The wastewater sample (substrate) was collected from a biofiltration sample point of a local South African municipality wastewater treatment plant in the KwaZulu-Natal province. The activated sludge sampled from the anaerobic digester point source was used as the inoculum. An inoculum to substrate ratio of 3:5 (volume basis) was used for each 1 L bio-digester. Standard methods for the examination of water and wastewater [43 ] were employed to characterise the substrate and inoculum in triplicates with the results shown in Table 2 . Color and turbidity were analysed with the spectrophotometer (HACH DR3900, Hach Company, Loveland, CO, USA) and turbidity meter (HACH 210, Hach Company, Loveland, CO, USA), respectively. Using the COD high range vials (HACH), 0.2 mL of the samples were measured and poured into the COD vials. It was then digested at 150 °C for 2 h. After the digestion, the vials were cooled at room temperature and the COD was measured using the spectrophotometer (HACH DR3900, Hach Company, Loveland, CO, USA).
Dr3900
Manufactured by HACH
Sourced in United States, Germany
The HACH DR3900 is a laboratory spectrophotometer designed for accurate and reliable measurement of a wide range of water quality parameters. It features a high-resolution color display, intuitive user interface, and pre-programmed methods for common water testing applications.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Hq40d, by HACH (3 mentions)
Hach2100n, by HACH (2 mentions)
7890a gas chromatograph, by Agilent Technologies (2 mentions)
Db ffap column, by Agilent Technologies (2 mentions)
Multi n c 3100, by Analytik Jena (2 mentions)
Hq30d, by HACH (2 mentions)
Phc301, by HACH (2 mentions)
Hq40d multi, by HACH (2 mentions)
Infinite m200 pro, by Tecan (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dr900, by HACH (1 mentions)
Nova nanosem 450, by Thermo Fisher Scientific (1 mentions)
Hi98130, by Hanna Instruments (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch, by Bio-Rad (1 mentions)
Avanti j 20 xp, by Beckman Coulter (1 mentions)
Optima 8300, by PerkinElmer (1 mentions)
Ph meter, by Metrohm (1 mentions)
Lck 314, by HACH (1 mentions)
99 protocols using dr3900
1
Characterization of Wastewater Substrate and Inoculum
2
Wastewater Characterization Methods
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In this study, all parameters such as pH, COD, turbidity, colour and TSS were measured according to APHA Standard Methods for the Examination of Water and Wastewater [38 ]. For pH and turbidity, a pH meter (Metrohm, Switzerland) and a turbidimeter (HACH 2100AN, USA) were used, respectively. The other parameters COD and colour were determined according to the Reactor Digestion Method (Method 8000) (HACH DR3900, USA) [39 (link),40 (link)] and the ADMI Weighted Ordinate Method (Method 10048) [41 (link)] (HACH DR3900, USA) while TSS was analysed using the Photometric Method for suspended solids in a liquid (Method 8006) [41 (link)].
3
Biomass growth and nutrient consumption
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Biomass growth was monitored spectrophotometrically by absorbance at a wavelength of 660 nm (DR3900, Hach, Germany) 4–5 times a week (Supplementary Material 3 ), and gravimetrically by quantifying the concentration of volatile suspended solids (VSS) as described in experimental methods for wastewater treatment (van Loosdrecht et al., 2016 ). For the 40-h batch and SBR1, absorbance measurements were adequate since the biomass was low concentrated and in suspension. For SBR2 and SBR3, the biomass aggregated and VSS measurements were much more accurate.
The consumption of the dissolved nutrients was monitored by sampling the mixed liquor at the beginning and end of the reaction phase, after centrifugation (5 min, 17000 × g) and filtration of the supernatant on 0.45-μm filters (Millex-HV, PVDF, Germany). The concentrations of COD, ammonium (as N-NH4+) and orthophosphate (as P-PO43–) were measured by colorimetric assays (LCK kits no. 114/614/COD, 302/303/ammonium, 348/350/phosphate; Hach-Lange, Dusseldorf, Germany) followed by spectrophotometry (DR3900, Hach, Germany). The COD colorimetric method measured all oxidizable substances (here notably acetate, yeast extract, and EDTA from the trace element solution). As technical control, samples were measured in triplicates and the relative standard deviation was 0.5 – 1.9%.
