2.1. Microbial isolation and growth curve measurement
During the stable operational phase of the reactor, sludge samples were collected to identify individual microbes within the HRCS microbial consortia. A solid medium for culturing was prepared by mixing synthetic wastewater with 1.5% agar (Miller, Difco, B&D, Denmark). The composition of the synthetic wastewater is outlined in Table 1. This mixture was sterilized at 121°C for 20 min before being poured into Petri dishes to form solid media. The HRCS sludge underwent dilution between 101 and 105 and was then evenly spread on the agar surface. Following 24 h of incubation at 25°C, eight distinct colonies were picked based on their unique visual features like size and color. These colonies were then transferred onto fresh agar plates for additional analysis. Cultivation of these microbial isolates was done in 2.5% Luria-Bertani (LB) (Miller, Difco, B&D, Denmark) liquid medium overnight. DNA extraction from these cultures was performed using the SPINeasy DNA Kit for Soil (MP Biomedicals, USA). Polymerase chain reaction was conducted on DNA extracted from the eight isolated strains. This process utilized the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). To identify the V3-V4 regions of the 16S rRNA genes, amplicon sequencing was performed using sanger sequencing method (Macrogen, SEOUL, Republic of Korea). Subsequent analysis to ascertain the nucleotide sequences of the 16S rRNA genes from each isolate was carried out using the BLAST tool, accessible through the NCBI website (http://www.ncbi.nlm.nih.gov/).
Table 1
Characteristics and concentrations of synthetic wastewater.
Composition | Unit | Concentration and value |
Total COD | mg/L | 335.2 ± 25.0 |
Particulated COD | mg/L | 166.8 ± 14.1 |
Soluble COD | mg/L | 168.4 ± 10.8 |
Total nitrogen | mg/L | 52.6 ± 3.5 |
Ammonia nitrogen | mg/L | 47.9 ± 0.6 |
Total phosphorus | mg/L | 6.2 ± 0.6 |
Total suspended solids | mg/L | 137.2 ± 22.1 |
Volatile suspended solids | mg/L | 92.3 ± 24.8 |
pH | - | 6.82 |
To preserve the microbial colonies cultured from the HRCS consortia, they were stored at 4°C. Growth curve analysis involved inoculating a single colony in a 500 mL Erlenmeyer flask with 250 mL of sterilized 2.5% LB medium. The flask was covered with a sterile cotton plug for aeration and placed in a shaking incubator at 150 rpm and 25°C. Sampling occurred every 3 h to measure optical density at 600 nm (OD600) using a spectrophotometer. When OD600 reached 1, the culture was diluted to an OD600 of about 0.25, followed by 12 h of incubation with hourly OD600 measurements for constructing a comprehensive growth curve. The specific growth rate (µ) and doubling time were determined using equations (1) and (2) as described15.
µ (h− 1)= \(\frac{ln{X}_{2}-ln{X}_{1}}{{t}_{2}-{t}_{1}}\) (1)
In Eq. (1), µ represents the specific growth rate, X2 is the cell concentration at time t2, and X1 indicates the cell concentration at time t1.
Doubling time = \(\frac{ln2}{{\mu }}\) = \(\frac{0.693}{{\mu }}\) (2)
2.2. Reactor setup and synthetic wastewater characteristics
Figure S1 shows the flow diagram of the HRCS reactor, which was inoculated either with AS or with Klebsiella pneumoniae for separate experiments, though both setups utilized the same reactor design and system. The source of the initial sludge inoculum was the Suyeong WWTP, located in Busan, South Korea. The design of the reactors involved cylindrical acrylic containers, each with an operational volume of 2 L. The reactors had dimensions of 30 cm in height and 11 cm in diameter, which included the headspace. Key components such as level sensors, stirrers mounted above, and pumps for both peristaltic action and aeration were integrated into each reactor setup. The operation of the HRCS system involved an HRT of 2 h and a SRT of 1.04 d. This was achieved through an inflow rate of 24 L/d and a 50% exchange ratio. The sludge disposal process took place during the reaction phase, which included continuous mixing. The entire cycle of the system lasted 60 min. The assigned times for each phase were 20 min for stabilization, 20 min for contact (including filling and reacting), 14 min for settling, and 1 min for idling. In the stabilization phase, the system did not receive additional substrate and maintained a working volume of 1 L. Both reactors received consistent and sufficient aeration at 1 L/min. The influent from the storage tank was stirred periodically for consistency. Operational temperatures of the reactors varied between 14°C and 21°C without the application of temperature control.
