Low-Temperature and High-Efficiency Solid-Phase Amplification Based on Formamide.

The thermal stability of DNA immobilized on a solid surface is one of the factors that affects the efficiency of solid-phase amplification (SP-PCR). Although variable temperature amplification ensures high specificity of the reaction by precisely controlling temperature changes, excessively high temperatures during denaturation can negatively affect DNA stability. Formamide (FA) enables DNA denaturation at lower temperatures, showing potential for SP-PCR. Research on FA's impacts on DNA microarrays is still limited, necessitating further optimization in exploring the characteristics of FA in SP-PCR according to particular application needs. We immobilized DNA on a chip using a crosslinker and generated DNA microarrays through bridge amplification based on FA denaturation on our automated reaction device. We optimized the denaturation and hybridization parameters of FA, achieving a maximum cluster density of 2.83 × 104 colonies/mm2. Compared to high-temperature denaturation, FA denaturation required a lower template concentration and milder reaction conditions and produced higher cluster density, demonstrating that FA effectively improves hybridization rates on surfaces. Regarding the immobilized DNA stability, the FA group exhibited a 45% loss of DNA, resulting in a 15% higher DNA retention rate compared to the high-temperature group, indicating that FA can better maintain DNA stability. Our study suggests that using FA improves the immobilized DNA stability and amplification efficiency in SP-PCR.

[Pre-precipitation of Sewage-SNAD Granular Sludge Process Test].

Using artificial water, the simultaneous partial nitrification, ANAMMOX (anaerobic ammonium oxidation), and denitrification (SNAD) granular sludge process was started in a sequencing batch reactor (SBR), and then the ammonia concentration in the influent was reduced gradually. After stable operation for a period of time under the low ammonia concentration, sewage treated by a pre-precipitation process was used as a substrate to investigate the performance and stability of the SNAD granular sludge process. The results show that after the SNAD process was successfully started, the ammonia removal rate was greater than 98%, and total the nitrogen removal rate was about 89%. As the influent ammonia concentration decreased, the nitride-oxidizing bacteria (NOB) activity was increased and the total nitrogen removal rate gradually decreased to 75%. When the pre-precipitated domestic sewage (NH4+-N 52-63 mg·L-1, COD 99-123 mg·L-1) was used as the inflow, the average effluent removal rate of the total effluent was 73.2%, the effluent COD concentration was below 35 mg·L-1, and the maximum effluent ammonia nitrogen and total nitrogen concentration were 0.7 mg·L-1 and 12.8 mg·L-1. The ammonia and total nitrogen concentration in the continuous 30 day effluent reached the 1A level of the integrated discharge standard of water pollutants for municipal wastewater treatment, indicating that the removal of organics and nitrogen from domestic sewage was achieved efficiently and synchronously.

[Startup Strategies for the SNAD Granular Sludge Process at Low Temperature].

To study the effect of the startup strategies on the simultaneous partial nitrification, ANAMMOX, and denitrification (SNAD) granular sludge processes, these processes were initiated by starting the completely autotrophic nitrogen removal over nitrite (CANON) process and anaerobic ammonia oxidation-denitrification (SAD) process at 12.7℃ and 18.3℃, respectively. The results show that the ammonia nitrogen was almost completely removed and the total nitrogen removal rate reached 86.7% after the R1 reactor was successfully started. When the ammonia concentration was low, the total nitrogen removal rate in the effluent decreased to 75.3%, the total nitrogen concentration in the effluent was~10 mg·L-1, and excessive proliferation of the NOB was observed. The total nitrogen concentration in the effluent exceeded the 1A level of the integrated discharge standard of water pollutants applied in Beijing City. After the R2 reactor was successfully started, the effluent contained almost no ammonia nitrogen and the total nitrogen removal rate was~89.1%, that is, slightly higher than that of the R1 reactor. When the ammonia concentration was low, the concentration of ammonia nitrogen in effluent was less than 1.0 mg·L-1 and the total nitrogen concentration in the effluent was less than 6 mg·L-1. The concentrations of ammonia nitrogen and total nitrogen in the effluent reached the 1A level of the integrated discharge standard of water pollutants applied in Beijing City. First, the startup of the SAD process gradually eliminated the NOB from the system through anaerobic operation in the initial stage of the startup, maintained the stability of the system, provided a good basis for the subsequent aeration to start the SNAD process, maintained the stable operation of the reactor, and the long-term discharge of total nitrogen reached the standard.

[Lab-scale ANAMMOX Process in a Wastewater Treatment Plant].

