to more than 479 mm/month. This makes the influent concentration varies a lot and the nitrogen removal percentage of partial nitrification and Anammox below than 50%. Moreover, the sensitivity index of ammonium in 2010 has significant effect on the performance of SNAD system.
The comparisons between full-scale and lab-scale SNAD system: (1) Despite of heavy rain in 2010, the full-scale SNAD system demonstrated a stable and high treatment performance for nitrogen removal from actual landfill leachate. Due to the small volume of lab-scale reactor, the buffer capacity of lab-scale SNAD system is way more sensitive than full-scale system, and (2) The operation of lab-scale SNAD system can be more precise on controlling different parameters, such as pH value and DO. pH value in the optimal range to maintain the concentration of free ammonia between 3.5
to 10 mg NH3-N/L, which made sure the nitrification process stop at ammonium oxidation step and DO concentration in a range of 0.3 to 0.4 mg/L in case nitrite accumulate in the reactor.
Overall, the SNAD process will offer a great future potential for removing nitrogen and organic compounds, it can save energy consumption and cost of adding extra chemical, from wastewater in the industrial application, especially from optoelectronics industrial wastewater in Taiwan.
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Table 9. Performance of the full-scale SNAD system under different years
Years
TN removal (%) Sensitivity Index (SI)b*
Partial nitrification
+ anammox denitrification NH4 NO2-
NO3
-COD
2009 71.6% 22.2% -（224） -（-） -（160） -（492）
2010 47% 23% 2.3（468） -（-） 1（125） -0.14（491）
2011a 65.2% 13% 1.8（393） -（-） 1（125） 0.16（319）
aThe average and maximum concentrations is in 2011/1/1-2011/5/31.
bSensitivity index based on the species concentration in 2009
* The values within ―{}‖ and ―()‖ indicates average and maximum concentrations in mg/L, respectively
4.5 Diversity of the bacterial community in SNAD system
Biotechnological analysis such as FISH and real-time quantitative PCR (qPCR) were conducted to verify the presence of microbial community in SNAD system. Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction, is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, real time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. The procedure follows the general principle of PCR; its key feature is that the amplified DNA is detected as the reaction progresses in real time. This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.
The seed sludge was inoculated from a landfill leachate treatment plant. Figure 10 (a) shows the red granules from aeration tank which found to be typical in Anammox reactor s [10]. Fig. 10 (b) shows the attached growth of Anammox bacteria on the aeration tank wall.
46
(a)
(b)
Fig. 10 Pictures of red granules from aeration tank (a) Granules in the aeration tank, (b) attached growth of Anammox bacteria on the aeration tank wall.
Red granules taken from landfill leachate treatment plant were analyzed by FISH to confirm the occurrence of Anammox bacteria. Fig.11 (a) shows that all bacterial cells were stained with DAPI；Fig 11 (b) shows that all Anammox bacteria hybridized with probe Amx820.
(a) (b)
Fig. 11. Fluorescence micrographs of bacteria granules collected from the aeration tank (a) DAPI, (b) Amx820
Moreover, 16S rRNA clone analysis revealed that all clones from aeration tank were related to Kuenenia stuttgartiensis, Candidatus Kuenenia Stuttgartiensis and Anaerobic ammonium oxidizing planctomycete KOLL2a with 99% sequence similarity (Table 9).
Similarities with other species are also listed in Table 9. Furthermore, the presence of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were confirmed by using qPCR and Terminal Restriction Fragment Length Polymorphism (TRFLP or sometimes T-RFLP). TRFLP is a molecular biology technique for profiling of microbial communities based on the position of a restriction site closest to a labeled end of an amplified gene. The method is based on digesting a mixture of PCR
10μm 10μm
48
amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer.
The result is a graph image where the X axis represents the sizes of the fragment and the Y axis represents their fluorescence intensity.
The R2 value of qPCR is greater than 0.97 for all curves and amplification efficiencies with slopes of -3.44 and -3.17. Two standard curves were constructed using cloned 16S rDNA sequence of eubacteria and Anammox bacteria into pGEM-T (Promega, USA) cloning vector respectively.
Table 10 and 11 provide the detail experimental outcomes of qPCR and the ratio of different bacteria to eubacteria and Anammox bacteria. The relatively quantification
Table 10 and 11 provide the detail experimental outcomes of qPCR and the ratio of different bacteria to eubacteria and Anammox bacteria. The relatively quantification