The formulation of the synthetic wastewater, which included both particulate and soluble elements such as organic compounds, nitrogen, and phosphorus, followed the guidelines by Aiyuk and Verstraete (2004)16. Primary constituents of this synthetic wastewater included Urea, NH4Cl, CH3CO2Na·3H2O, peptone, MgSO·7H2O, KH2PO4, CaCl2, starch, milk powder, dried yeast, and soy oil. The wastewater also contained crucial trace elements for bacterial growth, such as Cr(NO3)3·9H2O, CuCl2·2H2O, MnSO4·H2O, NiSO4·6H2O, PbCl2, and ZnCl2. The amounts of these compounds were adjusted to achieve desired levels of chemical oxygen demand (COD), total nitrogen (TN), and total phosphorous (TP). Both influent and effluent underwent concurrent analysis during each evaluation phase to account for potential organic compound degradation over time. To maintain accuracy in analysis, especially due to the particulate nature of the wastewater, continuous stirring was ensured for all samples. Table 1 presents detailed compositions of the synthetic wastewater.
2.3. Biochemical methane potential assessment
The exploration of biogas production through the anaerobic breakdown of sludge from HRCS systems led to conducting a biochemical methane potential (BMP) test. The inoculum, consisting of mesophilic anaerobic sludge, originated from the Suyeong WWTP in Busan, South Korea. This inoculum was initially stored at 4°C and then warmed to 35°C for 2–4 d before the experiment to reduce any pre-existing methane. The experiment design included a substrate-free control to monitor background methane production. Additionally, a test with active sludge as the substrate served as a positive control, assessing the seed sludge's effectiveness. The ratio of inoculum to substrate in each experiment was maintained at 2:1, based on volatile solids (VS)17. Throughout the study, the pH naturally ranged from 6.8 to 8.2. The anaerobic digestion tests involved three replicates in 155 mL serum bottles, each with a working volume of 100 mL and a 55 mL headspace. The inoculum and substrate were introduced into these bottles, which were then sealed with butyl rubber stoppers and aluminum caps. An anaerobic environment was established by purging with N2 gas for 3 min. The bottles were incubated under mesophilic conditions at 35°C. Biogas production was measured every 2–3 d using a syringe, with methane concentration determined by GC-MS (Nexis GC-2030, Shimadzu, Japan) featuring a GC-TCD. The calculation of methane production took into account the headspace volume in the serum bottles, as detailed in Eq. (3)17.
$${\text{V}}_{\text{C}\text{H}4}\left(35 ^\circ \text{C}\right)={\text{C}}_{1}\left({\text{V}}_{0}+{\text{V}}_{1}\right)-{\text{C}}_{0}{\text{V}}_{0}$$
3
Where, VCH4 designates the methane volume produced (mL). Variables C1, V0, V1, and C0 represent the methane content in the sampled biogas (mL), the volume of the headspace (mL), the volume of biogas produced during the sampling (mL), and the methane content in the biogas from the prior sampling (mL/mL), respectively.
The derived methane volume from Eq. (3) underwent normalization for standard temperature and pressure conditions. This adjustment adhered to standard gas laws, as stipulated in the German standard protocol (VDI: 4630, 2006) and is depicted in Eq. (4)18.
$${\text{V}}_{\text{C}\text{H}4}\left(\text{S}\text{T}\text{P}\right)={\text{V}}_{\text{C}\text{H}4}(35 ^\circ \text{C}) \times \frac{(p-{p}_{w})\times {T}_{0}}{{p}_{0}-T}$$
4
In this context, VCH4 denotes the dry gas volume under standard conditions (mLN). Parameters VCH4 (35°C), p, pw, To, po, and T stand for gas volume at 35°C (mL), gas pressure during reading (hPa), water vapor pressure as a function of ambient temperature (hPa), standard temperature (273 K), standard pressure (1013 hPa), and ambient or gas temperature (K) respectively.
2.4. Analytical methods and calculations
To evaluate organic matter removal efficacy, consideration was given to both biodegradable and non-biodegradable substrates through COD measurements. The concentrations of COD, TN, TP, and NH4+–N were analyzed from influent and effluent samples using HS–COD–MR, HS–TN(CA)–H, HS–TP–H, and HS–NH3(N)–H kits, respectively, provided by HUMAS, Republic of Korea, and analyzed with a water analyzer (HUMAS, HS-3300, Republic of Korea). Samples were measured from a uniformly mixed state and analyzed without filtration. After passing samples through 1.2 µm glass microfiber filters (GF/C filters; Whatman, UK), soluble COD was measured. The difference between total COD and soluble COD provided the particulated COD values. For NH4+–N, 0.45 µm filters excluded fine particle and color influences. Concentrations of solid entities like total suspended solids (TSS) and volatile suspended solids (VSS) were analyzed using standard methods19. The SRT was calculated using the formula as Eq. (5).