A lab-scale, completely anaerobic ammonium oxidation (ANAMMOX) process was operated in a municipal wastewater treatment plant (WWTP). Sewage effluent treated by an A/O process and nitrification process was input as the substance to start up the up-flow ANAMMOX filter reactor. After the 109th day, the ammonia removal rate and nitrite removal rate were greater than 90% for 15 successive days and the nitrogen removal rate was higher than 70%. The ANAMMOX filter reactor successfully started up. From days 245 to 333, the reactor was running during the winter. The weight of biomass reached 12.24 mg·g-1, and the average nitrogen removal rate was 54.3%. Backwash was adopted at day 461, and the weight of biomass decreased to 8.01 mg·g-1. From days 605 to 693, the reactor was running in the winter again. The weight of biomass was 10.41 mg·g-1, and the average nitrogen removal rate was sustained at 69.7%. Compared with the previous winter, the weight of biomass was lighter but the total nitrogen removal loading was 23% greater. For the entire operation, the ANAMMOX rate at high temperature was stable but that at low temperature increased from 1.5 kg·(kg·d)-1 to 3.6 kg·(kg·d)-1. The results show:Long-term domestication at low temperature was in favor of improving treatment efficiency of ANAMMOX process in cold environment and realized ANAMMOX process operated efficiently in winter.

[Effect of Carbon Source on Lab-scale SAD Process in a Wastewater Treatment Plant].

Lab-scale anaerobic ammonia oxidation and denitrification (SAD) processes were operated simultaneously in a municipal waste water treatment plant (WWTP). Sewage treated by the A/O and nitrification process was used as the substance to start up an anaerobic ammonia oxidation filter reactor. Adding glucose and sodium propionate to influent was used as the substance to start up the SAD filter reactor after the successful start-up of the ANAMMOX reactor. The SAD process performed well with an average total nitrogen concentration in the effluent of 6.41 mg·L-1 when 30 mg·L-1 glucose was added to the effluent sewage at ambient temperature. Compared with the ANAMMOX process, the total nitrogen concentration in the effluent from the SAD process decreased 42%. The stability of the SAD process was destroyed and the SAD process turned into a denitrification process when 30 mg·L-1 glucose was added in the influent sewage in a low temperature environment. In normal and low temperature environments, the SAD process functioned well, and the average total nitrogen concentration of the effluent was 6.54 mg·L-1 when 30 mg·L-1 sodium propionate was added in the influent sewage. Compared with glucose, sodium propionate had little influence on the SAD process.

[Lab-scale SNAD Process in Wastewater Treatment Plant].

Outside the municipal waste water treatment plant(WWTP) which located in Mentougou District, Beijing, the effluent of the anoxic/oxic(A/O) phosphorus removal process served as the substrate to operate a completely autotrophic nitrogen removal over nitrite(CANON) filter reactor.. After the reactor was successfully activated, glucose was added to the influent as the organic carbon source. The simultaneous partial nitrification, anaerobic ammonium oxidation (ANAMMOX), and denitrification (SNAD) process was started to study the effect of SNAD filter on sewage treatment. The results showed that from 119 d to 128 d, the ammonia removal rate of the CANON process was more than 95%, and the maximum total nitrogen concentration in the effluent was 13.0 mg·L-1. Total nitrogen concentration surpassed the 1A level of the Integrated Discharge Standard of Water Pollutants applied in Beijing City. The SNAD process was started by adding glucose to the influent at 129 d. The total nitrogen removal rate of this process was about 85% at 133-187 d, and the total nitrogen concentration in the effluent was 5.5-7.3 mg·L-1. The filter plugged up at 195 d, and backwash was utilized at 196 d. During the subsequent 30 d, the total nitrogen removal rate of the reactor was greater than 85%, and the total nitrogen concentration in the effluent remained at 6.2-7.2 mg·L-1. Compared with the CANON process, the SNAD process improved the total nitrogen removal rate and reduced the total nitrogen concentration of the effluent by 6 mg·L-1. The ammonia and total nitrogen concentrations in effluent satisfied the 1A level of the Integrated Discharge Standard of Water Pollutants.

[Laboratory-scale CANON Processes Applied to Wastewater Treatment Plants].

A laboratory-scale completely autotrophic nitrogen removal over nitrite (CANON) process was operated in a municipal wastewater treatment plant (WWTP). Sewage effluent treated by the anaerobic/oxic (A/O) process and was used to operate a WWTP to obtain the initial substance for the start-up of a CANON filter reactor. On the 48th day, the ammonia removal rate was measured at greater than 90% in successive 10 d samples and the nitrogen removal rate was greater than 70%. The CANON filter was successful at start up. From the 49th to the 129th day, the dissolved oxygen in the reactor was maintained at fairly low concentration of 0.2-0.5 mg·L-1. The effluent contained nearly no ammonia and the maximum total nitrogen (TN) concentration was 15.6 mg·L-1, which exceeded the national Class 1A Discharge Standards for pollutants from municipal wastewater treatment plants. Nitrite oxidizing bacteria (NOB) proliferated excessively in the reactor. Backwash was implemented on 129th, 169th and 213th days. The nitrogen removal rate was more than 70% for a long time and TN concentration in effluent was below 12 mg·L-1. The nitrogen concentration in effluent fitted the national Class 1A Discharge Standards and the NOB were effectively inhibited. These results show that backwash has negligible on the structure of filter and its impact on the thickness of the bio-membrane and its functional bacteria was small, however, it is capable of effectively inhibiting the activity of the NOB. Periodically backwashing can be utilized as an engineering application to maintain stable operation of the CANON process.