The consumption of the dissolved nutrients was monitored by sampling the mixed liquor at the beginning and end of the reaction phase, after centrifugation (5 min, 17000 × g) and filtration of the supernatant on 0.45-μm filters (Millex-HV, PVDF, Germany). The concentrations of COD, ammonium (as N-NH4+) and orthophosphate (as P-PO43–) were measured by colorimetric assays (LCK kits no. 114/614/COD, 302/303/ammonium, 348/350/phosphate; Hach-Lange, Dusseldorf, Germany) followed by spectrophotometry (DR3900, Hach, Germany). The COD colorimetric method measured all oxidizable substances (here notably acetate, yeast extract, and EDTA from the trace element solution). As technical control, samples were measured in triplicates and the relative standard deviation was 0.5 – 1.9%.
4
Polysaccharide Quantification in EPS
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Polysaccharides were quantified following a method developed by [26 (link)]. In short, the freeze-dried EPS extracts were diluted in a sodium hydroxide solution (0.01 M in ultrapure water) to a final concentration of 1000 mg/L. For the standards, a sugar mixture of 1000 mg/L was prepared with equal amounts of fucose, rhamnose, galactose, glucose, xylose, mannose, and ribose. Sugars were selected due to their previous detection in bacterial EPS [27 (link)]. The sugar mixture was diluted to concentrations of 0, 10, 25, 50, 75, 100, and 200 mg/L in order to establish the calibration curve. Then, 200 µL of each sample or standards were pipetted in a glass reaction tube, followed by 200 µL of 5% w/v phenol solution and 1000 µL of 95% sulfuric acid. Tubes were vortexed, left at room temperature for 20 min and vortexed again before analyses. The absorbance of the samples and standards were measured at 482 nm with a spectrophotometer (DR3900 Hach, Loveland, CO, USA).
5
Wastewater Characterization and Biogas Analysis
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Physicochemical characteristics of the wastewaters from the Organic Export Abattoir, HR and MR effluents were analyzed following standard methods (APHA 2017 ). TCOD, SCOD, TN, NH4+-N, TP, PO4− 3, S− 2, and SO4− 2 were analyzed following HACH instructions using a spectrophotometer (HACH DR/3900 HACH, Germany). Oxidation–reduction potential (ORP) and pH were analyzed using a pH meter (JENWAY, Manchester, UK). Resistivity, salinity, electrical conductivity (EC) and total dissolved solids (TDS) were analyzed by multi-meter (EUTECH Instruments, Madrid, Spain). TS and VS were analyzed according to Standard Methods for the Examination of Water and Wastewater (APHA 2017 ) using an oven at a temperature of 105°C and 550°C, respectively. TVFA and TotA were analyzed using titration according to (APHA 2017 ) standard method. Total biogas production was measured by sucking the biogas collected in 2-L glucose bag using a 100-mL airtight syringe. The biogas composition was measured using a gas analyzer (Geotechnical instrument gas analyzer, Leamington Spa, UK).
6
Comprehensive Water Quality Analysis Protocol
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The research team ensured that the collected samples are analyzed within the same day of collection. Chemical parameters were mainly measured using the combination of spectrophotometer (Hach DR3900, HACH/LANGE, Berlin, Germany), colorimeter (Hach DR900, HACH/LANGE, Berlin, Germany), with standard reagents as well as the test kits provided by Hach Company, [31 ]. The Standard Operating Procedure for GLNPO Turbidity (The U.S. Environmental Protection Agency Great Lakes National Program Office, Washington, D.C.) was used for the analysis of turbidity [32 ], and the American Public Health Association (APHA) 4500-Nor, Washington, D.C., United States, was used for the analysis of total phosphorous, while lab pH-meter (Hach Co, HACH/LANGE, Berlin, Germany) was used for pH as well as the ultraviolet-visible (UV-V) spectrophotometer (PE-5400UV) pr-in ECOCHEMICAL, St. Petersburg, Russia was used for color measurements. The TSS in the samples was determined using Hach TSS portable hand-held turbidity meter. In general, the analyses of all the studied samples were accomplished following the recommendations in the APHA Standard Methods for the Examination of Water and Wastewater [33 ].
