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Complete nitrogen removal over nitrite (CANON) was used to treat reject water with ammonia concentrations ranging from 70 to 154mg·L-1. Two experimental sequential batch reactors, SBR_A and SBR_B, differed in the time of the reject water inflow (6h40min vs 40min), process temperature (25 vs 29°C), and the © UNIVERSITY OF WARMIA AND MAZURY IN OLSZTYN nitrogen concentrations in raw reject water on the biodiversity of anammox bacteria is not entirely clear. Anammox bacteria need very specific environmental conditions to grow, and their growth is inhibited in nitrogenrich wastewater, which has restricted the application and industrialization of the anammox process (Jin et al. 2012). In general, these bacteria grow very slowly (doubling time at 30-40°C is 10-14 days (Strous et al. 1998; van der Star et al. 2007)), their cell yield is low (Strous et al. 1998, 1999), and they are highly sensitive to changes in environmental conditions, which makes them very difficult to cultivate. Here, we use the completely autotrophic nitrogen removal over nitrite process (CANON) to enable simultaneous partial nitrification and the anammox process in two sequencing batch reactors (SBRs) fed with diluted reject water from anaerobically digested sludge dewatering. We report that the biodiversity of the bacterial community was higher in the SBR with the higher temperature (29°C), shorter time of wastewater inflow (40 min), and six aeration periods per day than in the SBR with the lower temperature (25°C), longer periods of wastewater inflow (6 hours and 40 minutes), and 3 aeration periods per day. IN TRO DUC TION In wastewater treatment, reject water from sludge dewatering has a high ammonium-nitrogen concentration, which if recirculated to the main treatment stream, can cause problems with nitrification and denitrification, and affect the balance of the microbial community. Separate treatment of reject water is a solution to this problem that reduces the nitrogen load of the main stream and improves nitrogen removal (Fux et al. 2002). For treating wastewater with a high concentration of ammonium nitrogen and a low C/N ratio, a combination of partial nitrification with the anammox (anaerobic ammonium oxidation) process is a promising method (Van Loosdrecht and Jetten 1998). In the first stage, about 50% of the influent ammonium can be oxidized to nitrite, reducing the amount of oxygen required by almost a half (Furukawa et al. 2009). This helps to make the combination of processes a cost-effective solution. Many researchers have studied the process from a technological point of view (Fux et al. 2002; Galí et al. 2007; Vega–De Lille et al. 2015; Zhang et al. 2010), but the impact of the high and unstable Karło et al. Bacterial biodiversity in CANON systems 27 MATERIALS AND METHODS Experimental systems Reject water was treated in two laboratory-scale SBRs, the volume of each SBR was 10L, the hydraulic retention time was around 1.6 days, and the sludge age was 50-100 days. In both SBRs, the CANON process was conducted, but the reactors differed in technological conditions. In SBR_A, the temperature was 25.0 with a range of ±0.5°C (25.0±0.5°C) and the feeding time was 6 hours and 40 minutes. In SBR_B, the temperature was 29.0±0.5°C, and feeding time was 40 minutes. In both reactors there were three 8-hour cycles per day (Figure 1). For inoculation of both reactors, two-thirds of the final reactor volume was filled with regular activated sludge from municipal wastewater and one-third with digested sludge. Total suspended solids (TSS) content was 0.5g·L-1. The SBRs worked from July 2013 to January 2014 (181 days). The influent (1.5±0.2L·cycle-1) contained NH4-N between 70-154mg·L-1, NO2-N between 0.4-24.0mg·L-1, and NO3-N between 2.5-15.0mg·L-1. The oxygen concentration was 0.25-0.30g·m-3 during the aeration phase and dropped to 0.00-0.05g·m-3 in the mixing phase of each cycle. Samples of sludge were collected from both SBRs at Figure 1. Different working conditions in the two experimental bioreactors: in SBR_A, the temperature was lower (25°C), the inflow of wastewater was longer (6h and 40min), and there were 3 aeration periods per day, whereas in SBR_B, the temperature was higher (29°C), the wastewater inflow was shorter (40min), and there were 6 aeration periods per day. Concentration of nitrogen forms To measure the concentrations of nitrogen forms (NH4-N, NO2-N, NO3-N) in the influent (raw, diluted reject water) and in the effluent, colorimetric methods based on Merck Spectraquant® quick tests were used. approximately two-week intervals. To prevent RNA degradation, RNA Later (Sigma-Aldrich) was added to each sample according to the manufacturer’s instructions. Then samples were frozen and stored at -45°C until examination. Figure 2. Concentrations of nitrogen forms in two experimental SBRs in which the CANON process was conducted. In the influent, NH4+‐N concentration varied between 70‐154mg·L‐1. The anammox process had started after day 65-70 of the experiment, resulting