SRT = \(\frac{{V}_{reactor} \times {\left[VSS\right]}_{reactor}}{{Q}_{waste} \times {\left[VSS\right]}_{waste}+ {Q}_{effluent} \times {\left[VSS\right]}_{effluent}}\) (5)
Where, SRT stands for sludge retention time (d), Vreactor denotes the reactor volume (L), and [VSS]reactor represents the VSS concentration inside the reactor (g-VSS/L). Qwaste is the waste flow rate (L/d), with [VSS]waste indicating the VSS concentration in excess sludge (g-VSS/L). Qeffluent signifies the effluent flow rate (L/d), and [VSS]effluent represents the VSS concentration in the effluent (g-VSS/L). To ensure consistent SRT and mitigate biofilm formation potential, daily scraping of reactor interior walls occurred. During reactor operation, the observed sludge yield (Yobs) was calculated using Eq. (6)20.
Yobs = \(\frac{{\text{Q}}_{\text{w}\text{a}\text{s}\text{t}\text{e}} \times {\left[\text{V}\text{S}\text{S}\right]}_{\text{w}\text{a}\text{s}\text{t}\text{e}}+ {\text{Q}}_{\text{e}\text{f}\text{f}\text{l}\text{u}\text{e}\text{n}\text{t}} \times {\left[\text{V}\text{S}\text{S}\right]}_{\text{e}\text{f}\text{f}\text{l}\text{u}\text{e}\text{n}\text{t}}}{{\text{Q}}_{\text{i}\text{n}\text{f}\text{l}\text{u}\text{e}\text{n}\text{t}} \times {\text{t}\text{C}\text{O}\text{D}}_{\text{i}\text{n}\text{f}\text{l}\text{u}\text{e}\text{n}\text{t}}- {\text{Q}}_{\text{e}\text{f}\text{f}\text{l}\text{u}\text{e}\text{n}\text{t}} \times {\text{t}\text{C}\text{O}\text{D}}_{\text{e}\text{f}\text{f}\text{l}\text{u}\text{e}\text{n}\text{t}}}\) (6)
Where, Yobs denotes observed sludge yield (kg-VSS/kg-COD), with Qwaste, Qeffluent, [VSS]waste, and [VSS]effluent defined as earlier. Qinfluent is the influent flow rate (L/d), tCODinfluent refers to total COD concentration in the influent (mg/L), and tCODeffluent pertains to the total COD concentration in the effluent (mg/L). The assumption made was that the VSS exiting the reactor comprised solely sludge, excluding organic matter. The specific methane yield (SMY) was calculated using Eq. (7).
SMY = \(\frac{{\text{V}}_{\text{c}\text{u}\text{m}\text{u}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}} \left(\text{L}\right) \times (2\times 32 \text{g}/\text{m}\text{o}\text{l}) \times (1/{\text{V}\text{S}}_{\text{r}\text{e}\text{m}\text{o}\text{v}\text{e}\text{d} \text{s}\text{l}\text{u}\text{d}\text{g}\text{e}}) }{22.4 \text{L}/\text{m}\text{o}\text{l}}\) (7)
Where, SMY indicates the specific methane yield (g-CODCH4/g-VSremoved sludge), Vcumulative (L) is the cumulative gas volume produced in the BMP test, and 2×32 g/mol is the oxygen mass consumed during methane gas production. 1/VSremoved sludge is the VS content of the discarded sludge (g), and 22.4 L/mol represents molar volume of methane gas under standard conditions.
The carbon recovery rate (CRR) is linked to the SMY and quantifies the amount of methane COD produced per unit of organic COD present in the influent to the reactor. The CRR was determined using Eq. (8).
CRR = \(\text{S}\text{M}\text{Y}\times {\text{Y}}_{\text{V}\text{S}/\text{C}\text{O}\text{D}}\) (8)
Where CRR represents the carbon recovery rate (g-CODCH4/g-CODinf) and YVS/COD is the VS yield per influent COD in the reactor (g-VS/g-CODinf).