7
Wastewater Sludge Characterization Protocol
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The pH was quantified with a Hannah pH–meter (HI98130, Hanna Instruments, Woonsocket, RI, USA). A HACH 2100N turbidity meter (Hach Company, Colorado, CO, USA) HACH DR 3900 within the wavelength of 455–635 nm (Hach Company, Colorado, CO, USA) was used for the COD and TKN measurements. The sludge samples were characterized using scanning electron microscopy and energy dispersive X-ray (SEM/EDX, FEI Nova NanoSEM 450 coupled with EDT and TLD detector) equipment based at the University of Cape Town, South Africa. The biogas composition analysis was carried out with a Geotech Biogas 5000 Portable Biogas Analyser (ISO17025) supplied by Keison Products, (Chelmsford Essex, UK).
8
Wastewater-Dye Solution Characterization
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A stock solution of 2.5 M BB41 was prepared by diluting 30.16 g of the dye in 5 L deionized water coupled with 20 L of local wastewater [3 ]. HCl and NaOH were used for pH adjustment, measured with a Hannah pH—meter HI98130 (Hanna Instruments, Woonsocket, RI, USA). The main composition of the synthesized effluent was pH (7.55), COD (176.87 mg/L), color (54.8 Pt.Co) and turbidity (12.28 NTU). The COD and color was measured with a HACH DR 3900 (Hach Company, Loveland, CO, USA), while turbidity was measured by using a HI 98703 Portable Turbidimeter (Hanna Instruments, Veneto, Italy).
9
NTR1 and TNF-α Modulate Gene Expression
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Gene expression changes caused by NTR1 and TNF-α were analyzed using RT-qPCR. HAOBs (1×106) were pre-treated with or without 2.5 and 20 µmol/l of NTR1 and then placed into an inflammatory microenvironment induced by TNF-α. Total RNAs were extracted from the cells using TRIzol® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols, and then the concentration of the total RNAs was determined using UV spectrophotometer DR3900 (Hach). A TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific, Inc.) was used according to the manufacturer's instructions to synthesize first-strand cDNAs. RT-qPCR was then performed with BlazeTaq™ SYBR Green RT-qPCR Mix 2.0 (BioCat GmbH) according to the manufacturer's protocols using PCR instrument CFX96 Touch (Bio-Rad Laboratories, Inc.), and the reaction system with 10 µl total volume was prepared. The conditions for RT-qPCR were 1 cycle of 95°C for 30 sec, followed by 40 cycles of at 95°C for 10 sec and at 60°C for 30 sec. The comparative cycle threshold method (2−ΔΔCq) was used to calculate the relative expression of each mRNA (22 (link)). The experiment was repeated three times. The primer sequences of the genes used in the experiment are listed in Table I .
10
Wastewater Characterization after Chlorination
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Wastewater samples were collected after chlorination process at a local wastewater treatment facility in KwaZulu-Natal, South Africa. The samples were characterized according to APHA [34 ]. Table 1 shows the characterization of the wastewater used in this study. A turbidimeter (Hach, 2100N, Loveland, CO, USA) was used to analyse turbidity, while a spectrophotometer (HACH, DR 3900, Düsseldorf, Germany) was used to analyse color, phosphate, COD, TSS, and absorbance at a wavelength of 465 nm, 880 nm, 620 nm, 810 nm and 620 nm, respectively.
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