in a decrease in the concentration of nitrite and an increase in that of nitrate in the effluents from both SBRs. 28 ENVIRONMENTAL BIOTECHNOLOGY 12 (1) 2016 template was transcribed to cDNA with a TranScriba Kit (A&A Biotechnology) according to the manufacturer’s instructions. Amplification of the 16S rRNA gene of all bacteria was performed using the following primers: 338F-GC and 518R. The sequence of the forward primer was 5’-(CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC) TAC GGG AGG CAG CAG-3’ and that of the reverse primer was 5’-ATT ACC GCG GCT GCT GG-3’ (Muyzer et al. 1993). The amplification of PCR products was performed in a C1000TM thermal cycler (BioRad) in a 30μL reaction containing 1.5 U GoTaq Flexi Polymerase (Promega), 1×buffer, 2mM MgCl2, 5pmol·μL-1 of each primer, 20pmol·μL-1 of dNTPs and 0.5μL template DNA or cDNA. The temperature cycling conditions for the amplification of the 16S rRNA partial gene were previously described by Muyzer et al. (1993). PCR products were electrophoresed in 1% agarose gel with a 1kb DNA Ladder (Promega) and visualized under UV light. Molecular analyses Isolation of genetic material Total genomic DNA was extracted from 0.2g of the sludge samples using a mechanical method described by Ziembiƒska et al. (2014). Total RNA was extracted from samples using a commercial Total RNA Mini Plus Kit (A&A Biotechnology) according to the manufacturer’s instructions. The amount of DNA and RNA isolated from samples was measured spectrophotometrically (in triplicate) using Qubit (Invitrogen) and stored at -20°C until PCR was performed. Reverse transcription reaction and PCR conditions Total RNA isolated from the samples was used as a template for reverse transcription. In the first step, the inactivation of DNases was performed with RQ1 RNase-Free DNase (Promega) according to the manufacturer’s instructions. Next the RNA Figure 3. Total nitrogen concentrations and the efficiency of nitrogen removal in two experimental SBRs. SBR_A performed in an unstable manner after 50 days; its removal efficiency ranged between 40 and 80%. SBR_B performed in a stable manner. After 77 days of adaptation, its removal efficiency ranged from 52 to 90%. On the basis of the PCR-DGGE fingerprints, dendrograms were created using the Neighbor Joining with Dice coefficient method. The number of OTUs (operational taxonomic units) was defined as the number of lanes in the gel. RESULTS AND DISCUSSION Efficiency of nitrogen removal During the whole experiment, the NH4-N concentration fluctuated from 70 to 154mg·L-1, according to the composition of the digested sludge and the fermentation efficiency. In the beginning of the experiment (adaptation phase), nitrogen removal was very low (5-23%). After 30 days of the experiment, the concentration of nitrites peaked in the effluent, indicating that the balance had shifted towards nitrification. The anammox process, for Denaturing gradient gel electrophoresis (DGGE) PCR products obtained with the 338F-GC and 518R primers were electrophoresed in the Dcode Universal Mutation Detection System (BioRad). Polyacrylamide gel (8%, 37.5:1 acrylamide-bisacrylamide, Fluka) with a gradient of 30-60% denaturant was prepared according to the manufacturer’s instructions. Electrophoresis was performed in a 1×TAE buffer at a constant temperature of 60°C for 16 hours at 40V. Gels were stained with SYBR Gold (1:10 000, Invitrogen) in MiliQ water for 30min and destained in MiliQ water for the next 30min, then visualized under the UV light. DGGE fingerprints were analyzed using Quantity One 1D software (BioRad). The Shannon-Wiener diversity index was calculated using the relative intensity of the bands in each sample, as previously described by Watanabe et al. (2004). Karło et al. Bacterial biodiversity in CANON systems 29 which nitrites are the substrate, had occurred after 65-70 days of the experiment (Figure 2). At this stage the concentration of nitrites had decreased and that of nitrates began to increase in the effluents from both SBRs. SBR_A performed in an unstable manner (Figure 3). After 50 days, its nitrogen removal efficiency ranged between 40 and 80%, and tended to decrease after 90 days of the experiment. In SBR_B, adaptation lasted for 77 days, after which its nitrogen removal efficiency was 50-90%, without any clear trend (Figure 3). Nitrogen removal was stable in SBR_B for the final 104 days of the experiment. Chu et al. (2015) showed that 20 Figure 4. DGGE fingerprints of bacterial 16S rRNA gene fragments that were amplified from total DNA samples taken from the two experimental SBRs. S inoculum; A, B, C, D, E locations of dominant bands. Figure 5. Shannon-Wiener bacterial biodiversity index for all bacteria in samples taken from SBR_A and SBR_B (based on the DGGE results presented in Figure 4). The structure of the bacterial community was more stable in SBR_B than in SBR_A. days were needed to achieve 20-30% nitrog

Evaluation with scanning electron microscopy of Cd, Cu, and Zn removal from aqueous solutions by ash from gasification of poultry feathers