Chapter 4 Result and Discussion
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 percentage of different bacteria to eubacteria are shown in Table 12.
Table 10. Outcomes of Sequence analysis [10].
Species Identified Similarity (%) NCBI No. Reference
Kuenenia stuttgartiensis 99 CT573071.1 [50]
Candidatus Kuenenia Stuttgartiensis
99 AF375995.1 [51]
Anaerobic ammonium oxidizing planctomycete KOLL2a
99 AJ250882.1 [52]
Candidatus Brocadia fulgida 93 EU478693.1 [53]
Planctomycete KSU-1 93 AB057453.1 [40]
Candidatus Brocadia fulgida 93 DQ459989.1 [54, 55]
Candidatus Jettenia asiatica 92 DQ301513.1 [56]
Table 11. Detail outcomes of qPCR.
Average Ct of eubacteria 18.54 23.20 17.32
Copy number 2.63*106 1.17*105 5.93*106
Average Ct of total Anammox bacteria
16.69 22.42 18.46
Copy number 7.90*105 1.24*104 2.19*105
＊Each value was calculated from triplet
Table 12. Relatively quantification of different bacteria to eubacteria
DNA Red granule
AOB /EB 0.075198236 0.019569671 0.227247028
Average Ct (NOB) 22.01 23.90 18.84
SD (NOB) 0.2735 0.1180 0.2828
NOB /EB 0.0896855 0.613658437 0.348210915
Average Ct (KS) 0.0897 0.6137 0.3482
SD (KS) 0.4494 0.0394 0.1800
KS / TA 0.828922332 18.0136671 0.040009267
*AOB: ammonium oxidizing bacteria; EB: eubacteria; NOB: nitrite oxidizing bacteria
Table 13. Relatively quantification of different microbial community in SNAD system
*BA: Brocadia anammoxidans
Red granule (LWP/fresh)
KS / EB AOB / EB NOB / EB BA / EB
Ratio (SD) 65.25 (2.78) 7.42 (1.12) 9.71 (2.88) 11.05 (4.1)
50
TRFLP with primers amoA-1F and amoA-2R was also carried out to confirm the presence of AOB. The results of TRFLP analyses obtained are shown in the figure 12 which indicated the presence of Nitrosomonas europaea and Nitrosomonas oligotropha.
Fig.12. The experimental outcomes of TRFLP analysis.
Chapter 5 Conclusion
The SNAD process was successfully developed in the SBR and the effect of HRTs on the performance of SNAD system were investigated. Around 96% NH4+
-N removal and 87% COD removal were achieved under the NLR and OLR of 22.2 and 11.1 g/m3-d, respectively. The increases in NLR and OLR up to 66.7 and 33.3 g/m3-d have produced little impact on the performance of the reactor; whereas, the sudden reduction/shock in the operating DO, pH and temperature has produced a major drop in the SNAD performance. However, the performance was able to recover from disturbances when NLR and OLR were decreased to 33 g/m3-d and 16 g/m3-d, respectively. The removal of nitrogenous compounds in each of the SNAD process was modeled using the stoichiometric relationship. The presence of Anammox bacteria in the SNAD process was confirmed using FISH, qPCR and TRFLP. Overall, SNAD process has potential to successfully remove the nitrogen and carbon compounds from wastewater that can be applied in industrial application for the future.
52
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